Cornus florida (flowering dogwood) and C. nuttallii (Pacific dogwood) are North American native tree species that belong to the big-bracted group of dogwoods. Cornus species are highly valued for their ornamental characteristics, and have fruits that contain high fat content for animals. Also, they are an important understory tree in natural forests. Dogwood anthracnose, caused by Discula destructiva, was observed in the late 1970s on the east and west coasts of the United States and by 1991 had quickly spread throughout most of the native ranges of C. florida and C. nuttalli. We investigated the genetic diversity and population structure of 93 D. destructiva isolates using 47 microsatellite loci developed from the sequenced genome of the type strain of D. destructiva. Clone-corrected data indicated low genetic diversity and the presence of four genetic clusters that corresponded to two major geographic areas, the eastern United States and the Pacific Northwest, and to the two collection time periods when the isolates were collected (pre- and post-1993). Linkage disequilibrium was present in five out of six subpopulations, suggesting that the fungus only reproduced asexually. Evidence of population bottlenecks was indicated across four identified genetic clusters, and was probably the result of the limited number of founding individuals on both coasts. These results support the hypothesis that D. destructiva is an exotic pathogen with independent introductions on the east and west coasts of North America. We also tested the cross-amplification of these microsatellite primers to other Discula species. Genomic DNA from 17 isolates of four other Discula species and two isolates of Juglanconis species (formerly Melanconis species) were amplified by 17 of 47 primer pairs. These primers may be useful for investigating the genetic diversity and population structure of these Discula species. Cornus florida L. (flowering dogwood) and C. nuttallii Aud. (Pacific dogwood) are tree species native to North America and classified in the big-bracted group of dogwoods [Cornus florida is widespread throughout eastern North America from southeastern Ontario, to Canada in the north, to northern Florida in the south, and as far west as eastern Texas [C. nuttallii is endemic to western North America and ranges from southwestern British Columbia to northern California [Cornus florida is a major component of the understory in eastern hardwood forests [Cornus florida nursery sales in the U.S.A. exceeded $27 million in 2014 with more than $6.5 million from Tennessee nurseries [dogwoods . Cornus rn Texas , whereaslifornia  with smalifornia . Cornus  forests  and prov forests , 5. The C. florida cause only cosmetic damage requiring no intervention. However, powdery mildew and dogwood anthracnose can be very problematic for commercial production and cause considerable harm to individual trees as well as entire populations. Powdery mildew, a fungal disease caused by Erysiphe pulchra [Discula destructiva, is a very devastating disease that was first observed on C. nuttallii during 1976 in Washington State [C. florida was in 1977 in New York [C. florida trees within New York and Connecticut were either killed or extensively damaged by what was then considered a new “mysterious” disease [Most foliar diseases of  pulchra , typical pulchra , 9. Althon State , whereasNew York . Thousan disease .C. nuttallii [C. nuttallii in the west and C. florida in the east were being affected by the same disease [Discula destructiva has several known modes of dispersal. Localized dissemination has been demonstrated by splashing water in the form of rain or overhead irrigation [C. florida trees were exported from the U.S.A. to the United Kingdom in 1995 [The disease spread across the natural western range of uttallii  includinuttallii , 14. In uttallii . In 1988 disease . Discularigation . Insectsrigation . Animalsrigation . Humans  in 1995 , Italy [ in 1995 , and Swi in 1995 .C. florida trees had been killed by dogwood anthracnose, and only 3% of trees were free of symptoms [C. florida trees by 1992 and 75% just two years later [C. kousa Hance), which is non-native to North America, displays some resistance to dogwood anthracnose [C. kousa trees may exhibit small leaf spots, but shoot dieback is not as severe, and tree mortality rarely occurs [The first signs and symptoms of the disease typically are found on leaves and young shoots, and infection may progress into twigs, which die back. The hyphae may infect the main trunk through progression from the base of infected epicormic shoots to cause sunken, necrotic annual cankers; the lesions primarily affect phloem tissues .Canker fsymptoms . In a fosymptoms . Surveysrs later . Japaneshracnose . Infectey occurs , 29.Gloeosporium species in the west [Colletotricum gleosporioides in the east [Discula species as the cause of the disease, which was later characterized as a new species, D. destructiva [D. destructiva, and hence two types of Discula species associated with dogwood anthracnose were designated as Discula Type I (D. destructiva) and Type II, an unnamed species of Discula [D. destructiva isolates produced phenol oxidases that oxidized gallic acid and other phenolic substrates, whereas none of the Discula type II species could not oxidize any of these substrates [Discula destructiva is a haploid, ascomycetous fungus in the order Diaporthales, and the Gnomoniaceae [Discula species are anamorphs of Apiognomonia, but a teleomorph has not been observed for D. destructiva on diseased materials or in the laboratory [Discula for dogwood anthracnose has been argued because of the lack of a sexual stage [The causal agent of dogwood anthracnose was initially thought to be a the west  and Collthe east . Fulfilltructiva . However Discula , 30. Besoniaceae , 32. Somboratory . Conserval stage .Discula destructiva may be either an indigenous species that had gone unnoticed until the late 1970s or an introduced pathogen [D. destructiva did not arise from a native population [D. destructiva isolates, further supporting the exotic invader hypothesis. The DAF method generates an abundance of data, but amplicons cannot be linked to specific genomic sites or loci. Furthermore, a single band in a gel may contain multiple amplicons, which may or may not be present among all the isolates. Analysis of arbitrary signatures from amplification profiles (ASAP) [D. destructiva likely have separate origins [D. destructiva was identified by qPCR from Cornus specimens collected before the first report of the dogwood anthracnose in North America from China and Japan, the hypothesized center of origin, where it may exist as an endophyte [pathogen . The forpulation . DNA amppulation  reporteds (ASAP)  detecteds (ASAP) . Banding origins , 39. Amp origins  also revndophyte .D. destructiva using microsatellite loci. Microsatellite loci or simple sequence repeats (SSRs) are repeating units of nucleotides that occur throughout the genome of eukaryotic organisms that can be used as genetic markers [D. destructiva. They facilitated greater detection of genetic diversity by amplifying discrete or defined loci compared to arbitrary primers [D. destructiva; 2) to identify spatial structure and genetic diversity of D. destructiva isolates collected across North America from different time periods; and 3) to test the cross-amplification of these microsatellite loci in other Discula species.To elucidate the pathogen origin and spread in the U.S.A., we investigated genetic diversity and spatial structure of  markers . Compare primers , and theD. destructiva collected from 1989–2000 across eastern and western North America were used in this study. Nineteen isolates of D. destructiva were obtained from a collection at Rutgers University  and fifty isolates were supplied from a collection at the University of Tennessee . Genomic DNA of 24 other D. destructiva isolates, as well as genomic DNA from isolates of five other Discula and Juglanconis species were available from the UTK collection  for 10–14 days at room temperature (ca. 20°C) before being used to inoculate medium to grow mycelia for DNA isolation. Stock cultures were transferred to fresh medium every two months. Small pieces (ca. 5 mm2) of mycelium were harvested from the periphery of 14-day-old fungal colonies and used to inoculate sterilized cellophane  [Ninety-three isolates of llection . Stock c U.S.A.)  placed oD. destructiva by amplifying the ITS region of fungal ribosomal DNA using the polymerase chain reaction (PCR). Each 30 μl reaction contained the following: 6 ng genomic DNA template, 1.5 μl of 5 μM each of ITS1 (5’—TCCGTAGGTGAACCTGCGG—3’) and ITS4 (5’—TCCTCCGCTTATTGATATGC—3’)[2, 3 μl of 10X PCR Gold Buffer , 1.5 μl dimethyl sulfoxide , 0.24 μl of 5 U/μl AmpliTaq Gold® DNA polymerase (Applied Biosystems), 3 μl of 2.5 mM deoxynucleoside triphosphate (dNTPs), and 10.26 μl sterile distilled water. The thermal cycler  was programmed with initial denaturation of 95°C for 3 min followed by 35 cycles of 95°C for 1 min, 50°C for 30 s, 72°C for 80 s, and a final extension at 72°C for 10 min. Five μl of ITS PCR products were visualized with ethidium bromide on 2% agarose gels using a 2000 Gel Documentation System (BIO-RAD). PCR amplicons were purified using Qiagen QIAquick purification kit  according to the manufacturer’s instructions. The purified DNA was quantified using NanoDrop ND-1000 Ultraviolet-Vis Spectrophotometer and sequenced at the UTK Genomics Core (http://mbrf.utk.edu/). Fungal isolates were confirmed as D. destructiva by comparing the ITS sequences to known sequences in the GenBank database using the Basic Local Alignment Search Tool (BLAST) available from the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/). The ITS sequences of the 66 isolates in live culture were aligned using BIOEDIT v7.2.5 (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The 24 pre-1993 D. destructiva isolates (DNA only) and 21 Discula species were confirmed previously using the same procedure except that the primers ITS1 and IST2 [All living isolates from the UTK and RU collections were verified as TATGC—3’), 3 μl ofand IST2  were useDiscula destructiva type isolate MD235 was used for genome sequencing. A paired-end DNA library (Illumina) was made according to manufacturer’s instructions and sequenced to produce 74.9 million 100 bp x 2 read pairs. The reads were filtered by requiring a minimal quality score of 20 in at least 70% of the bases. Filtered reads were assembled using SOAPdenovo2 [https://github.com/statonlab/disculaSSRs). Di-, tri-, and tetra- nucleotide perfect repeats were only reported if they met the following criteria: di- nucleotide motifs with 8–40 repeats, tri-nucleotide motifs with 7–30 repeats, and tetra-nucleotide motifs with 6–20 copies. Sequences were masked for low complexity regions with DUSTMASKER [Pdenovo2  with kmeSTMASKER  and primSTMASKER . The folSTMASKER ; primer 2, 1 μl of 10X PCR Gold buffer (Applied Biosystems), 0.5 μl DMSO, 0.1 μl of 5 U/μl AmpliTaq Gold® DNA polymerase, 1 μl (2.5 mM) dNTPs, and 4.4 μl sterile, distilled water. Reactions were completed in an automatic thermal cycler under the following conditions: initial denaturation at 96°C for 4 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min.Primer pairs for 50 microsatellite loci  with annD. destructiva isolates and other Discula species isolates. Reactions that did not produce a product were repeated at least once before assuming that the loci of these isolates were either a null allele or considered missing data for the analyses.PCR products were analyzed on a QIAxcel Capillary Electrophoresis System  using an internal 25-bp size standard to provide raw allele length data. Forty-seven primer pairs  that proD. destructiva haplotypes were identified using POPPR v2.1.1 [FLEXIBIN v2  was usedR v2.1.1  a packagR v2.1.1 . All isoR v2.1.1 , 54.D. destructiva subpopulations were assessed using ARLEQUIN v3.5.2 [A, but is considered less biased because it is independent of the number of loci used in the study. In addition, the value of The program GenAlEx v6.5 , 56 and N v3.5.2  where no.2 [r¯d)  was estihan zero .C. florida, C. nuttallii, and C. kousa, and lastly, partitioned based on STRUCTURE findings. For AMOVA analyses, the following variance components were calculated: i) among groups (Fct), ii) among subpopulations within groups (Fsc), and (iii) among subpopulations  (Fst). All AMOVA analyses were computed using 10,000 permutations at P<0.05 significance level.Genetic clustering and population structure were analyzed using the program STRUCTURE v2.3.4  with a BPrincipal coordinate analysis (PCoA) implemented in GenAlEx, and dendrograms created in POPPR using Nei’s genetic distance  and BruvThe program BOTTLENECK v1.2.02  was usedD. destructiva from RU and UTK. The amplicon sequences were matched to D. destructiva . The DNA of pre-1993 isolates from the UTK collection was previously confirmed as D. destructiva in a similar manner except ITS1 and ITS2 [Discula-like or Type II isolates with ITS1 and ITS4 demonstrated that isolates NC2 and VA17b were Juglanconis species.  [A single band of ~550 bp was amplified using ITS1 and ITS4 primers for the previously uncharacterized 69 isolates of and ITS2  were usedentity) .The final genomic assembly spanned 49 Mb with scaffold N50 of 126.6 kb (111 scaffolds) and contig N50 of 33,311 bp (429 contigs). A total of 23,334 sequences produced 2,560 perfect microsatellite loci . Forty-seven (18 di- and 29 tri- nucleotide repeat motif) out of 50 primer pairs were optimized to successfully amplify DNA and consistently produce a single product per locus . Three pClone-correction removed 13 individuals from the original data set (n = 93): 3 from the pre-1993 North, 2 from the pre-1993 South, one from the post-1993 North, 6 from the post-1993 South, and one from the post-1993 West subpopulation. Clone-corrected data  was used in all subsequent analyses with i) 47 SSRs that include both polymorphic (n = 40) and monomorphic (n = 7), and ii) only the 40 polymorphic microsatellite loci. Here, we present data only for 47 microsatellite loci because data for 40 loci produced similar results (data not shown).Discula destructiva isolates included in the study were assigned to one of six subpopulations based on common geographical region  and year of isolation (pre-1993 and post-1993). The mean number of effective alleles (Ne) per subpopulation ranged from 1.03 in the post-1993 North subpopulation to 1.53 in the pre-1993 West population. Shannon-Weiner index of multilocus genotype (MLG) diversity (H) ranged from 1.1 in post-1993 North, to 3.47 in the pre-1993 South. Genotypic diversity corrected for sample size (Hexp) across all subpopulations was 0.18, ranging from 0.03 in the post-1993 North to 0.31 in the pre-1993 West  best explains the population structure of llection , S2 Fig.llection . The posllection . Only onllection . When ΔKllection . Under sD. destructiva subpopulations into the following: i) one hierarchical group, ii) two time periods (pre-1993 and post-1993), iii) two geographic regions (east and west), iv) three groups based on different D. destructiva hosts–C. florida, C. nuttallii, and C. kousa, and v) four clusters identified by program STRUCTURE. When data were analyzed as one hierarchical group, the majority of variation was within six D. destructiva subpopulations (70.90%) rather than among sampling locations  (P<0.01). However, the majority of variation was found within Discula groups from these two time periods  (D. destructiva subpopulations (eastern and western North America), the majority of the variation was found among individuals within six Discula subpopulations , whereas 5.40% of variation was explained by differences among Discula subpopulations within eastern and western groups . Although more than 42% of variation was identified between these two geographical regions, the differences were not significant . When partitioned by the host, 39.21% and 60.79% of the variation was attributed among three host groups and within these groups, respectively   was performed by grouping  P<0.01) . When pa P<0.01) . Furtherectively . However P<0.01) .PT) indicated significant (P< 0.05) subpopulation differentiation except between the North and South subpopulations, and ranged from 0.09–0.88  in all four genetic clusters, which indicated that a recent population bottleneck has occurred. The mode-shift indicator showed allele frequency distortion for all genetic clusters  and Fagus sylvatica (beech), whereas only 4 loci amplified from D. umbrinella isolates obtained from Castanea (Ct.) sativa (chestnut). An average of 1.3 loci were amplified from isolates of D. quercina from Q. garryana (Oregon white oak) and C. florida, D. fraxinea from Fraxinus americana (white ash), and D. campestris from Acer saccharum (sugar maple). Four primer pairs amplified loci from isolate NC2, Juglanconis sp. (formerly Melanconis species) [Juglanconis sp. isolate, VA17b, no loci were amplified. Amplification was observed for an average of 3.8 Discula species isolates among the 17 D. destructiva primer pairs. The greatest number of Discula species isolates to which D. destructiva microsatellites loci were cross-amplified was 12 (DD25), whereas there were several primers  that only amplified DNA from one other Discula species isolate.Seventeen of forty-seven primer pairs were demonstrated to successfully cross amplified DNA from iother  species . An averspecies) , whereasD. destructiva subpopulations from different geographical regions and two time periods. We also detected signatures of a recent population bottleneck and evidence of asexual reproduction among most D. destructiva subpopulations. The low genetic diversity among D. destructiva isolates found here supports previous studies that utilized DAF [D. destructiva is an exotic species, which was recently introduced into east and west coasts of North America [The results of our study indicated significant population structure, low genetic diversity with limited gene flow, and high genetic differentiation among ized DAF , ASAP [3ized DAF , and AFLized DAF . The cur America , 35, 40.D. destructiva exhibited significant population structure represented by four genetic clusters that correspond to historical and geographical distribution of the pathogen and times of collection. Genetic clusters defined by time of collection (pre- and post-1993) can be explained by multiple introductions of the pathogen occurring on each coast of North America. Distinct genetic clusters and population differentiation separated eastern and western isolates, which corroborates the findings of previous D. destructiva studies [Using Bayesian approaches,  studies , 38–40, D. destructiva sampling localities; the majority of variation is individually based (71%) (P<0.01). Approximately 89% and 53% of variance was identified within subpopulations between two time periods (P<0.01) and different geographical origin (P<0.01), providing evidence that the majority of differentiation was individually based, rather than among these groups. Although our data indicated that 42% of variation could be explained by eastern and western groups, the results were not significant and could be explained by unequal sample size in our dataset. When grouped by host species, 39% of variation was attributed to the differences among three host groups (P<0.01). However, these results should be interpreted with caution because the majority of individuals used in our study were collected from C. florida, possibly resulting in biased estimations. When D. destructiva subpopulations were grouped based on STRUCUTRE findings, the majority of genetic variation was found among the clusters . Only 34% of variation could be explained by variation within each of the six subpopulations among the four identified clusters.Our analysis indicated that 29% of genetic differentiation was attributed to variance among Phytophthora infestans that resulted in millions of people either being forced to migrate or die from starvation [D. destructiva, are introduced is vital to the prevention of future invasions and potentially devastating consequences [According to the Millennium Ecosystem Assessment , one of arvation , 76. Undequences .D. umbrinella that is indigenous to Europe had high genetic variability among isolates as measured with RAPDs [D. destructiva isolates in previous studies [Low genetic diversity is commonly found in introduced species because of founder effects, in which a small number of representatives, or even a single genotype of the organism, becomes the founder(s) of a population in the new area. Higher genetic diversity would be expected if this species were native to North America , 78. In th RAPDs . Low lev studies –36 sugge studies . These r studies , 78.D. destructiva subpopulations. The lack of gene flow between east and west coast isolates indicated that D. destructiva did not spread from eastern North America to western North America or vice versa. Discula destructiva isolates from pre- and post- 1993 in North and -South showed significant gene flow that suggested multiple independent introductions into these areas. However, due to limited and unequal sample size in some subpopulations, we cannot exclude the bias in our migration assumptions. This issue can be mitigated with a larger collection of data that includes the source population, which is believed to be predominantly in Japan and to some extent in China [D. destructiva subpopulations. Nevertheless, a single pre-1993 West isolate (M10 from UTK), clustered with the pre-1993 eastern group (both North and South), which suggests the possibility of movement between coasts, probably due to the importation of infected trees from the western to the eastern U.S.A. The introduction of dogwood anthracnose into Missouri and Kansas was reported to have occurred due to the movement of infected nursery stock [Geographical barriers, climatic factors, and the great distances that separate these genetic clusters can be a plausible explanation for limited gene flow and divergence among in China . In orgain China , which wry stock , demonstD. destructiva and the native dogwood species of North America [An exotic fungus may not exhibit a high degree of virulence on its native host, but when it comes in contact with a new host, the fungus can become extremely virulent because of the lack of host resistance that develops over time through coevolution . This co America . Underst America . Dogwood America .P < 0.05) was present among D. destructiva isolates due to linkage among loci indicating asexual or clonal reproduction [D. destructiva are indicative of an introduced pathogen [D. destructiva. Our findings may suggest that sexual reproduction of D. destructiva may have occurred infrequently in the past, although it has never been observed in a laboratory or natural environment in North America. In fact, the complete lack of sexual reproduction has led investigators to propose conservation of the imperfect or asexual stage (D. destructiva) as the permanent scientific name [The standardized index of association  described in Trigiano et al. (1995) were identified as Juglanconis species (formerly Melanconis species) [Discula species [Discula species, including D. destructiva, are classified in the Gnomoniaceae, whereas, Juglanconis species are placed in Juglanconidaceae [Discula species included in this study confirmed their identity as Discula.Redlin  provided Type II , 30. Add did not  confirmespecies) , which a species , 84. Botnidaceae . A BLASTD. destructiva and other Discula species, indicating they were related, and thus, allowed their relationships to be analyzed [D. destructiva appears to be most closely related to D. umbrinella isolates from oak and beech, followed by D. umbrinella from chestnut. Discula quercina, D. fraxinea, and D. campestris are less closely related to D. destructiva, with the Juglanconis species being more distantly related. This relationship is similar to that described in a previous study [D. umbrinella was not included in their analyses. The UPGMA strongly supported the divergence of D. destructiva from other Discula species and found similar genetic relationships to those revealed by previous studies in which eastern and western D. destructiva isolates were separated from each other [Discula species [D. destructiva loci  amplified DNA from most D. umbrinella isolates and are probably suitable for studying genetic diversity and population genetics of this related species. However, because of an insufficient sample size of other Discula and Juglanconis species isolates and the apparent lack of the same microsatellite loci, we cannot study the genetic diversity and population genetics of these other species.The test for cross-amplification of the microsatellite primer pairs revealed shared loci between analyzed , 86. Basus study , but D. ch other , 40 and  species . MoreoveD. destructiva and other Discula species. Sufficient allelic data were produced by these primers to analyze genetic diversity, population structure, genetic differentiation and linkage disequilibrium of D. destructiva isolates. Discula destructiva has been considered an introduced pathogen because of the sudden appearance of dogwood anthracnose near ports of entry on both the east and west coasts of North America, which rapidly spread across the distribution of native dogwoods and caused significant losses of trees. The results of our study support this scenario and provide a new evolutionary perspective that may be valuable in future analyses and models. Future population studies involving Asian specimens could elucidate the origin of D. destructiva and provide novel insight into the spread and distribution of this invasive pathogen.In conclusion, the 47 microsatellite primer pairs used in this study successfully amplified loci in S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigData was obtained after 10,000 permutations.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigNei's genetic distance was utilized to construct and visualize phylogenetic tree using 1,000 bootstraps with support values greater than 50%.(TIF)Click here for additional data file.


Natrix helvetica) was most likely located in southern France, outside the classical refuges in the southern European peninsulas. One genetic lineage of the common grass snake (N. natrix), distributed in Scandinavia, Central Europe and the Balkan Peninsula, had two distinct glacial refuges. We show that one was located in the southern Balkan Peninsula. However, Central Europe and Scandinavia were not colonized from there, but from a second refuge in Central Europe. This refuge was located in between the northern ice sheet and the Alpine glaciers of the last glaciation and most likely in a permafrost region. Another co-distributed genetic lineage of N. natrix, now massively hybridizing with the aforementioned lineage, survived the last glaciation in a structured refuge in the southern Balkan Peninsula, according to the idea of ‘refugia-within-refugia’. It reached Central Europe only very recently. This study reports for the first time the glacial survival of a thermophilic egg-laying reptile species in Central Europe.Extra-Mediterranean glacial refugia of thermophilic biota, in particular in northern latitudes, are controversial. In the present study we provide genetic evidence for extra-Mediterranean refugia in two species of grass snake. The refuge of a widely distributed western European lineage of the barred grass snake ( The extant phylogeographic structure of thermophilic European biota is often explained by their retreat to refugia in southern peninsulas  during the last glacial and postglacial recolonization of northern regions5. However, there is increasing evidence for extra-Mediterranean refugia6, and this is also true for thermophilic species like reptiles8. Grass snakes  are widely distributed across the western Palaearctic12. The range of the barred grass snake (N. helvetica) extends from the Pyrenees to the Rhine region, and it includes the Italian Peninsula, Sicily, Corsica and Sardinia. In the north, the range reaches to Northumberland, close to the Scottish border9. The common or eastern grass snake (N. natrix sensu stricto) occurs from the Rhine region eastwards to Lake Baikal in Siberia12. Its northern range extension is controversial, with unambiguous records up to central Sweden and southern Finland and debated records close to the Arctic Circle further north in Fennoscandia14. In any case, these two grass snake species are cold-tolerant compared to many other egg-laying reptile species9. This suggests that extra-Mediterranean refugia might have existed, like recently shown for the more thermophilic wall lizard . For this species, extra-Mediterranean refugia were inferred for southern France, northern Italy, the eastern Alps and the Central Balkans8.Climatic oscillations in the Pleistocene induced large-scale range shifts of many animal and plant species all over the world15 revealed glacial refugia for grass snakes in each of the southern European peninsulas, Corso-Sardinia, North Africa, Anatolia and the neighbouring Near and Middle East, with multiple microrefugia in continental Italy plus Sicily, the Balkan Peninsula, Anatolia and the Near and Middle East. In continental Italy, one refugium was inferred for the Padan Plain, south of the Alps, which qualifies for an extra-Mediterranean refugium. However, the location of the glacial refugia of some of the most widely distributed mitochondrial lineages of N. helvetica and N. natrix remain unclear. In N. helvetica, the concerned lineage is distributed from the Pyrenees across France, the Benelux countries, Switzerland and westernmost Germany to the Rhine region. Its range also includes Britain up to Northumberland, but not Ireland  and microsatellite data for grass snakes. However, nuclear gene pools turned out to be highly admixed, especially in the south of the range of N. natrix11, with several genetic lineages involved . Here we focus on the latter mitochondrial marker because more data are available and differentiation patterns were found to be the same15. Microsatellite data could not be used for examining the location of glacial refuges because in the south massive admixture of distinct genetic lineages11 causes high diversity indices that have nothing to do with the original diversity in former refuges. The same is true for the Rhine region, where a narrow hybrid belt of the two grass snake species results in high nuclear genomic diversity11.Geographically broad sampling of 1,372 grass snakes covered the distribution ranges of the blue lineage of N. helvetica15, 526 to the yellow lineage and 443 to the red lineage of N. natrix. An additional sequence from a N. helvetica from northwestern Italy, generated according to laboratory procedures described before15, was added to our previously published data set11. Sequences of obviously non-native grass snakes from the study regions11 were excluded from calculations. All samples studied are listed in Supplementary Table Among the used mtDNA sequences of grass snakes, 403 corresponded to the blue lineage of The new sample (from a roadkill) was obtained and methods were carried out according to relevant guidelines, regulations and best ethical and experimental practice of the Senckenberg Nature Research Society.popart (www.popart.otago.ac.nz) and the implemented parsimony network algorithm of tcs18, a network was built for the 1,372 mtDNA sequences for displaying relationships of haplotypes. In addition, Principal Component Analyses (PCAs) were calculated using the same data set and the R package adegenet19.Using 16. To examine this expectation, samples were arbitrarily assigned to northern and southern groups. For the blue lineage of N. helvetica, the divide was the Massif Central in southeastern France  were calculated. To verify the validity of the expansion model, a parametric bootstrap approach was used, comparing the fit of the observed and 100 simulated mismatch distributions. The fit to the expected mismatch distribution was quantified by the sum of square deviations (SSD). SSD values are non-significant when the data do not deviate from the expectation of population expansion. Raggedness indices (rg)27 were computed quantifying the smoothness of the observed mismatch distribution. Non-significant results indicate an expanding population27. Finally, changes in population size were examined by calculating Tajima’s D and Fu’s FS values in dnasp. An increasing population size (or purifying selection) is indicated by significantly negative D and FS values, whereas positive values suggest a recent bottleneck29.To assess genetic diversity of each group, the following diversity indices were calculated using N, a subsample of N individuals was drawn randomly (without replacement) and diversity indices were calculated. The procedure was repeated five times for each sample, and then the mean subsample diversity for each sample was calculated.Sample sizes differed considerably among populations. In order to eliminate the positive bias of sample size on inferred diversity, a rarefaction procedure was employed. For each sample larger than the smallest one with size diyabc 2.0.330. This software allows for rapid testing of different scenarios by calculating summary statistics rather than exact likelihoods31. For each lineage, three alternative scenarios were examined that represented either gradual diversification from refuge areas or sudden expansion . For N. natrix, the yellow lineage was divided into the groups North (Scandinavia), Centre , and Balkan. The red lineage was divided into the groups North, Carpathian Basin, and southern Balkans. Priors were set to follow uniform distributions ranging from 10 to 10,000 for effective population sizes (Ne) and from 10 to 10,000 for diversification times . For each lineage 300,000 simulations were run, after which summary statistics were drawn for: (i) number of haplotypes, (ii) number of segregating sites, (iii) mean of pairwise differences, (iv) private segregating sites, (v) mean of pairwise differences (W), (vi) mean of pairwise differences (B), and (vii) FST. According to previous studies33, the priors for the mutation rate were set to a minimum of 10−8 and a maximum of 10−6. All other settings were left as suggested in the diyabc 2.0 handbook. Posterior probabilities for scenarios were calculated by logistic regression considering the 3,000 simulated datasets that were closest to the observed values. To convert the obtained estimates of divergence times from generations to years, a generation time of 10 years was used, based on life history data 9.For comparing specific hypotheses for the demographic history of the three studied lineages , we utilized the Approximate Bayesian Computation approach implemented in ion Fig. . Three gAll data analysed during this study are included in this published article and its Supplementary Information files. DNA sequences have been uploaded to the European Nucleotide Archive (ENA) and are available under the accession numbers listed in Supplementary Table Natrix helvetica (blue lineage) were separated by a minimum of 40 mutation steps from the haplotype cluster of the yellow lineage of N. natrix, and by a minimum of 54 steps from the haplotypes of the red lineage of N. natrix. Between the two clusters of N. natrix, a minimum of 40 mutations occurred. Haplotypes of all lineages had star-like genealogies with a common internal haplotype surrounded by rare haplotypes differing only in one or a few mutations from the central haplotype. However, only for N. helvetica (blue lineages) this topology was unambiguous, with the internal haplotype h1 and eleven rare haplotypes (h2–h11) differing in only one mutation step from h1. There was no obvious pattern of a geographically correlated distribution of any haplotypes.The haplotypes of each grass snake lineage corresponded to a highly distinct cluster in parsimony network analyses Fig. –S5. HaplN. natrix consisted also of a common central haplotype (y1) surrounded by many rare haplotypes that differed in one or two mutations from y1. Yet, there were some network reticulations, and the more distinct haplotypes y15-y17 differed by four to five mutations from y1. Notably, these haplotypes y15-y17 and the rare haplotypes y12, y21 and y23 occurred only in the southern Balkan region, whereas the vast majority of other haplotypes was found only north of the Carpathians. There was only one shared haplotype (y20) between the two geographical groups. However, some unique southern haplotypes  differed in only one or two mutations from the most common central northern haplotype y1.The haplotype cluster of the yellow lineage of N. natrix corresponded not to one, but to two star-like subclusters, with tip haplotypes differing in up to three mutations from the two frequent central haplotypes r3 and r4. The latter two haplotypes differed by two mutation steps from one another. There was no clear pattern in geographical distribution of haplotypes of the red lineage, with haplotypes of both subclusters occurring north and south of the Carpathians.The haplotype cluster of the red lineage of In accordance with the network analyses, the PCAs revealed three highly distinct clusters corresponding to the blue, yellow and red lineages  and the raggedness index (rg) indicating that the data do not deviate from the expectation of population expansion  and the red lineage of the common grass snake (N. natrix) glacial refugia in the south, close to the Mediterranean Sea or inland of the southern Balkan Peninsula. This is indicated by higher genetic diversities in the former refuges and supported by our diyabc analyses. Typically, such refugia are thought to be located in the southern peninsulas37. This was not the case for the blue lineage of N. helvetica, because the two peninsulas adjacent to its range, the Iberian and Italian peninsulas, are occupied by genetically distinct grass snakes . From there, the blue lineage seems to have rapidly expanded its range approximately 12,000 BP . These putative microrefuges, and the occurrence of the yellow and other mitochondrial lineages in the southern Balkan Peninsula, are in line with the idea of multiple glacial refuges of grass snakes15 and other reptiles51 there. The microrefuges of the red lineage must have been in very close proximity, so that haplotypes of each microrefuge came rapidly into contact, spread together and are now co-distributed across the entire range  and coincides with the time when the ice sheets first reached their local last glacial maxima and the sea level dropped first to glacial lowstand52, suggesting that the range interruption was caused by corollary environmental changes. The divergence of the Scandinavian and Central European populations was dated to only 7,700 BP, arguing for a colonization of Scandinavia from the Central European source populations during the Holocene climatic optimum.In the face of the wide distribution gap in the northern Balkans, yet another possibility comes into mind, namely two glacial refugia, one north of the Alps and one in the southern Balkan Peninsula. It seems plausible that the last glaciation interrupted an originally continuous population of the yellow lineage, with survivors in two distinct refuges, one in the north and one in the south. This hypothesis is supported by the high genetic diversity indices of northern Central European and Scandinavian grass snakes compared southern Central European ones Table  and by oes Table . The diy14 and our ABC modelling, which suggests that the red lineage expanded its range into Central Europe only 3,300 BP , a species widely co-distributed with grass snakes, has been doubted for permafrost regions40. Yet, even today, grass snakes occur in regions with discontinuous and continuous permafrost, for example near Lake Baikal in Siberia76, and the same is true for the adder76. Based on fossil and genetic evidence, Bhagwat & Willis72 examined glacial refuges of more than 50 European vertebrate species and concluded that there is a positive correlation between present-day distribution (northern range limits beyond 60°N) and persistence in northern glacial refugia. Species with northern range limits beyond 60°N are likely to have survived in northern refuges, and N. natrix qualifies for this category. Even if the northernmost questionable records14 are disregarded, there are unambiguous records of grass snakes beyond 60°N (for instance in Finland at 61.9°N14).Also for other taxa, there is growing evidence for extra-Mediterranean glacial refugia, in particular in the Alpine region, the Carpathian and the Caucasian regionsa Tables  suggest ion Fig. . In this77. For instance, forest land snails have been recorded from the late glacial of the Bohemian Massif, providing evidence for the persistence of small patches of broadleaf forest in Central Europe78.With respect to refugia in permafrost regions it has to be kept in mind that this does not necessarily imply life on permanently frozen ground. The surface of permafrost soils thaws during the warm season, and permanent frost remains only in different depths beneath. Moreover, it has been suggested that thermophilic biota have persisted in sheltered valleys with suitable microclimates, and there is hard evidence for some northern refugia up to the Norwegian coastN. helvetica was most likely located in southern France, outside the Mediterranean peninsulas. The red lineage of N. natrix seems to have survived the last glacial in a classical southern refugium in the Balkan Peninsula. Haplotype networks, mismatch distributions and demographic analyses of mtDNA sequences suggest that its refuge was geographically structured. From the Balkan, the red lineage has spread late to Central Europe and the Carpathian Basin and admixed en route with the yellow lineage of N. natrix11. For the yellow lineage, there is strong evidence for two distinct refugia during the LGM, one in the southern Balkan Peninsula and another one in Central Europe. This is supported (1) by the wide distribution gap in the northern Balkan Peninsula, (2) largely distinct mitochondrial haplotypes of grass snakes from the disjunct southern and northern parts of the distribution range, (3) by the higher genetic diversity of grass snakes from northern Germany and Scandinavia compared to snakes from southern Central Europe, (4) the lack of the red lineage in Scandinavia and northern Central Europe, and (5) by modelling using Approximate Bayesian Computation. From the Central European refugium, the yellow lineage colonized Scandinavia during the Holocene climatic optimum, long before the red lineage arrived in Central Europe.In conclusion Fig. , our stuSupplementary Information


Ramphocelus  located in southwestern Colombia. We evaluated whether this hybrid zone originated as a result of secondary contact or of primary differentiation, and described its dynamics across time using spatial analyses of molecular, morphological, and coloration data in combination with paleodistribution modeling.Characterizations of the dynamics of hybrid zones in space and time can give insights about traits and processes important in population divergence and speciation. We characterized a hybrid zone between tanagers in the genus Models of potential historical distributions based on climatic data and genetic signatures of demographic expansion suggested that the hybrid zone likely originated following secondary contact between populations that expanded their ranges out of isolated areas in the Quaternary. Concordant patterns of variation in phenotypic characters across the hybrid zone and its narrow extent are suggestive of a tension zone, maintained by a balance between dispersal and selection against hybrids. Estimates of phenotypic cline parameters obtained using specimens collected over nearly a century revealed that, in recent decades, the zone appears to have moved to the east and to higher elevations, and may have become narrower. Genetic variation was not clearly structured along the hybrid zone, but comparisons between historical and contemporary specimens suggested that temporal changes in its genetic makeup may also have occurred.Our data suggest that the hybrid zone likey resulted from secondary contact between populations. The observed changes in the hybrid zone may be a result of sexual selection, asymmetric gene flow, or environmental change.The online version of this article (doi: 10.1186/s12862-017-1096-7) contains supplementary material, which is available to authorized users. Characterizations of hybrid zones allow one to make inferences about traits and processes relevant to understanding the origin and maintenance of differences between populations and species , 2. A clInferring hybrid-zone origins from current patterns of variation is challenging because, with time, genetic signatures of secondary contact or primary intergradation tend to erode , 7. AlteSetophaga warblers, coupled with behavioral experiments showing aggressive superiority of males of one species over the other, indicate that movement of this zone has likely been driven by competition-mediated asymmetric hybridization [Another focus of studies on hybrid zones is the analysis of their temporal dynamics, which can allow understanding the role played by different evolutionary forces in such scenarios. When hybrid genotypes are less fit than parental genotypes, ‘tension zones’ are formed, which are maintained by a balance between the homogenizing effect of dispersal into the hybrid zone and the diversifying effect of selection against hybrids . If therdization –21. Tempdization , 23.Ramphocelus , whereas the smaller and yellow-rumped form icteronotus occurs along the costal plains west of the Andes extending north into Costa Rica and south into northern Peru. Females and immatures are similar to their respective males, but are less strongly colored. Along a c. 140 km transect running approximately northwest from the city of Cali downslope along the western flank of the Cordillera Occidental, the two forms hybridize, forming a gradient in coloration and body mass , butaupidae;  and was aupidae; . In the dy mass the diet , 44; or the diet ).Ramphocelus hybrid zone in southwestern Colombia originated as a result of secondary contact or of primary differentiation, and to examine the zone’s dynamics over nearly a century to make inferences about the action of different evolutionary processes. To accomplish this, we (1) reconstructed the biogeographic and demographic history of the hybridizing populations based on ecological niche modeling and coalescent analyses of mtDNA sequence data, (2) characterized genetic, morphological and plumage variation across the hybrid zone, and (3) compared spatial patterns of variation in morphometrics, plumage coloration, and genetic structure between specimens collected at different times to assess possible changes in the position and width of the hybrid zone.Here, we sought to evaluate whether the Ramphocelus hybrid zone historically by examining specimens collected from 1894 to 1986 in the ornithological collections of the American Museum of Natural History, the Cornell University Museum of Vertebrates, Universidad del Valle, and the Instituto de Ciencias Naturales at Universidad Nacional de Colombia. To describe current patterns of variation, we collected 73 new specimens in 2007-2010: 65 of them are from localities ranging across the hybrid zone over a distance of 140 km by road connecting the cities of Cali and Buenaventura in department Valle del Cauca [We characterized the el Cauca ; the remflammigerus and icteronotus at present. We conducted analyses in which localities of both forms and presumed hybrids were considered together to build potential distribution models, and also built separate models using data for flammigerus-like and icteronotus-like specimens; intermediate individuals were excluded from the latter analyses. Model performance in predicting present distributions  was satR. flammigerus). To reduce overprediction, we cropped maps of current and historical distributions to include only areas within the Andes Ecoregion [We projected models based on current climate data onto historical climate surfaces for 6000 years ago and for the Last Glacial Maximum  to determine whether the distributions of our study taxa were likely disjunct in the past as predicted by the secondary contact hypothesis, or have likely been continuous as predicted by the primary intergradation hypothesis. This approach requires assuming ecological niche conservatism and that climate represents a long-term stable constraint on potential distributions. Because ecological niche models are based only on climatic data, they tend to overpredict potential distributions into areas where the study species do not occur owing to historical limitations to dispersal  and one15719.1; ; Additiohttp://www.geneious.com). The mean length of these sequences was 988.8 bp (range 888-1008 bp).DNA was extracted from tissue samples or toepad samples taken from specimens using a Qiagen DNeasy Tissue Kit or a phenol-chloroform method . PCRs usb gene .We also analyzed mtDNA from historical toepad samples of 87 specimens collected by C. G. Sibley in 1956 and housed at the Cornell University Museum of Vertebrates . DNA extflammigerus and icteronotus at present using a maximum-likelihood (ML) phylogenetic analysis employing the GAMMA model. Nodal support was estimated using 1000 bootstrap replicates in RAxML, run from the RAxML BlackBox Web-Server [Ramphocelus carbo  and R. CT), among localities within sectors (FSC), and among localities among sectors (FST). Given that we lacked extensive sampling within point localities that would allow us to examine changes in genetic structure at a fine scale, this analysis allowed us to examine whether there has been any change in the way in which genetic variation is grossly distributed within and among different parts of the hybrid zone; if spatial genetic structure has become eroded , then one would expect an increase in genetic variation existing within sectors and a decrease in that existing among sectors over time . To make data comparable across time periods, sequences for 2010 specimens were trimmed to match the 210 bp available for the 1956 specimens. As an additional way to visualize potential changes in genetic structure over time, we constructed median-joining haplotype networks for the 1956 and 2010 samples using the program PopART (http://popart.otago.ac.nz/index.shtml).To assess potential changes in the genetic makeup of the hybrid zone over time, we examined population structure separately for the 1956 specimens and for our samples collected in 2007-2010 (hereafter 2010 specimens) employing procedures implemented in the program ARLEQUIN v3.5 . Based oflammigerus and icteronotus have experienced demographic expansions or if these taxa have exhibited historically stable population sizes, we examined trends in effective population size through time using the Extended Bayesian Skyline Plot (EBSP) method implemented in Beast v1.7.4 [b substitution rate of 2.08% divergence per million years [flammigerus-like and icteronotus-like specimens. As with niche modelling analyses, intermediate individuals were not included in analyses conducted separately by taxon. Skyline plots were built in R [et al. [To determine whether populations of t v1.7.4  using set v1.7.4 . We ran on years . We usedilt in R  with cod [et al. .n = 139 males, 83 females) with dial calipers to the nearest 0.1 mm: wing length (chord of unflattened wing from bend of wing to longest primary), exposed culmen, bill depth (at the base), bill width (at the base), tail length , and tarsus length . To describe morphological variation, we reduced variation in these characters using a principal components analysis (PCA).To characterize the hybrid zone phenotypically, we measured six morphological characters on museum specimens  using an Ocean Optics USB4F00243 Spectrometer with the SpectraSuite software (Ocean Optics). Three color measurements were estimated for each reflectance spectrum based on segment classification analysis [We characterized plumage coloration based on reflectance spectra from 400 to 700 nm measured on the rump of adult museum specimens  across the hybrid zone in different time periods using log-logistic models, commonly known as Hill functions [To compare patterns of morphometric and plumage color variation among adult specimens collected at different times in a geographical context, we defined three periods based on temporal sampling gaps: prior to 1911, 1956-1986, and 2010. Hybrid zones are often studied using cline-fitting algorithms that employ Bayesian or maximum-likelihood methods to estimate parameters like cline center and width , B is a coefficient denoting the steepness of the curve, and E corresponds to the distance along the X axis where 50% of the value in Y is observed . We usege for R .Due to differences in sampling effort over the hybrid zone across time periods, we evaluated the sensitivity of our estimates of cline center and width to sampling effects and calculated confidence limits for these parameters based on a bootstrapping procedure. For each time period and for both morphology and plumage reflectance data, we generated a bootstrap distribution of cline center and width estimated from 1000 data sets constructed by keeping sample size constant while resampling individuals with replacement. Because in some cases sample size was small, bootstrapping resulted in some samples in which variation was not clinal or in which estimates of cline parameters were unrealistic given the geographic extent of the hybrid zone; therefore, we only retained bootstrap samples in which the estimated cline center took values between 0 and 140 km. We compared estimates of cline center and width across the tree time periods using analyses of variance (ANOVA) and post-hoc Tukey tests treating estimates obtained in bootstrap samples as replicates. Likely due to a low number of individuals with morphological data for the western extreme of the hybrid zone in 2010, bootstrap estimates of cline width for this time period were highly variable and unrealistic; therefore, we do not report confidence limits for this parameter and did not include it in the ANOVA.Manacus hybrid zone in Panama [To validate the use of Hill functions to describe phenotypic and genetic clines and to infer cline center and width parameters, we compared our estimates based on Hill functions to estimates obtained by widely used cline-fitting algorithms  on a published data set that has been subject to cline analyses, namely the molecular data of seven loci of the n Panama . We usedn Panama  to estimn Panama . Consistflammigerus + icteronotus, with an area under the ROC-curve score of 0.987. Because this suggests that the assumption that climate limits distributions in these taxa is reasonable, we projected models onto past climatic conditions to estimate the potential historical distributions of flammigerus + icteronotus as well as those of each taxon separately at 6000 and 21,000 years ago.A potential distribution model developed under current climatic conditions in Maxent accurately predicted the present-day distributions of flammigerus + icteronotus has not been stable over time. The modeled potential range of these taxa combined at present in the Northern Andes Ecoregion extends for c. 420,000 km2. The predicted potential distribution based on climate for 6000 years ago was of similar size, with c. 460,000 km2 ; samples from Ecuador and Colombia were 1.6% divergent, and samples from Panama and Colombia differed by only 0.4% on average. Except for a separation between samples from Ecuador and Colombia, relationships among haplotypes were not clearly resolved by the ML phylogenetic analysis, in which most nodes lacked bootstrap support and no clades associated with specific geographic regions or with plumage coloration were identified , indicating that a significant fraction of genetic variation (19%) was apportioned among sectors of the study transect. This was not the case for the present-day data, in which no genetic structure across the transect was detected .AMOVAs suggested that patterns of genetic structure across our study transect differ between specimens from 1956 and 2010, with greater genetic structure among sectors in the 1956 data  but strongly suggested a marked population expansion in flammigerus . As with the distribution modeling analyses, these results are consistent with the hypothesis that the hybrid zone originated as a result of secondary contact following demographic expansion from formerly disjunct areas.Although credibility intervals for population size in Bayesian skyline plots were wide, the analysis including all sequence data  suggested that populations show a genetic signature of demographic expansion Fig. . ConstanReduction of morphometric variation using PCA resulted in a first component (PC1) describing body size in both males and females. In both sexes, variables loading most heavily on this axis  were tail length and wing chord. Thus, in the following we use PC1 as a general measurement of body size. We did not consider other principal component axes  in additional analyses because they did not vary gradually across the hybrid zone.Manacus [Our estimates of cline parameters estimated using Hill functions were highly concordant with published estimates obtained using HZAR and Analyse in Manacus , especiaicteronotus-type) being smaller than those from the east  along the hybrid zone, with birds from localities to the west  than in the 1956-1986 (22.0 km) and 2010 33.1 km) specimens , but were potentially in contact by 6000 ya, likely following a marked expansion of the geographic distribution of R. f. flammigerus. This scenario is supported by the historical demography analysis based on mtDNA sequence data, which indicates that populations have likely experienced significant range expansions, a pattern that awaits confirmation with multilocus data (see below) that should allow for lower uncertainty in estimates of population genetic parameters. Despite the broad credibility intervals around estimates of population size through time , taken together with results of niche modelling, the pattern observed in skyline plots is consistent with a scenario in which forms likely diverged in isolation  and then met as distributions expanded, presumably tracking the influence of Pleistocene climate change on vegetation [Ramphocelus populations likely occurred in the Pleistocene, as indicated by low levels of mtDNA divergence suggesting recent differentiation. However, because Quaternary climatic oscillations started well before the LGM [Based on patterns of genetic variation, fossil pollen data, and ecological niche modeling, several studies in the north temperate zone indicate that the origin of hybrid zones can be explained as a result of population expansions from isolated refugia during the Quaternary , 13, 71.getation , 77. A rgetation . Our dat the LGM , it is pRamphocelus hybrid zone may date to at least 6000 before present, Sibley [flammigerus and icteronotus resulted from recent anthropogenic deforestation and expansion of crops creating scrub and second-growth habitats, which are favored by these tanagers over dense rain forest. Although our analyses suggest that climatic conditions were suitable for contact between these forms thousands of years prior to major human-caused alterations in the area, it is likely that anthropogenic activities have facilitated contact between them, possibly leading to an increased incidence of hybridization in recent times.In contrast to our proposed scenario suggesting the origin of the , Sibley  hypothesHeliconius butterflies located in the same geographic region where we studied hybridization in Ramphocelus also provided evidence consistent with the hypothesis of origin via secondary contact [Heliconius butterfiles [Oophaga poison frogs [A recent study on a hybrid zone between  contact . Becauseterfiles , Oophagaon frogs ), work oon frogs , 15.Ramphocelus hybrid zone: there is clinal variation in coloration and body size, with males exhibiting clearer trends than females . With tRamphocelus is 1-2 years, and that dispersal distances per generation lie somewhere between 1 and 20 km, the expected width of clines under a neutral diffusion model  woutions in , the estene flow  conclusi context .Ramphocelus hybrid zone, but this interpretation needs to be tempered because samples were not taken at exactly the same localities at each time period and because the centers we estimated in some cases had wide support limits. In addition, our results should be interpreted with care given that the inferred geographic displacement of cline centers has been slight, corresponding to only c. 3 km between consecutive time periods. Nonetheless, our field observations indicate that the current patterns of variation are, in fact, different from those described by Sibley [icteronotus phenotype  has indeed extended to the east. Several previous studies have also reported on moving tension zones [That cline centers do not appear to be coincident across different periods for each of the characters suggests movement of the y Sibley . Specifion zones –87 althoon zones , 89.Ramphocelus hybrid zone may have been driven by competition or sexual selection favoring the icteronotus phenotype. We believe it is unlikely that aggressive superiority of icteronotus explains this pattern as shown in other studies of hybrid zones owing to its smaller body size, although we note that in a Manacus (Pipridae) hybrid zone in Panama, the smaller M. vitellinus is more aggressive than the larger M. candei [icteronotus phenotype has increased reproductive success as a result of sexual selection via male-male dominance or female choice [A possible explanation for the concurrent movement of clines for different characters in hybrid zones is competitive advantage of one phenotype mediated by aggression or by sexual selection . Thus, m. candei . Thus, ie choice , 92.R. flammigerus, then it would represent a plausible explanation for the likely movement of coloration in concert with others traits.If sexual selection is based on carotenoid plumage color, which presumably is strongly influenced by the environment , then iticteronotus phenotype would introgress across the hybrid zone as a consequence of increased gene flow from coastal to interior populations resulting from larger populations sizes in the former. This could be studied in the future with multilocus estimates of effective population sizes and of the magnitude of gene flow in both directions [Ramphocelus hybrid zone.An alternative explanation for zone movement was proposed by Sibley , who prerections . In addirections , then morections , 89, anyWe note that the inferred slight movement of the hybrid zone involves not only a west-east displacement, but also a shift in its center to higher elevations in the Andes. Because the elevational ranges of tropical birds may shift upslope in response to global warming , it is aRamphocelus hybrid zone may have resulted from a short duration of the allopatric phase relative to effective population sizes. Because no clinal mtDNA variation was observed across the Ramphocelus hybrid zone, we were unable to estimate cline parameters for different time periods to examine zone movement as done in a few studies on hybrid zones examining genetic variation in specimens collected at different times [Ramphocelus hybrid zone appears especially well-suited for analyses of variation in functionally important genes, which may provide important insights about the genetic basis of phenotypic variation. Recent work on other avian systems has revealed that phenotypic divergence in plumage traits controlled by relatively few loci may persist despite high overall genomic similarity [In contrast to multiple studies on hybridization in birds finding significant mtDNA divergence between populations located away from the center of hybrid zones and clinal variation in haplotype frequencies across them  would rmilarity –110.Ramphocelus hybrid zone over time, with repeated sampling of multiple specimens at fixed locations; rather, specimens were collected by numerous researchers, often opportunistically, for different purposes, and with no common sampling schemes at different times. Therefore, we did not feel confident in assigning individuals to discrete localities as required for cline-fitting algorithms which rely on calculating means and variances for samples of specimens from the same sites [Finally, our study serves to illustrate that Hill functions and bootstrap resampling are useful statistical tools to characterize patterns of variation across hybrid zones. In contrast to other studies , our worme sites –68. Our Ramphocelus hybrid zone we studied originated following secondary contact between populations that expanded their ranges out of isolated areas in the Quaternary, likely as a consequence of climatic change. Concordant patterns of variation in phenotypic characters across the hybrid zone and its narrow extent are suggestive of a tension zone, maintained by a balance between dispersal and selection against hybrids. In addition, our estimates of phenotypic cline parameters obtained using specimens collected over nearly a century revealed that the zone has likely moved to the east and to higher elevations, and has likely become narrower. These observed changes in the hybrid zone may be a result of sexual selection, asymmetric gene flow, or environmental change. Our data represent a baseline for a variety of ecological, behavioral, and genetic studies that could shed further light on the forces involved in speciation and hybrid-zone dynamics in this system.Models of potential historical distributions based on climatic data and genetic signatures of demographic expansion suggested that the Additional file 1: Table S1.Ramphocelus flammigerus included in the study. (DOC 450 kb)Information on the samples of Additional file 2: Figure S1.Clines for morphological and plumage data estimated across 1000 bootstrap samples. The vertical line in each plot corresponds to the mean value of cline centers for the 1911 , 1956  and 2010  periods. (PDF 4613 kb)Additional file 3: Figure S2.Ramphocelus flammigerus hybrid zone in southwestern Colombia. Data are shown separately for historical specimens  and recent specimens . (PDF 538 kb)Variation in morphology  and plumage chroma in female specimens across the


Consequently, HSF-1 hyperphosphorylation mediated by JNK operation causes transcriptional inactivation of HSF-1, and supported ROS-mediated autophagy induction. Interestingly, in HSF-1 immunoprecipitated and silenced HCT-116 cells, co-expression of apolipoprotein 1 (ApoL1) and JNK was observed which promoted the caspase independent autophagic cell death accompanied by p62 downregulation and increased LC3-I to LC3-II conversion. Finally, in vivo findings confirmed that vitexin suppressed tumor growth through activation of autophagic cascade in HCT-116 xenograft model. Taken together, our study insights a probable novel association between HSF-1 and ApoL-1 was established in this study, which supports HSF-1 as a potential target of vitexin to improve treatment outcome in colorectal cancer.Heat shock transcription factor-1 (HSF-1) guards the cancerous cells proteome against the alterations in protein homeostasis generated by their hostile tumor microenvironment. Contrasting with the classical induction of heat shock proteins, the pro-oncogenic activities of HSF-1 remains to be explored. Therefore, cancer's fragile proteostatic pathway governed by HSF-1 could be a potential therapeutic target and novel biomarker by natural compounds. Vitexin, a natural flavonoid has been documented as a potent anti-tumor agent on various cell lines. However, in the present study, when human colorectal carcinoma HCT-116 cells were exposed to vitexin, the induction of HSF-1 downstream target proteins, such as heat shock proteins were suppressed. We identified HSF-1 as a potential molecular target of vitexin that interact with DNA-binding domain of HSF-1, which inhibited HSF-1 oligomerization and activation ( Cells encounter stress from various environmental cues which causes profound alterations in proteome homeostasis known as proteotoxic stress. They are well equipped with sophisticated adaptive mechanisms to combat these stresses. At molecular level, heat shock response (HSR), a ubiquitous transcriptional response driven by heat shock transcription factor HSF-1), governs the expression of heat shock proteins (Hsps) for cytoprotection , governs, 3. DuriMacroautophagy (hereafter referred autophagy) is a well conserved, highly orchestrated lysosomal catabolic process in eukaryotes that facilitates the degradation or recycling of superfluous or impaired cellular constituents via autophagolysosome formation . The duaHSR and autophagy represent two ends of the protein homeostasis spectrum, which are likely to complement each other during cellular stress. Earlier studies reported that Hsp70 overexpression prevented p62/sequestosome/SQSTM1 degradation and decrease LC3-II protein expression, indicating autophagy regulation by HSR. . HSF-1 nbona fide BH3-only pro-death protein apolipoprotein L1 (ApoL1) has been characterized for induction of ACD independent of caspase-mediated apoptosis which can be a general autophagy mediator  Figure . Vitexinin silico analysis to identify the interaction and binding sites between HSF-1 and vitexin that caused disruption of HSF-1-DNA interaction. Human HSF-1 consists of N-terminal DNA-binding domain (DBD), followed by coiled-coiled oligomerization domain (HR-A/B), a variable regulatory domain (RegD) and a transactivation domain (TAD) /2, where W represents the width (shortest tumor diameter) and L represents the length (longest tumor diameter). Body weight of tumor-bearing animals was recorded to determine compound toxicity. Tumors were dissected and stored in liquid nitrogen or fixed in formalin for further analysis.Male BALB/c pathogen free athymic nude mice,  were purchased from Orient Bio, Inc. . Mice were housed in a sterile temperature controlled-room on a 12 h: 12 h light: dark schedule with standard rodent chow diet and water 2O2 to block endogenous peroxidase activity. After antigen retrieval, sections were incubated with HSF-1, p-JNK and ApoL1 antibodies followed by PBS washes. Appropriate secondary antibodies were used for detecting immunoreactivity with DAB substrate . Background counterstaining was performed with hematoxylin and slides were analyzed under light microscope (×400). The percentage of IHC stained area in tissue sections were calculated by ImageJ  software.Formalin fixed paraffin-embedded tumor specimens were sectioned (4 μM). After being deparaffinized followed by a hydration process, the slides were treated with 3% Hp < 0.05.Statistical analysis was performed with SPSS  22.0 for windows. Results are expressed as the mean ± standard deviation (SD) of three independent experiments. The data were subjected to one-way analysis of variance (ANOVA) and the significance of difference between samples means was calculated by Duncan's multiple range test . Differences were considered statistically significant for values of


Lasiopodomys brandtii) differs from that of most other species of the genus Microtus. The demographic history of this species and the patterns shaping its current genetic structure remain unknown. Here, we explored patterns of genetic differentiation and infered the demographic history of Brandt’s vole populations through analyses of nuclear microsatellite and D-loop sequences.The colonial habit of Brandt’s vole , clinal patterns in the allele frequencies, alleles that were rare in the original area that have become common in the newly occupied regions, and higher genetic differentiation in the expanded range compared with the original one.L. brandtii most likely originated from the southeastern part of its current geographic range, and subsequently colonized into the northeastern and western parts by expansion. The genetic patterns among the derived populations and with respect to the original population are consistent with that expected under genetic surfing models, which indicated that genetic drift, rather than gene flow, is the predominant factor underlying the genetic structure of expanding Brandt’s vole populations.Our results indicate that The online version of this article (doi:10.1186/s12862-017-0995-y) contains supplementary material, which is available to authorized users. The evolutionary history of most species generally is characterized by an interplay among environmental changes, episodes of range expansion and migration, population admixtures, and local extinctions , 2. ThesLasiopodomys brandtii)  is a steppe-dwelling rodent species that is currently distributed from the central parts of Inner Mongolia, through the central and eastern of Republic of Mongolia, and to the southern borders of Mongolia in Trans-Baikalia, Russia  and maximum (ΔK = 226.2) at K = 2  matrix . For the two clusters , 3.3% (P < 0.0001) of the total variation accounted for the among clusters  of total variation emerged between clusters  if DR was assigned to the western cluster (Table P < 0.0001) of the total variation was assigned to the among clusters  of the total variation was distributed among populations for all samples; 75.99% (P < 0.0001) of the total variation was distributed among the two haplogroups; and 59.7% (P < 0.0001) of the total variation was distributed between the eastern and western clusters, resulting in maximization of genetic differences  of the total variation was distributed among clusters  appears to have diverged earlier than Hg2 Fig. .We applied MIGRATE-N to the microsatellite data and found that Model 1 had the highest likelihood Fig. ; Table 3P < 0.05) and Fu and Li’s F  were both significantly negative   to the entire mtDNA data, and Hg1 and Hg2 (see above). The MJ network Fig.  already 05) Fig. . When ha05) Fig. ; Table 105) Fig. –f. The lFST values computed for the microsatellite data between sampling sites from the southeastern, northeastern and western range of the species varied, 0.042 (range 0.015–0.1), 0.044 (0.025–0.099), and 0.059 (0.023–0.099), respectively  and geographical distances (lnGD) were significantly correlated for the microsatellite dataset from entire samples , which is consistent with IBD. Significant IBD was supported for the eastern samples if analyzed separately , but was not significant for samples from the western samples . If we considered three, rather than two, regional genetically split populations none of the IBD analyses emerged as significant   and expected heterozygosity (He) significantly decreased with increasing FST values to the probable start or arrival site with the expection of the starting site of BO in the northeastern . Seven and five allele frequencies showed significant variations with increasing FST values to the probable arrival site BT and the starting site BO in the northeastern, respectively. In the southeastern, eight allele frequencies were significantly correlated with the FST to BX  of the most primitive ancestral haplotype inferred by RASP and Lagrange  [FST values instead of the geographical distance to test the range expansion of Brandt’s vole. The results that the He values significantly decrease with increasing FST values to the probable arrival (BT and AL) or starting (BX and BO) sites for the species  . Howeveries Fig.  supporteLasiopodomys was present in the late Early Pleistocene [L.brandtii happed earlier than 0.041 Mya (about middle-late Pleistocene), which is for the species’ diversion into Hg1 clade  or the probable arrival site centroid (BT and AL) within each of the three regions  in the habitat , 111. PoWe hypothesized that Brandt’s voles originated from the extant southeastern distribution. Under the integrative effect of various factors, such as temperature, rainfall, vegetation and landscape traits, this species colonized from the original area surrounding BX in Xilinguole region, propagating by wave within the region to reach the probable arrival site BT in the northeastern and expanding thereafter within the area; the subsequent expansion of Brandt’s voles population to the western at the arrival site AL from the starting site BO in the northeastern followed the same surfing pattern. Landscape heterogeneity, a low density of individuals in the front of the wave and a low dispersal capacity promote the strong genetic structuration of the three clusters. Brandt’s vole thus formed the current patterns, which are characterized by isolated, patchy, unstable habitats with some genetic characteristics.Additional file 1: Table S1.Table S2. Summary statistics for twelve microsatellite loci over all Brandt’s vole samples. Table S3. Genetic diversity measures calculated over 12 microsatellite loci and across all geographic locations. Table S4. Summary of Chi-Square Tests for Hardy-Weinberg Equilibrium for each geographic location sampled. Table S5. Geographic occurrences and frequencies of D-loop haplotypes H1–30. Table S6. Summary statistics on polymorphism and demographics inferred from analyses of D-loop sequences. Table S7. Pairwise FST values calculated based on microsatellite data between 23 geographic locations sampled. Table S8. Clades support and ancestral areas reconstruction as obtained with S-DIVA and Lagrange. (DOCX 115 kb)Sampling information for Brandt’s vole. Additional file 2: Figure S1.Brandt’s vole population structure based on 12 microsatellites loci as implemented by STRUCTURE. A) Values of lnP(D) from 20 independent runs for K = 1–23. B) Values of ΔK from 20 independent runs plotted for K = 1–23 and calculated by the Evanno method. (PDF 3121 kb)Additional file 3: Table S9.A) Allele frenquency of each locus for Brandt’s vole populations in southeastern and northeastern distribution; B) Allele frenquency of each locus for Brandt’s vole populations in northeastern and western distribution. (XLSX 62 kb)


Arrhenatherum elatius originating from a large latitudinal gradient. In general, populations from the North grew smaller and had a lower flowering probability. Toward the North, the absolute plastic response to the different winter conditions as well as heritability for biomass production significantly declined. Genetic differentiation in plant height and probability of flowering were very strong and significantly higher than under neutral expectations derived from SNP data, suggesting adaptive differentiation. Differentiation in biomass production did not exceed but mirrored patterns for neutral genetic differentiation, suggesting that migration‐related processes caused the observed clinal trait variation. Our results demonstrate that genetic diversity and trait differentiation patterns for A. elatius along a latitudinal gradient are likely shaped by both local selection and genetic drift.Across Europe, genetic diversity can be expected to decline toward the North because of stochastic and selective effects which may imply diminished phenotypic variation and less potential for future genetic adaptations to environmental change. Understanding such latitudinal patterns can aid provenance selection for breeding or assisted migration approaches. In an experiment simulating different winter temperatures, we assessed quantitative trait variation, genetic diversity, and differentiation for natural populations of the grass  Beauv. ex J. Presl & K. Presl  mild temperatures without frost, (2) an extended frost period, and (3) relatively warm temperatures followed by a short, sudden frost. The three mid‐winter climate scenarios were designed to simulate different possible mid‐winter conditions occurring across the locations of seed origins, that is, frozen soil toward the North, unfrozen soil toward the South, and random high‐temperature variability occurring across the latitudinal distribution. We assessed quantitative trait variation under the different experimental conditions and compared diversity and differentiation patterns between quantitative and putatively neutral molecular traits. More specifically, we asked (1) whether quantitative trait expression and genetic diversity, and the plastic response to the different mid‐winter treatments show latitudinal clines, and (2) whether the observed quantitative trait differentiation shows signatures of selection.Here we assess genetic variability and phenotypic plasticity in quantitative traits of the grass  Jenkin,  as well  Jenkin, . Further Jenkin, . Differe Jenkin, . Here, i22.1Arrhenatherum elatius is a very common, relatively drought resistant forage grass. It is an autotetraploid, wind‐pollinated perennial, and has been described as primarily self‐incompatible with a low degree of self‐fertility  before undergoing one of three simulated mid‐winter scenarios chosen to approximate common temperature regimes in Southern, Northern and Central Europe, respectively Figure : (1) a f2.2.3Growth performance following frost exposure has previously been used as a relative measure to evaluate the effectiveness of plant cold acclimation  follows a binomial distribution with parameters n and p, where p is the unknown frequency of allele A in the tetraploid genotype , we computed the value of p that maximizes the likelihood of k. Using likelihood ratio tests (at an α = .05 level) between the likelihood for the best p and likelihoods for all other possible p values, we obtained a measure of confidence for the best p. In case of nonsignificant comparisons, we called incomplete genotypes . Eventually, for further analysis only polymorphic loci scored for a minimum of two samples per population were retained. Molecular genetic diversity across loci was calculated at population level as gene diversity corrected for sample size , we screened the data for loci putatively under selection using BayesScan 2.1  were obtained from BayesScan 2.1.Leaf material from one offspring per seed family was sampled, and DNA was extracted from freeze‐dried material with the DNeasy 96 Plant extraction kit . Assessment of molecular diversity and differentiation patterns was carried out using SNP data obtained by a genotyping‐by‐sequencing approach (GBS). SNP genotyping was conducted at the Genomics Diversity Facility of Cornell University, Institute of Biotechnology, USA. In short, the GBS libraries were constructed using the PstI restriction enzyme and a protocol modified from Elshire et al.  and sequ2.42.4.1We used generalized linear mixed models to explain the observed trait variability using latitude of sample origin, mid‐winter treatment, and their interaction as fixed factors. Sample origin, seed family, and their interaction with treatment as well as block were entered as random effects. Model selection was based on the REML implementation in the package “lme4”  for each trait and population was estimated (a) across all experimental conditions and (b) for each condition separately. Therefore, models were fitted in “MCMCglmm” (a) with treatment as fixed, block and seed family, and the family x treatment interaction as random effects and for (b) with only block and seed family as random terms. Total phenotypic variance explained (VT) was partitioned into the additive genetic variance (VA) and the residual variance (VR) composed of the error variance and the variance explained by block and for a) additionally the variance for the family × treatment interaction. Heritability (h2) was then defined as the ratio h2 = VA/VT = VA/(VA + VR)  as VA = 4*VF  included a significant seed family × treatment interaction. If the collected open‐pollinated seed families consist indeed of half‐sibs only, the quantity h2 can be interpreted as narrow‐sense heritability. However, as the relatedness among sibs has not been assessed directly, our heritability estimates may include nonadditive and additionally maternal effects. Heritability as a measure of evolutionary potential has been criticized in general, and mean‐scaled additive genetic variances (evolvability) have been proposed as more suitable instead  was estimated across all experimental conditions, and for each condition, separately fitting models in “MCMCglmm” containing treatment as fixed or only the intercept, respectively, and block, population, and seed family as random effects. The total genetic variance was partitioned into among (VP) and within‐population (VA) components , and genetic differentiation was quantified as QST = Vp/(2*VA + Vp)  and quantitative differentiation were tested against pairwise spatial and climatic distances among populations. We tested also for covariation between quantitative and neutral genetic differentiation. Significance of the correlations was evaluated by  Mantel tests in R with 1,000 permutations. Climatic distances between sampled populations were obtained as Euclidean distances from scaled bioclimatic data (19 variables) for each sample location extracted from the Worldclim database [http://www.worldclim.org/]  Lande, . We are d Merilä . To test2.4.3QST) differed from neutral expectations, we followed the approach of Whitlock and Guillaume . Hence, a deviation from zero directly indicates divergent or homogenizing selection in case of a significant positive or negative deviation, respectively. A distribution of QSTn values was calculated by simulating a neutral among‐population variance 1,000 times as VPn = 2*FST * VA/(1‐FST) and then multiplying by a factor r/(npop‐1), with npop the number of populations considered and r being a random number drawn from a chi‐square distribution with npop‐1 degrees of freedom, to simulate the sampling distribution around this expectation. For each simulation, the FST value obtained from GBS data was used, and a VA value was sampled from the posterior distribution of the models described above. QSTn values were then computed using the observed within‐population variance. The test statistic was calculated as the difference between 1,000 QST values drawn from the posterior distribution of the model and 1,000 simulated QSTn values and considered to be significant if the 95% credible interval did not include zero.Adaptive divergence and signatures of selection can be detected by comparing differentiation at quantitative traits with that expected under neutrality  indicating maladaptive plasticity. Relationships for all other combinations of fitness approximations and trait plasticities were nonsignificant.Tests for the adaptive significance of relative plasticity for the different mid‐winter treatments revealed that plasticity in final height negatively affected fitness approximated by biomass production  showed a significant decline and increase with latitude, respectively . Heritabilities estimated for the different experimental winter conditions separately revealed an opposite trend for final height in the warmfrost scenario  but no other significant correlations (p ≥ .1). Using evolvability instead of heritability, significantly negative correlations with latitude were found for biomass only .For 37 of 40 samples, genotypic information could be obtained. For these samples, the GBS approach yielded 5,026 polymorphic SNP loci of which 51 were putatively under selection and hence, excluded from the neutral 3.2FST) was moderate as estimated from GBS data (FST = 0.090). Pairwise neutral molecular differentiation estimates did not follow an isolation‐by‐distance or an isolation‐by‐climate pattern (Mantel p > .33).Neutral genetic differentiation at the molecular level  varied substantially among traits . Differentiation for final plant height and flowering probability significantly exceeded neutral expectations suggesting a contribution of diversifying selection  but showed an isolation‐by‐climate pattern for initial and final plant height only .Across‐treatment quantitative genetic differentiation . We hence choose not to interpret results from this analysis.Relating pairwise 44.14.1.1A. elatius are more likely the result of neutral, migration‐related processes.Genetically controlled latitudinal clines in biomass accumulation have been observed by common garden studies of numerous herbaceous plant species  was not detectable in the traits assessed. It has been argued that under current and historical environments, selection keeps the reaction norm equally adaptive, and only in new or stressful environments a significant G × E effect might manifest .The raw data (quantitative and SNP data) underlying the main results of the presented study have been deposited at the dryad repository (doi: Click here for additional data file.