Importantly, for Aleksite nanoribbons with a width below 150 nm, a 1D weak antilocalization effect along with 1D universal conductance fluctuations emerges, which transforms into 2D behavior for larger ribbon widthsThree-dimensional topological insulators comprise topologically protected surface states displaying massless, Dirac-like linear dispersion with spin-momentum locking. Electrical conduction through such surface states has been documented to manifest itself in a two-dimensional character of the angle-dependent magnetotransport behavior. Here, we explore the size-dependent electronic properties of nanostructures made of the lead-containing mineral Aleksite, a naturally occurring topological insulator. Compared to its sister compound Kawazulite, a member of the well-studied Tetradymite crystal class, the crystal structure of Aleksite is distinguished by its lack of any counterpart within the group of synthetic topological insulators. Low temperature Hall measurements on thin Aleksite nanosheets reveal a significant carrier mobility on the order of 1000 cm Firstly, it has been predicted to possess a relatively large band gap of the order of 0.3 eVAccording to theoretical studies, minerals belonging to the Aleksite group represent alternative candidates for natural TIsgaps see . Thirdly1Bi2.06Te1.79S1.54 The Aleksite crystals investigated in this work were purchased from Mikon Mineralienkontor  and originate from the Kochkar district in southern Urals, Russia. By energy-dispersive X-ray spectroscopy (EDX), their detailed composition was determined to be Pb1.54 see . IndividExfoliated Aleksite flakes see  were subi.e., well within the bulk conduction band. Similar strong n-type doping has been experimentally observed for PbBi4Te72Te4 and PbBi4Te7 (both below 200 meV)An indication that Aleksite constitutes a natural TI could be gained by angle-resolved photoemission spectroscopy (ARPES) on a tiny single crystalline domain of the mineral. After cleaving the Aleksite crystal in air, it was directly transferred to the UHV chamber and cooled to 100 K. Owing to the very small sample size, only a blurred experimental surface band structure could be recorded . Nonethen in the range of 2.2–4.8 × 1025 m−3. This finding is in agreement with the ARPES results, thus confirming that the Fermi level is located within the bulk conduction band. Moreover, Hall mobilities between 810 and 2006 cm2/(Vs) were extracted from the data, which is slightly higher than reported for KawazuliteIn order to explore the electrical properties of Aleksite, we prepared thin nanosheets and -ribbons via micromechanical cleavage of the microcrystals. In B = 12 T). Consequently, the magnetotransport behaviour of such type of samples could not be evaluated. Nonetheless, this task could be achieved using thin Aleksite ribbons, which exhibit a larger resistance of several 100 Ω. The corrected low-field magnetoresistance ΔRxx(B) = Rxx(B) −  Rxx(B  = 0) of the ~140 nm wide ribbon in B-field, a pronounced weak anti-localization (WAL) resistance minimum can be observed.The very low four-terminal resistance of thin, 5–10 μm large Aleksite flakes on the order of several Ohms resulted in low resistance changes of only several 100 mΩ at the highest external magnetic field (B-field component B) = α(e2/h)[In(Bϕ/B) − Ψ(1/2 + Bϕ/B)], where h is Planck’s constant, Bϕ is the dephasing field, α is a parameter related to the number of conducting 2D channels, and Ψ(x) is the digamma function. The fit of the data in lϕ ≈ 700 nm from the relation α  = δσ(B) − δσ(B = 0):where lso. The corresponding fits of the magnetoconductivity at different temperatures are presented in ln = 496 nm and a phase coherence length of lϕ = 728 nm at T = 1.4 K. Analysis using different sizes of the fitting window  yielded similar values. Furthermore, using the conductance correction δσ at B = 0 T in connection with equation lso = 70 nm is estimated. The low spin–orbit length reflects strong spin–orbit interaction in Aleksite, in analogy to other TIs like Bi2Se3which is independent of T−1/2 if the system is 2D and T−1/3 if it is 1De2/h. For 1D systems where W < lϕ,tot < L the UCF amplitude should be determined by the ratio of the total phase coherence length L of the system, i.e., L = 500 nm and lϕ,tot = 295 nm at 1.4 K , where the phase coherence lengths were taken from With increasing temperature, the UCF amplitude decreases as a result of the reduced phase coherence length. In general, the root–mean–square of the oscillations and the phase coherence length are related by W, and rms[Δσxx] was found to scale linearly with the phase coherence length in wider ribbons as expected for 2D behaviour. All the above observations taken together provide evidence for a cross–over from 2D to 1D diffusive transport when the ribbon width falls below approximately the total dephasing length lϕ,tot which should be dominated by the smaller value of lϕ and ln.The results of further magnetotransport experiments on Aleksite ribbons of larger width are summarized in In summary, we have gained evidence that the mineral Aleksite is a naturally occurring TI that introduces an additional type of crystal structure within this rapidly expanding class of materials. Despite its pronounced deviation from non-stoichiometric composition, Aleksite displays electrical transport performance comparable to the thoroughly investigated synthetic TIs like the Tetradymite-type bismuth chalcogenides. The magnetotransport properties of Aleksite ribbons in dependence of width clearly signify a cross-over from 1D to 2D behaviour. However, it should be emphasized that this observation alone is no direct proof for the contribution of topological surface states to the electrical transport. Nonetheless, the existence of a low-dimensional transport channel with relatively large coherence makes Aleksite a very interesting material for future studies.For electrical characterization, the samples were placed in a pumped bath cryostat (Oxford Instruments) at 1.4 K. AC measurements were performed using two lock-in amplifiers . The DC Hall experiments were carried out using an ADwin Gold card combined with a voltage amplifier (Stanford Research SR560) and a current amplifier (Femto DLPCA-200).2, and then transferred on a TEM grid. Electron diffraction was studied by a Philips CM200 Transmission Electron Microscope using electron energy of 80 kV. Scanning TEM (STEM) imaging was carried out with a Nion UltraSTEM 100 operated at 60 kV.With the aid of a Scotch tape, a thin layer was exfoliated from a bulk Aleksite crystal, deposited onto a Si substrate covered with 300 nm of SiO2), thin (~0.25 mm) piece of Aleksite was set-apart from a bigger mineral piece using a scalpel. The piece was then glued using electrically conductive glue on the sample holder. Immediately before introduction into UHV, the sample surface was exfoliated with a Scotch tape. The ARPES measurements were performed with a hemispherical SPECS HSA3500 electron analyzer characterized by an energy resolution of about 10 meV. Monochromatized He I (21.2 eV) radiation was used as photon source. During measurements the sample was cooled with the aid of liquid nitrogen to 100 K.A small .


Leymus chinensis depended on climate variations or genetic differentiations at 18 wild sites along a longitudinal gradient from 114 to 124°E in northeast China and at common garden condition of transplantation. Demographical, morphological and physiological phenotypes of 18 L. chinensis populations exhibited significant divergence along the gradient, but these divergent variations narrowed significantly at the transplantation. Moreover, most of the phenotypes were significantly correlated with mean annual precipitation and temperature in wild sites, suggesting that climatic variables played vital roles in phenotypic divergence of the species. Relative greater heterozygosity (HE), genotype evenness (E) and Shannon-Wiener diversity (I) in western group of populations suggested that genetic differentiation also drove phenotypic divergence of the species. However, neutral genetic differentiation (FST = 0.041) was greatly lower than quantitative differentiation (QST = 0.199), indicating that divergent selection/climate variable was the main factor in determining the phenotypic divergence of the species along the large-scale gradient.Elucidating the driving factors among-population divergence is an important task in evolutionary biology, however the relative contribution from natural selection and neutral genetic differentiation has been less debated. A manipulation experiment was conducted to examine whether the phenotypic divergence of  It has long been recognized that geographic variation in phenotypes of an organism can include both environmental and genetic components, thus determining the mechanisms that cause such variation is one of the basic objectives of ecological and evolutionary studies24581012ST) and neutral genetic differentiation (FST) among populations could be effective1112ST) was the analogue of QST, and also informative to compare the relative influence of genetic adaptation, phenotypic plasticity and genetic drift as causes of population differentiationST/QST > FST, phenotypic divergence exceeds neutral expectation and is likely to have been caused by directional selection for environmental adaptability. For instance, the aridity gradient was demonstrated to be the main driving factor for the population differentiation in molecular markers was much lower than in quantitative traits in wild barleyST/QST ≈ FST, the inference is that phenotypic divergence could have been achieved by molecular differentiation among subpopulations. If PST/QST < FST, this pattern is suggestive of uniform selection across the populations12To determining the relative functions of environmental and genetic factors, the comparison of quantitative differentiation in common garden experiments (QLeymus chinensis (Trin.) Tzvel., a perennial rhizomatous C3 cosmopolitan grass, is widely distributed at eastern end of Eurasian steppe zone, from western part of the Northeast Plain to eastern part of the Mongolian Plateau, China. The xerophytic traits, e.g. thick rhizome systems and high plasticity in leaf traits, enable the species to successfully tolerate and/or resist drought when soil moisture is less than 4% in dry seasons in desert steppes1617L. chinensis, aiming to better understand the driving mechanism of plant phenotypic variations and adaptive strategies to stress conditions under broad scale climate change. Furthermore, our specific aims were to examine: (1) the obvious divergence of quantitative traits in L. chinensis and their relationships with climate variables along the gradient; (2) the roles of neutral genetic differentiation on phenotypic divergence.L. chinensis at the east end  were more than 3-fold of that at the west end  , mean annual temperature (MAT) (P < 0.05) and soil water content (SWC) (P < 0.01), but negatively correlated with elevation (P < 0.01). Vegetative shoot densities and reproductive shoot densities from eastern sites  were nearly 2-fold as those from western ones . The vegetative shoot densities were significantly and positively correlated with MAP  and SWC . The reproductive shoot differentiation ratio (RSDR) in desert steppes  was 57.9% lower than that in meadows (01–10 sites) and typical deserts   .2 = 0.787, P < 0.01). Both vegetative shoot heights and stem node numbers were significantly and positively correlated with MAP in wild sites (P < 0.05) (P < 0.05), but negatively with elevation (P < 0.01). Average reproductive shoot height was 40.1 cm in desert steppes, reduced by 34.7% comparing to 61.4 cm in meadows (P < 0.01)  to 25.1 cm at the west end . Corresp < 0.05) , MAT (P  meadows . In tran < 0.01) .P < 0.05) (P > 0.05). Average leaf length at transplantation (27.4 cm) was 31.1% higher than that of wild sites  (P < 0.05) (P < 0.01) (Leaf lengths at meadows (20.7 cm) were 12.6% higher than that at typical steppes and desert steppes (18.6 cm) in wild sites ( < 0.05) . On the  < 0.05) . Leaf le < 0.05) , but neg < 0.01) .P < 0.01), but there was no significant difference between corresponding eastern (0.65 cm) and western (0.68 cm) populations at transplantation (P > 0.05) (2 (07 site) and minimum 5.1 cm2 (14 site) in wild sites. Average leaf area at transplantation (11.4 cm2) was 59.5% higher than that from wild sites  (−) was 22.4% higher than that of transplantation populations . LMA was significantly and negatively correlated with MAP and SWC, but positively with elevation in wild sites (P < 0.05).Unlike leaf length, leaf width in western sites (0.68 cm) was 18.8% higher than eastern ones (0.57 cm) in wild samplings ( > 0.05) . Leaf ar < 0.01) . Leaf maP < 0.01) and 51.3% (P < 0.01) greater than that from the desert sites, respectively. Seed weights and spike lengths were significantly and positively correlated with MAP and MAT in wild sites (P < 0.01)  .P < 0.05) (P < 0.05).Physiological adjustments/phenotypes of the species differed significantly along the broad gradient . Relativ < 0.05) . RWC was−1.DW) were about 2-fold of that in meadows and typical steppes  (−1.DW), then decreasing at the west end, and the average value in typical steppes (11.320 ug mg−1.DW) were 46.4% higher than those in meadows and desert steppes , respectively (P > 0.05), whereas soluble sugar content of western sites was 18.1% higher than that of eastern ones (P < 0.05). Proline and soluble sugar contents reduced by 33.7% and 35.7% in transplantation compared with their wild populations (P < 0.01) (P < 0.01) (P < 0.01) along the gradient.Both proline and soluble sugar contents of the species increased from the east to the west along the gradient. Proline contents from desert steppe sites (0.874 ug mg < 0.01) . Howeverectively . At tran < 0.01) . Proline < 0.01)  and SWC P < 0.01). Average Chl (a + b) content in transplantation (5.388 mg g−1) was more than 3-fold compared with the wild samples  (P < 0.05) along the gradient. Average Chl a/b ratio (1.281) in transplantation dropped by 41.1%, compared with the wild populations  (Leaf nitrogen (N) contents and Chlorophyll (Chl) (a + b) exhibited slight variation along the gradient . Leaf N  < 0.01) . Unlike  < 0.01)  and sign < 0.01) .E): 0.628, genotype evenness (E): 0.098 and Shannon-Wiener diversity (I): 1.105) were significantly higher than that in western ones , with average of 18 population HE = 0.606, E = 0.092, I = 1.046 (P < 0.01). There were 6.16% differentiation between eastern - western sites (P < 0.01) and 4.44% among three vegetation types , but not significant differentiation between soil pH groups   to 0.100 (02 and 11 site populations), with average 0.041  . Low FSTge 0.041 . Mean pa.041 (ST .ST = 0.199 at transplantation) for the 13 statistic traits  and Chl a/b) than that of neutral markers (FST = 0.041 for 15 microsatellite loci) among the 18 populations (ST = 0.299) was 49.5% higher than QST value (0.199) in transplanted ones (P < 0.01).There was a more pronounced level of differentiation in phenotypes  with MAP indicated that obvious divergence of quantitative traits in L. chinensis was strongly related with moisture conditions along the longitudinal gradient  supported our first hypothesis that the phenotypic divergence of L. chinensis was strongly driven by climate forces along the large-scale gradient.Physiological regulations in gradient . Free pradient 26. More soadient 26. Throughle sugar . The sigle sugar , as wellle sugar , showed hinensis . ConcostL. chinensis populations  and genetic differentiations were probably attributed to the uplift of Mongolian plateau at site 11: elevation suddenly lifted from 276 m to 421 m, SWC dropped from 12% to 9% and vegetation types transformed from meadows (site 10) to typical steppes (site 11)  . Both enST (0.041) from SSR datasets e.g. average FST = 0.25 for 13 widespread species; FST = 0.22 for 23 outcrossing speciesST values, 34e.g. in similar environment) via seeds had a positive effect on the population fitness by bringing in already selected genotypes, thus exaggerated the genetic differentiation36L. chinensis population differentiation between adjacent populations under similar environments.Selection and drift can promote genetic differences if populations are sufficiently isolated, however, gene flow prevents differentiation and local adaptation30datasets  in L. chST (0.199) estimated from L. chinensis quantitative traits at transplantation were more than 4-fold of the FST values (0.041) from microsatellite loci  in wild populations, demonstrating that quantitative differentiation in L. chinensis was significantly affected by the environmental changes  than FST (0.003) were observed in maize, suggesting quantitative traits appear to be under strong divergent selectionST–FST comparison and neutral molecular differentiation supported our second hypothesis that neutral genetic differentiation played an important role in phenotypic divergence, but divergent selection/climate variables was the main factor in determining the phenotypic divergence of the species along the large-scale gradient.Qite loci , suggest changes . There walues 0.0 from miche PST 0.99 in wilL. chinensis across the longitudinal gradient. This research highlighted the combination of phenotypes divergence and genetic differentiations and systematical analysis of quantitative traits in both wild sites and transplantation, providing reliable information about the driving factors for the phenotypic divergence which could not been addressed in wild Our findings suggested that climate changes (precipitation and temperature) and molecular genetic differentiation both drove quantitative differentiations of L. chinensis grasslands and greenhouse from 2012 to 2014. Eighteen populations/sites were selected along a longitudinal gradient in northern China, ranging from 43°24′ to 44°36′ N, 114°34′ to 124°16′ E, with approximate 900 km west-to-east . RSDR refers to the reproductive shoot density as a percentage of the total population densityAt least two ha of typical native nd fully expanded leaves  in each population were sampled randomly from mature plants. Leaf traits  were measured using a flatbed scanner connected to a personal computer running image analysis software. Leaf samples were placed in perforated paper bags and oven-dried at 80 °C for 24 h and then weighed to measure dry leaf mass. LMA was expressed as leaf dry mass per area (mg cm−2).Twenty 23) from each site and transplanted into a greenhouse in Institute of Botany, the Chinese Academy of Sciences (Beijing). Rhizomes were placed into pots (25 cm diameter) with near-neutral organic soil (pH 7.3–7.5). The temperature and humidity in the greenhouse were controlled between 20–30 °C and 50–60%, respectively. Each pot was watered to maintain the soil water content above 20% with running water every other day in the first month, then 100 ml twice a week. After 3 months of growth, leaves were collected for morphological and physiological measurements.Underground rhizomes of each population were taken from 10 monoliths (25 × 25 × 30 cmnd fully expanded leaves in each site were sampled randomly from mature plants at about 9:00 am. Fresh weight (FW), turgid fresh weight  and dry weight (DW) were measured. Relative water content45Twenty 2For proline, soluble sugar, leaf N and Chl content analyses, leaves from wild sites and transplantation were oven-dried under 105 °C for 20 min, then at 80 °C for 24 hr to a constant weight and grounded to pass through a 100-mesh screen. Proline content was estimated by the acid-ninhydrin methodThirty leaves from independent patches were collected randomly at each site then immediately dried in the silica gel and a total of 540 individuals were sampled. Genomic DNA was extracted and the concentration was examined by agarose gel electrophoresis. For microsatellite analysis, 80 pairs of primers were selected for candidates from previous publications5051The soil samples were taken with a cylindrical soil sampler  for the 0–15 cm layer. Among 7 and 14 soil samples were collected randomly per site (100 × 100 m) for determination of soil properties. Gravimetric soil water content was measured by oven-drying samples at 105 °C for 24 h, and the equation for calculating thehttp://www.worldclim.org/).Soil pH was determined by a Model HS-3C pH meter . Soil N contents were measured using Kieldahl methodOne-way analysis of variance (ANOVA) was performed to analyze the differences in morphological and physiological phenotypes among the 18 populations. Linear regression analyses were performed to evaluate the relationship of quantitative traits with environmental factors . All statistics for phenotypes were analyzed using SPSS 20.0 for windowsE, I, and E and population pairwise genetic distances were calculated with Atetra program53ST values were evaluated by Polysat package running on the R platformPopulation genetic analyses were performed on microsatellite dataset scored by the program GeneMarker v2.2.0Quantitative differentiation was estimated by the formula described belowST is a QST analogous that estimated from the wild sampling data12where How to cite this article: Yuan, S. et al. What drivers phenotypic divergence in Leymus chinensis (Poaceae) on large-scale gradient, climate or genetic differentiation? Sci. Rep.6, 26288; doi: 10.1038/srep26288 (2016).


Sphagnum magellanicum occurs in both the Northern and Southern hemisphere, and is commonly used as a model in studies of peatland ecology and peatmoss physiology. Even though it will likely act as a rich source in functional genomics studies in years to come, surprisingly little is known about levels of genetic variability and structuring in this species. Here, we assess for the first time how genetic variation in S. magellanicum is spatially structured across its full distribution range (Northern Hemisphere and South America). The morphologically similar species S. alaskense was included for comparison. In total, 195 plants were genotyped at 15 microsatellite loci. Sequences from two plastid loci (trnG and trnL) were obtained from 30 samples. Our results show that S. alaskense and almost all plants of S. magellanicum in the northern Pacific area are diploids and share the same gene pool. Haploid plants occur in South America, Europe, eastern North America, western North America, and southern Asia, and five genetically differentiated groups with different distribution ranges were found. Our results indicate that S. magellanicum consists of several distinct genetic groups, seemingly with little or no gene flow among them. Noteworthy, the geographical separation of diploids and haploids is strikingly similar to patterns found within other haploid Sphagnum species spanning the Northern Hemisphere. Our results confirm a genetic division between the Beringian and the Atlantic that seems to be a general pattern in Sphagnum taxa. The pattern of strong genetic population structuring throughout the distribution range of morphologically similar plants need to be considered in future functional genomic studies of S. magellanicum.Spore-producing organisms have small dispersal units enabling them to become widespread across continents. However, barriers to gene flow and cryptic speciation may exist. The common, haploid peatmoss  Truly cosmopolitan species occurring at every continent and in all biomes are rare . HoweverSpore-producing organisms, such as lichens and bryophytes, have microscopic dispersal units, generally less than 40 μm ,9. SporeNevertheless, wide distribution ranges of morphologically-defined species do not necessarily reflect high dispersal abilities. Cryptic species occur within widely distributed spore-producing organisms –28; phylSphagnum is a nearly cosmopolitan genus, found on all continents except Antarctica. Many species in the genus have circumboreal distributions in the Northern Hemisphere, and a few occur disjunctively in the Southern Hemisphere. Sphagnum magellanicum Brid. (subgenus Sphagnum) is one of them, being one of the most globally widespread peatmosses. It is frequently used as model to understand peatmoss physiology , whereas chromosome number for S. alaskense is currently unknown. Plants of S. alaskense in Alaska were previously misidentified as S. centrale C. Jens [S. alaskense might be gametophytically diploid like S. centrale [S. magellanicum and S. alaskense are dioecious; female and male gametangia  are separated on different gametophytes. Spore sizes ranges from 25 to 30 μm in S. magellanicum [S. alaskense, sporophytes have not been reported [in field . Howeverco-occur ,36. Sphacade ago . Later,  C. Jens , indicat n = 19, , whereasllanicum . Sporophreported , but havSphagnum species, including S. magellanicum [Sphagnum species and a fairly abrupt genetic break has been found in southeast Alaska, separating Alaskan specimens from conspecific plants to the south in western North America [S. magellanicum, as it also has a circumboreal distribution range in the Northern Hemisphere. Thus, we predict genetic similarity between European and eastern American populations [Eastern North American and European populations of several widespread llanicum , are onlllanicum –21,39,40llanicum . However America . We hypoulations , similarulations , but genS. magellanicum is spatially structured across its range, and if so, to evaluate how historical factors and long-distance dispersal might have shaped observed patterns. We also include plants of the morphologically similar S. alaskense to determine whether these two morphologically similar species are separated genetically and if they have different ploidy levels.We aim to assess whether genetic variation in Sphagnum magellanicum is common and often the dominant peatmoss in ombrotrophic mires in the southern arctic, boreal, and nemoral bioclimatic zones in the Northern Hemisphere. In the Southern Hemisphere, S. magellanicum occurs throughout South America. At higher elevations it occurs in tropical alpine [S. magellanicum mainly occurs in the northern antiboreal bioclimatic zone [S. magellanicum [S. magellanicum subsp. grandirete (Warnst.) A.Eddy has been reported from Madagascar [l alpine  and cloul alpine ,44, whiltic zone . The larllanicum , likely llanicum . The subdagascar , but itsS. magellanicum in the Northern Hemisphere is bog (ombrotrophic) and poor fen (minerotrophic) mire communities, and it is mostly absent from rich fens [Sphagnum magellanicum also occurs in moist heaths, on mineral soil in forests and on rock walls in oceanic regions of the Northern Hemisphere. In cloudy high altitude subalpine forests of Costa Rica it can form extensive carpets and small hummocks in small mires on shallow peat, but occurs more commonly around the margins of moraine lakes [The main habitat of ich fens ,49. It oich fens . Sphagnune lakes ,44. In tne lakes ,51.Sphagnum alaskense is found growing in poor to medium fens and mineral edges of ombrotrophic mires in western North America [S. alaskense from western Asia [S. alaskense from western North America in herb. TRH (10 specimens) are from poor and medium rich mire hummocks and lawns of mire margins, and a few collections are from hummocks in forested peatland. It seemingly avoids ombrotrophic (bog) mire sites. This is contrary to haploid S. magellanicum, which is a member of both ombrotrophic and minerotrophic mires, and grows in open mire expanses as well as along mire margins. America . The habern Asia  is more S. magellanicum from herbarium collections to cover as much of its geographical distribution as possible, and most of the species’ habitat range and morphological variation were covered as well. Broad sampling both spatially and ecologically increases the chance of finding genetically divergent lineages within S. magellanicum [S. magellanicum and 25 collections labelled S. alaskense from western North America were sampled. All samples collected were verified morphologically. From each collection, one shoot was picked for DNA analyses.We sampled plants of llanicum . Four heet al. [A small piece from the central part of the shoot apex was used for DNA extraction. Extractions were performed using either the CTAB protocol described in Shaw et al.  or DNeasSphagnum were amplified in S. magellanicum. Microsatellite names and primers are provided in Shaw et al. [et al. [™ Formamide  and 0.15 μL GSLizz500 were mixed for electrophoresis on an ABI 3730 sequencer. GENEMAPPER® software (Applied Biosystems) was used to genotype the alleles.Fifteen microsatellite markers developed for w et al.  and Sten [et al. . Three ttrnL (UAA) 59 exon-trnF (GAA) and tRNA(Gly) (UCC), hereafter trnL and trnG, respectively, were sequenced from a subset of samples from the microsatellite dataset. Thirty-two samples were chosen for DNA sequencing based on microsatellite variation (see below) and geographical distance. PCR amplifications were carried out using puReTaq Ready-To-Go PCR Beads (Amersham Biosciences) in solutions of 22.8 μL H2O, 0.1 μL forward primer, 0.1 μL reverse primer, and 2.0 μL DNA extract. The PCR cycle profile was as follows: 95°C for 5 minutes, 51°C seconds for 45 seconds, 72°C for 45 seconds, with step 2 and 3 repeated 35 times, 72°C for 5 minutes. For trnL, step 2 and 3 were as follows: 54°C for 45 seconds, 72°C for 190 seconds.Two loci from the plastid genome, K) with a maximum of 10. The Structure results were analysed, summarised, and visualised using the online version of Clumpak [K was also estimated by the Clumpak option “Best K”. This method uses the likelihood values of all K values to identify the most likely number of clusters in the dataset. The results of the Structure analyses were plotted on maps using the R packages maps and plotrix in the R Environment [Population structure was explored using clustering analyses implemented in Structure 2.3.4 –58. A Ba Clumpak . The besironment . Geneticironment ,62.FST and Nei’s genetic distances between the groups were calculated using GenAlEx [Genetic variation and distance measures were estimated for the data in two ways: (1) samples grouped by geographical origin and (2) genetically-based groups inferred from cluster analyses. For all geographical and genetic groups the percentage of polymorphic loci, expected heterozygosity, and mean number of alleles were estimated, and pairwise  GenAlEx ,62.S. magellanicum and S. alaskense. All sequences were aligned using ClustalW with default parameters in Mega 6.0 [Nucleotide sequences from two plastid loci were used to reconstruct the phylogenetic relationships among samples of Mega 6.0 . InsertiMega 6.0 . PhylogeMega 6.0 .T = tμ, where t is divergence time in years and μ is mutation rate per year) between the “orange” and “blue” genetic groups inferred by Structure (see below) using IMa [trnL sequences were included in the analysis. A preliminary test was performed following the recommendations in the user manual, while the full scale analysis was performed using 100,000 steps as burn-in followed by 20 mill steps. A geometric heating scheme with parameters set to 0.8 and 0.9 and 30 Metropolis-coupled chains was applied. The upper boundary for population sizes were set to 0.5 and divergence time to maximum 5. The migration parameters were excluded to increase statistical power.An isolation-with-migration model was used to estimate population divergence time  were diploid, as two alleles were found for each sample in 10 of 14 microsatellite loci. Only two loci were fixed for one allele among all diploid samples.One hundred-ninety-five samples were successfully amplified for 14 microsatellite loci see . Fifty-n alleles . SimilarS. magellanicum and S. alaskense were analysed together using the software Structure. With K = 3, the diploid formed one distinct genetic group, while haploid data were divided in two other groups (results not shown). One Chinese individual with only three heterozygous loci grouped with diploid samples at all K values in the Structure analysis. Thus, this sample was considered to be diploid, but with missing alleles. Four diploid samples grouped together with haploid genetic groups. However, these individuals were heterozygous in more than 50% of the loci and, thus, interpreted as diploids. The data were divided in two datasets (one haploid and one diploid), and further analysed separately. One hundred-eleven haploid plants and 82 diploid plants were analysed, respectively.All samples of S. magellanicum plants was inferred using Structure  = 0.99). The probability of K = 6 was 0.01, while the probability of all other K values was 0. A comparison of Structure results for K = 2–7 is shown in K = 5  inferred by Structure, group together in the PCA plot  = 0.99) across all diploid samples. Four samples form a separate group  and 0.50 (±0.08), respectively. The mean number of alleles per locus (NA) is 5.14 (±0.08) in S. alaskense and 6.14 (±1.32) in diploid S. magellanicum. The percentages of polymorphic loci are the same (86%). Nei’s genetic distance between the two is 0.02 and FST was 0.01.Genetic diversity is similar in trnG locus. Two “blue” haploid specimens differ from this haplotype by one substitution and one “purple” haploid plant differ by another substitution. Thus, trnG was not included in phylogenetic analyses. For trnL, five haplotypes  were found , and South American plants share two insertions in their sequences and are identical, except for a plant from Ecuador that differs in one substitution. All haploid samples in the Northern Hemisphere form a clade (no insertions). The three genetic groups, “blue”, “orange”, and “purple”, have different haplotypes, except one “orange” plant sharing the “purple” haplotype. The haplotype network . Using a mutation rate of 4.4x10Sphagnum  and a men mosses , convertS. magellanicum. Surprisingly, in this species the pattern is revealed at the ploidy level; diploid plants belong to the Beringian group whereas haploid plants form a broad Atlantic group. Haploid plants of S. magellanicum are further divided in five genetic groups based on microsatellite makers and these groups differ in distribution ranges. Our findings indicate that gene flow in the widely distributed S. magellanicum is limited between the various genetic groups, and little admixture is evident.Despite their apparently overall lack of worldwide morphological differentiation, many widely distributed Northern Hemisphere peatmosses are divided genetically into Atlantic and Beringian groups ,41. We fSphagnum magellanicum could potentially include several individual species based on our findings [S. magellanicum compared to other Sphagnum species with comparable distribution ranges and genetic diversity levels [findings . Geneticy levels . HoweverS. magellanicum samples have different geographical ranges. Only the “blue” and “orange” groups overlap and their distribution patterns resemble those of the two closely related species S. beothuk and S. fuscum, with the former restricted to the Amphi-Atlantic region, whereas the latter is found across the Northern Hemisphere [Sphagnum species are known: S. affine [S. angermanicum [S. beothuk [S. venustum [S. magellanicum could potentially be another amphi-Atlantic Sphagnum species. In Norway, the “orange” group seems to mainly occupy mire expanse sites, whereas the “blue” group usually is found along mire margins. However, based on field observations, the “blue” group probably has a wider habitat range than the “orange”, at least in areas where the latter is absent.The five genetic groups inferred among haploid misphere . Only fo. affine , S. angermanicum , S. beot beothuk , and S. venustum ,72. The Sphagnum species also have their main distributions in southeastern North America, for example S. fitzgeraldii and S. cyclophyllum [S. fitzgeraldii has a disjunct occurrence in Galapagos Islands, South America, and S. cyclophyllum is found further north along the eastern coast of North America than the “purple” S. magellanicum. The two South American groups within S. magellanicum are geographically allopatric, with the “green” group confined to the southernmost parts and the “pink” occurring in the northern parts. Similarly, the widespread lichen Cetraria acuelata forms one southern and one northern genetic group in South America [S. magellanicum.The distribution of the “purple” group is disjunct, with two specimens in Alaska, but the majority of plants occur in southeastern North America. Other ophyllum . However America . We haveS. magellanicum sampled from Alaska are diploid. Additionally, the majority of plants we examined from eastern Asia are diploid, suggesting that the haploid S. magellanicum probably is rare in the northern Pacific region. This supports the view of Maksimov and Ignatova [S. magellanicum plants in northeastern Asia as S. alaskense. One diploid plant collected in Iowa, U.S.A, together with two samples from southern Yamal Peninsula, Russia, are outliers in the otherwise amphi-Pacific distribution of the diploid plants.All but three plants of Ignatova  who reclS. magellanicum appear to have split before the last glacial maximum. As most genetic groups are differentiated at approximately the same level as the “blue” and “orange” genetic groups as shown by high FST values, the genetic groups may have differentiated because of separation in different glacial refugia with no gene flow among them. The genetic groups differ in their distribution ranges, thus, they may have had different abilities to disperse and colonise after the last glacial maximum. This could be due to differences in spore production, limitations to spore dispersal by for example wind currents, or limitations to the establishment of spores [The last glacial maximum influenced current species distributions and genetic diversity patterns in the Northern Hemisphere . The “orf spores .S. magellanicum survived the last glaciation in Beringia, as seen by their rarity in the region today. On the other hand, the present distribution of diploid plants indicates glacial survival in Beringia or eastern Asia with Holocene expansion into most of the Pacific region. The haploid “purple” group is currently found in an area that remained ice-free for the entire glacial period. The distinct alleles and high genetic diversity in this group indicate that it may well have survived in southeastern parts of North America. Also, survival in eastern North American refugia is likely for the “orange” group, with post-glacial colonisation of Europe across the Atlantic Ocean [Despite being a major refugium for many plants , it appeic Ocean . Alternaic Ocean . Both thSphagnum species usually have higher levels of genetic diversity than haploid species because of the fixation of two alleles at many loci, see for example [S. magellanicum is more diverse than any of the diploid groups. Allopolyploids are not as sensitive to reduction of genetic variation following bottlenecks because of fixed heterozygosity [S. magellanicum/S. alaskense compared to other allodiploid Sphagnum species could be caused by hybrid origin of few individuals of closely related species.Allodiploid  example . It is tzygosity . Thus, rS. magellanicum, indicating that the establishment in South America happened relatively recently [Sphagnum fragment has recently been found in the plumage of a bird migrating between the Northern and Southern Hemispheres [Tetraplodon reached South America [Both genetic groups found in South America have low levels of genetic diversity. Low genetic variation may be a consequence of recent establishment of one or few haplotypes following long-distance dispersal from the Northern Hemisphere . Howeverrecently . Dispersrecently ,80. A Spispheres , indicat America .Sphagnum are relatively young. Even though the clade is old, species diversification likely took place in the Northern Hemisphere during climate cooling in the late Tertiary [S. magellanicum comparing different genetic groups defined by microsatellite data. However, even though the genetic differences found may seem small, together with nDNA differentiation they may nonetheless indicate ongoing or recent speciation in this widespread species [Species within the genus Tertiary . We foun species .Frullania asagrayana is also divided in southern and northern groups in eastern North America based on microsatellites, but they do not differ in nucleotide sequences [F. asagrayana groups was hypothesised to be associated with the Pleistocene glaciations. This could also be the case for the Northern Hemisphere genetic groups we resolved within S. magellanicum; separation in different refugia with no gene flow during the last glaciation and secondary contact and/or overlapping distributions in the Holocene following post-glacial colonisation.The Northern Hemisphere haploid groups constitute one clade, while plants from South America share the exact same plastid sequence as diploid, except one specimen from Ecuador that differs with one substitution. The two “purple” individuals sampled from Alaska might not be as related to the plants from the southeastern United States as inferred from Structure based on microsatellites. Rather, they share plastid DNA with the diploid and South American plants. Plants from southeastern North America assigned to the “purple” group seem to be closely related to plants in the “orange” group based on plastid DNA. The distributions of these two groups overlap slightly in eastern North America. The sharing of one plastid DNA haplotype could indicate recent speciation, with too little time for complete linage sorting . The livequences . The divS. magellanicum and those named S. alaskense using microsatellite or plastid DNA markers. The fact that most plants of S. magellanicum from Alaska and northeast Asia are diploid and genetically similar to S. alaskense likely explains why the two can be difficult to separate in field, and indicate that they may belong to the same taxon. Preliminary morphological examinations indicate that S. alaskense plants seem to differ somewhat from diploid S. magellanicum. This is most easily seen by the more slenderly pointed branches in the outer part of the capitula of the former than the latter. Sphagnum alaskense also seems to have more imbricate branch leaves. However, this differentiation is not correlated with genetic patterns in any of the markers used here. Morphological differences with no genetic differentiation was similarly found within S. palustre L. [We were not able to find any distinction between the diploid plants named ustre L.  and phenSphagnum species have been confirmed using microsatellite markers; for example, S. troendelagicum [S. magellanicum and S. alaskense in Structure analyses did not resolve any potential parents among the haploid genetic groups. Haploid S. lescurii and the allodiploid S. missouricum also formed different genetic groups based on microsatellites [S. lescurii is the maternal parent of the diploid plants [S. magellanicum plants from Iowa and Alaska, U.S.A, and two plants of S. alaskense from British Columbia, Canada, were admixed between haploid genetic groups. Morphological examination shows that the Iowa sample is somewhat different from other diploid S. magellanicum, but still falls within that morphological group. These four samples might reflect independent hybridisation events.Other allodiploid elagicum , as theytellites  even thod plants . Two dipS. magellanicum and S. alaskense are conspecific or indeed different taxa, a thorough comparison of morphological characters has to be done together with molecular analyses using other molecular markers. Until then, all diploid plants of S. magellanicum should be considered to belong to S. alaskense.To further evaluate if the diploid Sphagnum species or even in spore-producing organisms in general. The wide distribution ranges of some Sphagnum species may be more limited than previously assumed based on morphological uniformity. Rather than circum-boreal distributions in Sphagnum, there seem to be main two ranges characterising genetic groups within morphospecies: one covering Asia and Alaska (except the southernmost part) and one mainly occurring in the Atlantic region, but with extensions into western North America (from southern Alaska and southwards) and through Russia into southeastern Asia.Our results provide further evidence that widely distributed peatmosses are genetically structured across their distribution ranges ,41. The S. magellanicum represent cryptic species, or merit formal taxonomic recognition at specific and/or infraspecific rank, requires examination of morphological characteristics that can be used to separate them. Especially, clarifying the status of the “orange” and “blue” genetic haploid groups is important as the groups overlap in the Atlantic region. Pooling them together in for example ecological or genomic studies could give misleading results if they indeed belong to different taxa. Our results show that widespread Sphagnum species may represent lack of morphological divergence and possibly cryptic speciation, rather than being the result of ongoing long-distance dispersal.Whether genetic groups of S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.


QTP), yet only a handful of studies have considered both population genetics and ecological niche context. In this study, we proposed and compared climate refugia hypotheses based on the phylogeographic pattern of Anisodus tanguticus  and present and past species distribution models (SDMs). We detected six plastid haplotypes in two well‐differentiated lineages. Although all haplotypes could be found in its western (sampling) area, only haplotypes from one lineage occurred in its eastern area. Meanwhile, most genetic variations existed between populations (FST = 0.822). The SDMs during the last glacial maximum and last interglacial periods showed range fragmentation in the western area and significant range contraction in the eastern area, respectively, in comparison with current potential distribution. This species may have undergone intraspecific divergence during the early Quaternary, which may have been caused by survival in different refugia during the earliest known glacial in the QTP, rather than geological isolation due to orogenesis events. Subsequently, climate oscillations during the Quaternary resulted in a dynamic distribution range for this species as well as the distribution pattern of its plastid haplotypes and nuclear genotypes. The interglacial periods may have had a greater effect on A. tanguticus than the glacial periods. Most importantly, neither genetic data nor SDM alone can fully reveal the climate refugia history of this species. We also discuss the conservation implications for this important Tibetan folk medicine plant in light of these findings and SDMs under future climate models. Together, our results underline the necessity to combine phylogeographic and SDM approaches in future investigations of the Quaternary evolutionary history of species in topographically complex areas, such as the QTP.Various hypotheses have been proposed about the Quaternary evolutionary history of plant species on the Qinghai–Tibet Plateau ( During the Quaternary climatic oscillations, plant species in the temperate region of the Northern Hemisphere have undergone repeated range contractions and expansions to track favorable habitats , and the Quaternary climatic oscillations probably affected species on the QTP more than those in other regions of similar latitude  may have experienced different Quaternary (late Neogene) histories and they may also exhibit different responses to future climate change  Pasher (Solanaceae) is a perennial octoploid  of the other three Anisodus species ca. 4.35 Mya , the last interglacial (LIG), and the future (2050 and 2070). We aimed (1) to survey the phylogeographic pattern of A. tanguticus, (2) to elucidate its Quaternary evolutionary history based on two lines of evidence, genetic data and SDMs, and (3) to discuss conservation implications for this folk medicine plant in light of phylogeographic pattern and future distribution dynamics.In this study, we employed three maternally inherited plastid DNA regions and the biparentally inherited nuclear internal transcribed spacer (ITS) region . The annealing temperature set for each pair of primers ranged from 53 to 56 °C. PCR was performed with initial denaturing of 7 min at 95 °C followed by 36 cycles of 1 min at 94 °C, 1 min of annealing, 1 min of elongation at 72 °C, and ending with an 8 min extension at 72 °C. PCR purification kits provided by CAS Array  were used to purify the PCR products. Sequencing reactions and successive purifications were performed, and capillary analyses were run on an ABI 3130XL  following the manufacturer's protocols.Total DNA was extracted from the silica gel‐dried leaves using the modified 2× CTAB procedure  was adopted to determine the size of the DNA fragments. The size of resulting fragments for all individuals was carefully checked so as to eliminate false positives.Direct sequencing of the ITS region was carried out for all specimens. Among these, successful sequencing results were obtained for 78 specimens only, most likely due to the wide occurrence of hybrid ITS sequences and the octoploid nature of the species. In these 78 sequences, we found ITS sequences that belong to two significantly different lineages, which differ from each other in a 9‐bp insertion (CCGCGGCGC), and more than 10 mutations that are strongly correlated with the presence or absence of the insertion. We therefore performed an RFLP analysis to discriminate between these two lineages for all populations. SacII was employed to cut the restriction site “CCGC|GG.” The restriction enzyme digestions were performed for 1 h at 37 °C in a 10 KM208637–KM208656.The sequences produced were gathered and aligned using Clustal X version 1.83  using PAUP* 4.0b10 Swofford . MP analP = 0.61). BEAST version 1.6.1  was applied to the age estimation. This substitution rate falls into the range 1.00 × 10−9–3.00 × 10−9 substitutions/site/year for an average synonymous substitution rate of plastid regions in plants  equivalent to expected heterozygosity for diploid data  and differentiation  were estimated following the methods described by Pons and Petit (http://www.pierroton.inra.fr/genetics/labo/Software/Permut). Two differentiation parameters  were compared to infer the occurrence of significant phylogeographic structure using a permutation test with 1000 permutations. Genetic structure was further examined by analysis of molecular variance (AMOVA) , the LGM (ca. 21–18 Ka before present), the present day, and in the future (2050 and 2070 A.D.) using the maximum entropy approach as implemented in the program MAXENT 3.3.3k . After eliminating potentially misidentified samples, decreasing sampling density in easier‐access areas, and including the above 35 points, a total of 157 points were used in subsequent modeling (herbarium samples plus field survey dataset). The altitude variable, and all bioclimatic layers with 19 bioclimatic variables at a resolution of 2.5 arc‐min during the LGM . To avoid strong collinearity of environmental variables, which could lead to model over‐fitting, Pearson correlation for bioclimatic variables was conducted and the less correlated variables were retained . In total, altitude and eight bioclimatic variables  were used to model the distribution of A. tanguticus.To reconstruct the past and current distribution ranges of Species distribution models were constructed according to current environmental factors and then projected for the three different time periods. We employed 20 replicates using 80% of the distribution coordinates for training and 20% for testing, and the model with the best AUC value was adopted. A jackknife test was performed to measure the percent contributions of bioclimatic variables to the prediction for the distributional models. At the same time, the “10 percentile presence” threshold approach was employed because presence‐only data were available. Graphics for each predicted SDM were drawn using DIVA–GIS 7.5.Among the 354 individuals, the total alignment length of the three plastid DNA sequences examined was 1660 bp. Nucleotide substitutions occurred at 10 sites, but no indel existed Table . In combPrzewalskia tangutica Maxim. and Atropa belladonna Linn. as out‐groups, the most parsimonious tree was reconstructed (results not shown) based on 1620 constant, 31 uninformative, and nine informative sites . Two well‐supported clades were apparent in the MP tree, and these were fairly well supported by bootstrap values (91% and 83%). One clade contained haplotypes A, B, and C with a bootstrap value of 91%; another clade contained haplotypes D, E, F, and the bootstrap value was 83%. Except for the infrequent haplotypes C and F, haplotypes comprising these two clades were geographically (mainly) distributed in the eastern and western groups of A. tanguticus, respectively  for each parameter of each run was well above 200, and the ESS of node ages are all above 1 × 10−9 substitutions/site/year , the West and the East Clades diverged from each other 1.69 Mya (95% HPD: 0.83–2.62 Mya). Within the West Clade, the divergence time between haplotype D and the MRCA of haplotypes E and F was estimated to be 0.63 Mya (95% HPD: 0.18–1.16 Mya), and the latter pair diverged from each other 0.19 Mya (95% HPD: 0–0.46 Mya). Within the East Clade, the divergence time between haplotype B and the MRCA of haplotypes A and C was estimated to be 0.47 Mya (95% HPD: 0.08–0.95 Mya), and the latter pair diverged from each other 0.24 Mya (95% 95% HPD: 0.01–0.56 Mya). When applying a substitution rate of 1.00 × 10−9 and 3.00 × 10−9 substitutions/site/year, the divergence between the two clades was estimated to have occurred earlier  or latter , respectively.According to BEAST age estimations based on the plastid dataset and a substitution rate of 1.64 × 10HE) was estimated based on haplotype frequencies in each population (Table NST = 0.823 ± 0.0656) and was higher than GST , indicating a phylogeographic structure across the entire sampling range. Total genetic diversity HT (0.661 ± 0.0643) across all populations was much higher than average within‐population diversity HS (0.131 ± 0.0426), and consequently, population differentiation across the sampling range of the species was high.Plastid haplotype diversity (on Table . PopulatFST = 0.822)  and populations 1–12 (western group) to independent groups, and the proportion of variation among groups was 75.38%  to independent groups, and the proportion of variation among groups was 79.45% , populations 5–8 , populations 9–12 (southwestern populations), and the eastern populations (13–32) to independent groups, and the proportion of variation among groups was 83.44%  Table . The gro2) Table ; Fig. 2. Table . Strong genetic barriers were detected among the eastern Fig. D, northwA. tanguticus have values of 0.984 ± 0.006, 0.978 ± 0.005, 0.983 ± 0.006, 0.980 ± 0.003, 0.982 ± 0.006, and 0.979 ± 0.006 for the present‐day model, the LGM‐MIROC model, the LGM‐CCSM model, the LIG model, the 2050 (rcp45), and the 2070 (rcp85) models, respectively, indicating that our models differed greatly from random expectation. Variable jackknife analyses suggested that Altitude (53.2%), Precipitation of Warmest Quarter (25.3%), Mean Diurnal Range (8.7%), and Mean Temperature of Warmest Quarter (6.0%) are the environmental variables that contributed most to potential distribution modeling. Both LGM models (MIRCO and CCSM) generated potential distributions (habitat suitability index > 0.15) that are largely consistent with each other  for A. tanguticus showed a significant southward contraction and predicted habitat suitability (>0.5) distributions are located in a narrow area close to the southeastern edge of the QTP  was also modeled based on the field survey dataset  predicted a more or less similar distribution, but the 2070 model  predicted a significant northward contraction compared to the present‐day model. Note that more than 26 populations that were sampled in this study fell out of the high habitat suitability (>0.5) predictions in the 2070 model, including all populations from the central‐western and southwestern sampling area.Meanwhile, the potential distribution of narios 200, 2070 wIt is noteworthy that the MAXENT models based on herbarium samples plus the field survey dataset  generate similar potential distributions compared to those based on the field survey dataset alone Figs. , 5.A. tanguticus, detected six plastid haplotypes. Phylogenetic analyses suggested that these six haplotypes were clustered into two well‐supported clades by phylogenetic analyses . SAMOVA suggested that the sampled populations could be divided into four groups, and that distribution of haplotype diversity is uneven among them . Such an age estimate is significantly younger than the widely accepted timescale of the latest QTP uplifts (ca. 3.4 Ma), but overlaps with the earliest known Quaternary glacial in the QTP, the Xixiabangma Glacial that occurred ca. 1.6 Mya , our SDMs for ROC Fig. B or CCSMROC Fig. C models A. tanguticus is an important driver for the relatively low level of genetic variation in this region  between this sampling area and the other sampling areas. As shown in Fig. A. tanguticus is to be integrated into SDMs, the locations of populations in the northwestern sampling area would be covered by the high habitat suitability envelope in both the LGM and LIG models. Under these circumstances, both genetic data and ecological niche support the presence of climate refugia in the northwestern sampling area.At the same time, it is highly likely that most populations in the northwestern sampling area  survived in situ through the LGM and LIG in climate refugia. An important indicator of climate refugia is higher than average haplotype diversity and the presence of private haplotypes , indicates that closely related haplotypes tend to be distributed within the same area; meanwhile, SAMOVA suggests that most genetic variation exists among four groups of populations (FCT = 0.834). Therefore, conservation and management programs should be undertaken in each of these four areas  is much lower than the total genetic diversity HT (0.661 ± 0.0643) across all populations, indicating that overall within population plastid diversity is low. However, many populations possess a higher level of haplotype richness, including populations 1–4, 6, 8, 23, 25; thus, these populations should be allocated preservation priority. In addition to these, our genetic survey will also provide useful genetic background when developing commercial cultivars.ain Yang . Howeverain Yang . To presain Yang . FollowiIn addition to the above suggestions based on genetic diversity, SDMs are also helpful when formulating conservation and management strategies, as they predict potential changes of geographic distribution under future climate change scenarios (e.g., Fordham et al. A. tanguticus, we reconstructed its Quaternary evolutionary history based on both phylogeographic pattern and SDMs. This species may have undergone intraspecific divergence during the early Quaternary, which might have been caused by survival in different refugia during the earliest known glacial in the QTP, rather than geological isolation due to the latest extensive QTP uplifts. Its two lineages are distributed mainly in the eastern sampling area and solely in the western sampling area, respectively, but were mixed with each other in the northwestern sampling area. Only one plastid haplotype was detected in most populations in the eastern sampling area, but five of six haplotypes were found in the northwestern sampling area. SDM models for the present, the LGM and the LIG, suggest that the interglacial period may have had a stronger effect than the glacial periods on the recent evolutionary history of this species. While LGM models suggest eastward contraction in the eastern sampling area and severe habitat fragmentation in the western sampling area, the LIG model predicted that this species probably experienced significant interglacial retreat and subsequent recolonization, especially in the eastern sampling area. The geographic distributions of plastid haplotypes and nuclear ITS‐RFLP types corroborate the LIG model, revealing that most eastern populations may have experienced postinterglacial recolonization, at least post‐LIG recolonization. At the same time, a combination of an asymmetric migration barrier (barrier I in Fig. In the present study, focusing on a QTP endemic octoploid herbaceous species In summary, SDMs are a very useful approach to complement and improve the testing of phylogeographic hypotheses based on population genetic approaches (e.g., Alvarado‐Serrano and Knowles None declared.Figure S1. Potential distribution of A. tanguticus during the present‐day, the LGM and the LIG based on presence locations according to both herbarium samples and field survey in this study.Figure S2. Potential distribution of A. tanguticus under future global warming scenarios based on presence locations according to both herbarium samples and field survey in this study.Click here for additional data file.


Salix amygdaloides, S. discolor, S. eriocephala, S. interior and S. nigra. Polymorphic microsatellite markers were used to characterize the allele data on the shrub Salix eriocephala, a North American species with economic potential. Eleven markers amplified and confirmed the potential of this species. Analysis of samples from six populations in eastern Canada showed that all markers were variable as well as polymorphic in at least one population. The number of alleles per locus ranged from 1 to 9 (mean 2.95) and showed that these microsatellite markers can be used to assess genetic diversity of North American willow species.Biomass produced from dedicated plantations constitutes a source of renewable energy and is expected to play an important role in several countries in the coming decades. The cultivation of woody crops such as willows therefore raises several environmental issues. In North America, several native willows are potentially interesting for biomass producers. Willow trees are diverse but few species used for environmental applications have been the object of molecular genetic studies. Based on the sequenced poplar genome, 24 microsatellite markers were assayed on five native North American willow species:  Salix L.  includes about 500 species worldwide, concentrated in temperate and cold regions of the Northern Hemisphere. Although it presents greater diversity in areas such as China , more tspecies;  using shspecies; ,5. In Nospecies; . Salix amygdaloides, S. discolor, S. eriocephala, S. interior and S. nigra, have morphological traits that suggest high biomass potential. These shrubs can grow rapidly to 6–8 meters and are widespread in eastern North America [Salix eriocephala is of particular interest because of its extensive North-South distribution and adaptation to a range of climatic conditions. Knowledge of the genetic diversity of populations is a prerequisite to breeding programs aimed at improving biomass production. At a broader scale, given that half the current range of this species was once covered by Quaternary glaciers, genetic information may provide clues to the species population structure following recolonization of glaciated areas [et al. [Salix, of which 17 were polymorphic . Two studies derived microsatellite markers for Salix from the related Populus trichocarpa  genome [et al. [Populus SSRs assayed in S. caprea (only 10% were polymorphic), each of which was functional on a variable number of species among the four species assayed; Lin et al. [Populus and the Barker study  to compare the genetic diversity of S. eriocephala to that of S. purpurea (introduced willow) at a regional level.The North American willow species,  America . Among ted areas . Barker ) genome  to evalu [et al.  successfS. eriocephala in widely distributed populations in eastern Canada. Our objectives were: (1) to determine whether these markers were present in the North American Salix species studied, and (2) to evaluate polymorphisms within the species S. eriocephala.Here, we assayed 24 microsatellite markers on five willow species and characterized 11 polymorphic microsatellite markers for Salix species of this study and five markers were shared in these willows  were present only in S. eriocephala.In total, twelve microsatellites were amplified with  willows . In addiS. eriocephala, population genetic statistics are summarized in Ho) and expected (He) heterozygosities, p-values for the Hardy-Weinberg Expected test, and details of each population. Eleven of the 24 markers were successfully amplified in the six populations of S. eriocephala , which falls within the range predicted by Tuskan et al. [et al. [Salix eriocephala, Lin et al. [vs. 1–9) for Populus-derived loci, but their population size was much larger than that of populations in our study, which may account for some of the difference. Furthermore, some of our populations were at the extreme northern edge of the species range of S. eriocephala and may have lost genetic diversity while migrating northward after glaciations [Tuskan et al.  using thr et al.  successfn et al.  used foun et al. ; the dif [et al.  study ann et al.  obtainedciations . In all S. eriocephala were selected to reflect the wide distribution of this species ; Hanlon Marsh, ON (4 trees); and Richmond Fen, ON (4 trees). The samples of S. discolor were collected at Levis, QC (10 trees); Montmagny, QC (8 trees); and Norton, NB (10 trees). The samples of S. interior were collected at Lafarge Pit, ON (5 trees); Limerick Forest, ON (5 trees); and Long Sault, ON (5 trees). The samples of S. nigra were collected at Gagetown, NB (4 trees); Pembroke, ON (4 trees); and Westmeath, ON (6 trees).Six populations of  species . These pn Canada . The samGenomic DNA was extracted from 25 mg of leaf material using a modified CTAB method . Leaf tiPopulus Genome Consortium identified 4,200 Populus SSRs [2, 1 U Taq DNA polymerase (GenScript). An Eppendorf Mastercycler® pro Thermal Cyclers (Eppendorf) under the following cycling parameters: initial denaturation at 94 °C for 3 min followed by 25–33 cycles  using GENEPOP v.4.2 [The International lus SSRs ,12,13. Alus SSRs  was used3 cycles  of 30 s OP v.4.2 ,16.S. eriocephala. If the loci used by Lin et al. [S. eriocephala, S. amygdaloides, S. discolor, S. interior and S. nigra. Studies of Salix microsatellites increased the molecular tools available for the investigation of willow species and the knowledge of this genus.In conclusion, we have added 11 new polymorphic microsatellite markers for n et al.  are adde


Icterus northropi) is a critically endangered, synanthropic island endemic with a declining population of fewer than 300 individuals. It now remains only on Andros Island (The Bahamas), which is riddled with waterways that past studies assumed did not hinder gene flow. We examined 1,858 base pairs of mitochondrial DNA sequenced from four gene regions in 14 birds (roughly 5% of the remaining population) found on the largest land masses of Andros Island (North Andros and Mangrove Cay/South Andros). We sought to discern genetic structuring between the remaining subpopulations and its relationship to current conservation concerns. Four unique haplotypes were identified, with only one shared between the two subpopulations. Nucleotide and haplotype diversity were higher for the North Andros subpopulation than for the Mangrove Cay/South Andros subpopulation. Analysis of molecular variance (AMOVA) yielded a Wright’s fixation index (Fst) of 0.60 (PFst = 0.016), with 40.2% of the molecular variation explained by within-population differences and 59.8% by among-population differences. Based on the mitochondrial regions examined in this study, we suggest the extant subpopulations of Bahama Oriole exhibit significant population structuring over short distances, consistent with some other non-migratory tropical songbird species.Bird species may exhibit unexpected population structuring over small distances, with gene flow restricted by geographic features such as water or mountains. The Bahama Oriole ( Bird species may exhibit unexpected population structuring, with gene flow limited by geographic features in ways that are sometimes unanticipated, given birds’ flight capabilities. In some cases, genetic structuring may result from ecological associations with forest or aquatic environments that lead to population structuring over small distances . For nonIcterus northropi), one of but a very few critically endangered synanthropic bird species  on North Andros (Molothrus bonariensis), a brood parasite that targets Icterus  to discern genetic structuring of the two potential subpopulations (North Andros and South Andros/Mangrove Cay); and (2) to relate our findings to current conservation concerns. One useful application, for example, would be to inform planning for possible translocation of the Bahama Oriole to Abaco, where it formerly occurred.N = 11), Mangrove Cay (N = 1), and South Andros (N = 4) during the years 2009–2010 (N = 12), or as nestlings briefly removed from two nests on North Andros . Captured birds were measured, sampled, banded using standard issue aluminum USGS identification bands, and immediately released. Two tail feathers were pulled from adult birds. For nestlings and adults, blood samples were obtained by pricking the brachial vein ; and NADH dehydrogenase subunit 2 (ND2), using the primers in We amplified mitochondrial DNA (mtDNA) sequences from the genomic DNA samples using standard polymerase chain reaction (PCR) methods, as detailed below. Four mtDNA gene regions were amplified: ATP synthase subunits 6 and 8 (ATP6/8); cytochrome N), haplotype diversity (h), and nucleotide diversity  ranges from 0 to 1, with values less than 0.01 indicating little divergence among populations, and values above 0.1 indicating substantial divergence among populations.To examine possible genetic structure, we compared two arbitrary subpopulations: North Andros and South Andros/Mangrove Cay. We pooled the South Andros and Mangrove Cay populations for analysis because of the small number of samples available, and because stepping stone cays with suitable foraging habitat are much closer together between Mangrove Cay and South Andros than between North Andros and Mangrove Cay. Multiple alignments for each gene region were performed in CLUSTALX version 2.1 . Sequencrsity π; . We explrsity π; . This nersity π;  allows frsity π; . Analysirsity π; , performIcterus northropi or close relatives  represent the intended mitochondrial targets rather than nuclear homologues.Using a BLAST search, we found that sequences obtained in this study were consistent with previously published sequences for b, and 331 bp from ND2, for a total of 1,858 bp. Of these, 1,852 (99.7%) characters were constant, and 6 (0.3%) characters were variable and informative. Variation within the combined four gene regions included 5 transitions and 1 transversion, consistent with the expectation that transitions outnumber transversions, and consistent with the degree of mitochondrial variation in members of the genus Icterus (N = 6) was found in both subpopulations, and the second most common haplotype  was found only in the northern subpopulation  of 0.60 (PFst = 0.016), with 40.2% of the molecular variation explained by within-population differences and 59.8% by among-population differences. Nucleotide and haplotype diversity  than for the southern one .The AMOVA for combined sequences  yielded iversity  were higb had the highest nucleotide diversity of the four gene regions examined for the southern islands, and ATP8 had the highest nucleotide diversity of the North Andros gene regions.Three of four gene regions, totaling 1,191 bp, lacked any variation in the southern subpopulation, and the fourth gene region (667 bp) lacked variation in the northern subpopulation . CytochrBased on examining four mitochondrial genes, the extant Bahama Oriole population on Andros appears to have significant population structuring. The two subpopulations we sampled appear to be relatively diverse compared with other bird species . Given tThe southern subpopulation of orioles, which is considerably smaller than the northern population , had subFst value  for the two subpopulations indicates significant genetic divergence between localities despite the relatively small sample size.We found some evidence for genetic differences between Bahama Orioles from North Andros and those from the southern islands of Mangrove Cay and South Andros. Although our sample sizes were relatively small, we collected samples from close to 5% of the estimated species population, with samples evenly distributed from north to south, and still found significant genetic structure. Only one haplotype was shared between regions, with two individuals on North Andros sharing a haplotype otherwise found only on the southern islands. A majority of the diversity was between the two subpopulations (59.8%), with 40.2% of the genetic variation explained by differences among individuals. The calculated Icterus, such as the Orchard Oriole (I. bullockii) and Baltimore Oriole , the Bahama Oriole and its close relatives on the islands of Cuba, Puerto Rico, and Hispaniola do not migrate (I. northropi. The degree of genetic differentiation over the relatively short distance between North Andros and Mangrove Cay that was found in this species is typical of other non-migratory avian species, and may be associated with gap avoidance (Despite the migratory capabilities of the continental members of the genus  migrate . Based ovoidance . Differevoidance , which gb. Haplotype networks either grouped most samples together, or separated samples into two haplotypes largely correlated with geographic location. The corroboration of haplotype networks for ND2, which evolves at a relatively fast rate, and ATP8, a comparatively conserved gene region, strengthens the case for managing these subpopulations as two management units, as does the extremely low diversity found in the southern subpopulation. Further studies should include nuclear genes for comparison.Examination of multiple gene regions offers several benefits in population-level studies, primarily because it provides the greatest possible explanatory power when examining relationships . CorroboSynanthropic species, which cohabit with humans and benefit from resources and modifications that exist in anthropogenic landscapes, present unique challenges for conservation management. Today, at least 14 highly endangered birds are recognized as synanthropic, including the Bahama Oriole . The extGiven the strong preference of Bahama Orioles for coconut palms in anthropogenic habitat during the breeding season, the arrangement of available nesting habitat may structure populations, even over small distances. This phenomenon has already been observed in natural systems, where ecological associations with forest or aquatic environments lead to population structuring over small distances . On AndrFor critically endangered species with restricted geographic ranges, stochastic natural disasters such as hurricanes pose a serious threat to species persistence . This th


Salamandra salamandra, was proved to be effective in identifying the hierarchical structure of populations living in broad-leaved forest ecosystems in Northern Italy. In this study, 477 salamander larvae, collected in 28 sampling populations (SPs) in the Prealpine and in the foothill areas of Northern Italy, were genotyped at 16 specie-specific microsatellites. SPs showed a significant overall genetic variation . The genetic population structure was assessed by using STRUCTURE 2.3.4. We found two main genetic groups, one represented by SPs inhabiting the Prealpine belt, which maintain connections with those of the Eastern foothill lowland (PEF), and a second group with the SPs of the Western foothill lowland (WF). The two groups were significantly distinct with a Global FST of 0.010 (P < 0.001). While the first group showed a moderate structure, with only one divergent SP , the second group proved more structured being divided in four clusters . This genetic population structure should be due to the large conurbations and main roads that separate the WF group from the Prealpine belt and the Eastern foothill lowland. The adopted methods allowed the analysis of the genetic population structure of Fire Salamander from wide to local scale, identifying different degrees of genetic divergence of their populations derived from forest fragmentation induced by urban and infrastructure sprawl.The multistep method here applied in studying the genetic structure of a low dispersal and philopatric species, such as the Fire Salamander  Habitat destruction and degradation are considered the most important threats for wild populations, but the effects produced by the overall habitat loss are often complex to understand because many impacting factors do not act separately, but rather they play cumulatively or interactively  genetic population structure because amphibians are usually model candidates for studies of fragmentation effects on connectivity  linked by dispersal  of 1–4 salamander larvae tail in each breeding site, all year round, from 2010 to 2013. We chose not to sample more than four individuals per site, in order to avoid possible bias deriving from full-sibling individuals when inferring population genetic structure by sampling larvae in breeding sites  for 2010–2012, and administrative order 964 decreed on 2013/02/11 by the Agriculture General Directorate of the Lombardy Region (D.G. Agricoltura della Regione Lombardia) for 2013–2014.μL of elution buffer , and stored at −20°C until subsequent handlings.DNA was extracted using the Quick-g DNA™ MiniPrep kit  according to the manufacturer’s instructions, eluted in 180 All samples were genotyped by polymerase chain reaction (PCR) for 20 species-specific microsatellite markers , 0.4 μL of dNTPs 10 μmol/L, and 0.2 or 0.3 μL of primer mix (forward and reverse) 10 μmol/L plus 0.25 units of Taq polymerase  and purified water. PCR conditions were optimized for each primer pair, amplifications were performed in a 9700 ABI thermal cycler using the following protocol: (94°C × 2 min’), a number of cycles ranged from 30 to 40 at (94°C × 30 s’’)  (72°C × 30 s’’), and a final extension at 72°C for 10 min’. Some primer pairs  were amplified using a touchdown PCR starting from Ta+8°C to Ta for 8 cycles.PCR amplifications were carried out in a 10-PCR products were analyzed in an Applied Biosystems 3130XL DNA sequencer , and allele sizes were estimated using the software GENEMAPPER 4.0 . Positive (known genotypes) and negative (no DNA) controls were used to check for laboratory contaminations, which never occurred. A 10% randomly selected subset of the other samples was PCR-replicated two times to check for allelic dropout and false alleles. Four of 20 microsatellite loci  were excluded from the analysis because we were not able to obtain PCR products that could be clearly interpreted.FST (Wright FIS), for each locus on all individuals. Before performing genetic population structure analyses, we tested each locus for Hardy–Weinberg Equilibrium (HWE), checking its FIS, in order to detect an excess of homozygosity, which can bias population structure analyses. All these analyses were performed using GenAlEx v. 6.501. Nevertheless, as suggested by Allendorf et al.  pertaining to the identified populations of origin. Populations were constructed by minimizing the departures from HWE and linkage equilibrium, which could result from recent admixtures, migration, or hybridization.Genetic population structure was performed analyzing the biparental multilocus genotypes by means of the Bayesian clustering procedure implemented in STRUCTURE 2.3.4  can be improved with the knowledge of the sampling location of each individual. In fact, including the sampling location as a prior in the model, the clustering algorithm assumes that the probability of assignment of each individual to a population of origin varies among locations, without finding population structure where it does not exist  may be further affected by the choice of the allele frequency model. Allele frequencies may show marked differences between distinct populations of origin and ancestral relationships between them is not expected , for each locus, are shown in Table FST statistics on a subsample  to allow convergence in GeneAlex algorithm, deleting locus Sal23 (which had a high number of no data) from the initial pool of 16 loci and four SPs  with fewer than five individuals. Significant overall genetic variation was observed . Pairwise FST between the 24 SPs ranged from 0.010 to 0.101, with 66 significant comparisons after Holm–Bonferroni correction. Sampling populations SP5, SP6, SP7, and SP12 showed the highest number of significant pairwise tests  resulted in values as low as 7.8 × 106 loci reK = 2; see Fig. Qp values, estimated by the four models, were always higher than 80% for both cluster 1 and 2, except for only one sampling population (SP12) for which the two “independent allele frequencies” models produced a Qp value of 58% for cluster 1. For the same SP, the two “correlated allele frequencies” models estimated a Qp value of 82% pertaining to cluster 1. For this reason, we considered the SP12 pertaining to cluster 1. The r LOCPRIOR parameter was always less than one, indicating that prior groups were informative in detecting population structure . See Table The first step of population structure analysis, performed on all samples , produced consistent results among the four parameter combinations. According to the Evanno method, we identified two distinct clusters . We first tested for HWE using GeneAlex and deleted from the analyses all loci not at equilibrium after Holm–Bonferroni correction and with null alleles. In the PEF group, only Sal23 was out of HWE, while in the WF group, SalE5 resulted monomorphic and SST-A6-I not at HWE. We therefore ran population structure without locus Sal23 for the PEF and without loci SalE5 and SST-A6-I for the WF. Neither of the “independent allele frequencies” models, ran in this step for both the PEF and the WF, converged, even increasing the length of Qp of 67%. All others SPs were assigned to a single cluster, with Qps always higher than 69%. The “no-admixture” model supported the presence of 2 clusters (K = 2), and the results were consistent with the “admixture” model, identifying SP12 as a single cluster (Qp = 94%) and all other SPs pertaining to a second cluster , except for SP10 which had a Qp of 67% . The two clusters showed a different number of private alleles, 33 in cluster 1 and 1 in cluster 2 (SP12). The latter also showed the locus SalE5 as monomorphic . Nonetheless, the Qps pertaining to cluster three or four were relatively low for all SPs. These two clusters were represented by two groups of genetically distinct individuals, each of which collected in a unique sampling location. The group assigned to cluster 3 was split from SP5 (Qis > 93% with K = 3 and Qis > 99% with K = 4), and the group assigned to cluster 4 was split from SP7 (Qis > 66%) . Pairwise FST among the four clusters of the WF group ranged between 0.007 and 0.083, but none of them were significantly different from zero after sequential Bonferroni correction  and the observed heterozygosity (Ho) were very similar between PEF and WF, suggesting that currently there was not a loss of genetic diversity on the whole.This research allowed us to detect a hierarchical population structure of the Fire Salamander in a wide area of the species range in Northern Italy. The 28 sampled populations were distributed in the Prealpine area, where forest cover is more continuous, and in the foothill lowland, where forests are more fragmented see Fig. . Some SPAfter the identification of these two genetically separated groups, the use of a multistep approach allowed us to identify substructured populations.FST. SP12 cluster is located in a foothill forest separated from Prealpine areas by both wide conurbation and a main road. In addition, this area was also found to be isolated by the lack of a stream network connection that may support the dispersal process and seems to be important in connecting other Eastern foothill populations to the Prealps.In the PEF groups, two more clusters were identified: A first one included all populations except SP12, which formed a second cluster whose separation was confirmed by both population structure analysis and pairwise K = 2, the group is divided into a Western (SP5 and SP6) and Eastern (SP7 and SP8) cluster, divided by large conurbations and a south–north main road , identifying two new clusters corresponding to two sampling locations, sampling location 5.5 (cluster 2), and sampling location 7.3 (cluster 4). Although these two new clusters are very close (with a distance of about three kilometers) to the other sampling locations of the same SP, they seem to represent separated demes. Cluster 2 appeared to be almost completely isolated by unsuitable areas for the Fire Salamander, largely represented by urban areas without stream network connections, while cluster 4 is isolated mainly by agricultural areas but with a stream connection, which could make the isolation less sharp  will maintain a high number of alleles in the long term, because it is represented by a large and mainly continuous population Fig. B.Despite the fragmentation process currently only led to an evident population structure without significant differences of genetic parameters among identified clusters, in the medium and long term, it is expected it could even produce a loss of genetic diversity . The effectiveness of this approach was proved by the use of the species here investigated, the Fire Salamander, an amphibian with rather strict ecological requirements (Di Cerbo and Razzetti In conclusion, the multistep approach can be useful to detect substructured populations at local scale which cannot be observed analyzing wide-scale data in one-step only. Thus, the screening of genetic population structure from a regional to local scale can be considered effective in detecting local populations of conservation concern which resulted imperilled by threats acting locally and also for a preliminary assessment of the ecological connectivity in fragmented landscapes.