Anguis) present in the Balkan Peninsula, and tested the relationship between genetic diversity and topographic heterogeneity of the inhabited ranges.Genetic architecture of a species is a result of historical changes in population size and extent of distribution related to climatic and environmental factors and contemporary processes of dispersal and gene flow. Population-size and range contractions, expansions and shifts have a substantial effect on genetic diversity and intraspecific divergence, which is further shaped by gene-flow limiting barriers. The Balkans, as one of the most important sources of European biodiversity, is a region where many temperate species persisted during the Pleistocene glaciations and where high topographic heterogeneity offers suitable conditions for local adaptations of populations. In this study, we investigated the phylogeographical patterns and demographic histories of four species of semifossorial slow-worm lizards  they inhabit. Areas with more rugged terrain harbour higher genetic diversity.We inferred phylogenetic relationships, compared genetic structure and historical demography of slow worms using nucleotide sequence variation of mitochondrial DNA. Four Anguis in the Balkans is concordant with the refugia-within-refugia model previously proposed for both several other taxa in the region and other main European Peninsulas. While slow-worm populations from the southern refugia mostly have restricted distributions and have not dispersed much from their refugial areas, populations from the extra-Mediterranean refugia in northern parts of the Balkans have colonized vast areas of eastern, central, and western Europe. Besides climatic historical events, the heterogeneous topography of the Balkans has also played an important role in shaping genetic diversity of slow worms.Phylogeographical pattern of the genus The online version of this article (doi:10.1186/s12862-016-0669-1) contains supplementary material, which is available to authorized users. Diversity of European biota has been strongly influenced by global climatic and environmental changes in the Quaternary. Toward the end of the Pleistocene, repeated climatic oscillations led to extinctions of many phylogenetic lineages from vast northern areas during glacial periods followed by re-colonisations during interglacials –3. Many In comparison to the Iberian and Italian peninsulas, the Balkans has remained much less studied in terms of the biogeographical history of the species distributed there, although it is richer both in biodiversity and paleoendemics –8. The BAnguis (family Anguidae) within its Western Palearctic range. This genus comprises five species, four of which occur in the Balkans [Anguis cephallonica Werner, 1894 and A. graeca Bedriaga, 1881 are Balkan endemics with rather restricted distribution in the south of the peninsula, ranges of A. fragilis Linnaeus, 1758 and A. colchica  are at the continental scale and cover vast areas of Europe and western Asia [We, and others –17 have  Balkans , 15. Whiern Asia , 17–19. ern Asia , 21, oneern Asia , 23. NevIn this study we collected and analysed data originating from the Balkan slow-worm populations with the aim to i) provide a detailed picture of distribution; ii) infer historical relationships of populations and describe genetic diversity; iii) reconstruct biogeographical histories of the Balkan slow-worm populations during the Quaternary. Finally, we tested iv) whether the genetic diversity observed in the Balkan slow worms is driven by specifics of topography. Dispersal barriers would most likely coincide with the extensive and variously rugged mountain ranges of the Balkan Peninsula, thus we expected the slow-worm genetic diversity to be correlated with topographic variation of this region.Anguis, we devoted this study to slow-worm populations from this region. Our sampling strategy focused on equally representing the whole Balkan region as well as all four Balkan species. Sampling effort also took into account that these species vary in distribution ranges and inter-specific genetic diversification and may have low population densities in some areas. Tissue samples were obtained mainly from road-killed individuals or alternatively from living animals as oral swabs, blood droplets, or miniature skin biopsies. This sampling procedure did not affect survival of the captured animals. No experimental research was carried out on these animals in this study. All samples were preserved in 96 % ethanol. A portion, 732 base pairs (bp), of the mitochondrial gene for NADH dehydrogenase subunit 2 (ND2) was targeted. Newly produced nucleotide sequences were supplemented to previously published sequences from the Balkans [Anguis species, namely 110 A. fragilis, 56 A. colchica, 49 A. graeca, 16 A. cephallonica , A. colchica (c1–c12), A. graeca (g1–g16), A. cephallonica , A. veronensis (v1–15); and [A. fragilis (AF01–AF07), A. colchica . The resulting dataset contained 271 sequences, excluding outgroup. Following previous works of our team [Pseudopus as outgroup .Since the Balkan Peninsula represents an important evolutionary centre of the genus  Balkans –17 to coour team , 15, we ND2 gene, five subsequent transfer RNA (tRNAs) genes and the light-strand replication origin using primers  and protocol following [A. cephallonica for both PCR amplifications (in degraded DNA) and sequencing. The final stretch contained 732 bp-long fragment of ND2 after trimming the low quality ends. The sequencing was performed by Macrogen Inc.  and new sequences have been deposited in GenBank under accession numbers KX020147–KX020322  were aligned manually. No stop codons were detected when the sequences were translated using the vertebrate mitochondrial genetic code in the program DnaSP 5.10 [p-distances among the main lineages or haplogroups within each taxon, and to estimate the number of haplotypes (h), haplotype diversity (Hd), number of segregating sites (S), nucleotide diversity (π), and Watterson’s theta (θW) for each of these lineages or haplogroups.The protein-coding aSP 5.10 . The samst codon position), HKY + I (2nd codon position), and HKY + G (3rd codon position); and for the ML analysis: GTR + G in each codon position. The ML clade support was assessed by 1,000 bootstrap pseudoreplicates. The MrBayes analysis was set as follows: two separate runs, with four chains for each run, 10 million generations with samples saved every 100th generation. The convergence of the two runs was confirmed by the convergence diagnostics . First 20 % of trees were discarded as the burn-in after inspection for stationarity of log-likelihood scores of sampled trees in Tracer 1.6 [For phylogenetic analyses we used the all-individuals dataset supplemented by distinct published haplotypes from outside the Balkans to obtain a complex picture of the phylogenetic relationships within the Balkan Peninsula and in the framework of the whole genus. The best-fit codon-partitioning schemes and the best-fit substitution models were selected using PartitionFinder v1.1.1. , accordiacer 1.6   using the 95 % limit of parsimony as implemented in TCS 1.21 [A. graeca, and one in A. cephallonica), we also applied a fixed connection limit at a higher number of steps allowing visualization of their likely connections to the networks constructed under the 95 % limit of parsimony.Haplotype-network approaches can be more effective for presentation of intraspecific evolution than the tree-based phylogenetic approaches . TherefoTCS 1.21 . To infeNe) through time directly from sampled sequences and does not require a specific a priori assumed demographic model. Main population groups correspond to monophyletic groups. In a single case of several basal haplogroups of A. fragilis, the population group was defined geographically (‘Slovenian’ populations). Preliminary analyses were run using both strict molecular clock and uncorrelated lognormal relaxed molecular clock. Since the parameter of the standard deviation of the uncorrelated lognormal relaxed clock was close to zero, the final analyses were run enforcing the strict molecular clock model. A uniform prior for the substitution rate with the initial value 0.0065 substitution/site/lineage/Myr  as impllizards; ) was setlizards; , all codlizards; . The resFS [D [R2 [In addition, the mismatch distributions (MD) were calculated as the distributions of the observed pairwise nucleotide differences and the expected values under a growing- or declining-population model using DnaSP 5.10 . The occFS  Tajima’sFS [D , and RamS [D [R2  calculatπ) with the terrain ruggedness index (TRI). TRI is a measure of topographic heterogeneity calculated as a sum change in elevation between a grid cell and its eight neighbour cells in a grid network [http://srtm.usgs.gov) with a spatial resolution of approximately 3 arc-sec (~100 × 100 m) with a final resample to 30 arc-sec (~1 × 1 km) using GRASS GIS 7.1 [2). The ‘extreme’ values of TRI were selected with the aim to preferentially study the influence of steeper terrain, presumably posing stronger barriers to gene flow and resulting thus in higher probability of lineage divergence. Due to the controversy about the biogeographical significance of the Apuseni Mts. as a separate unit within the Carpathians [Since a more complex topography is more likely to limit dispersal and gene flow, we hypothesized that regions with higher topographic heterogeneity (terrain ruggedness) will be inhabited by slow-worm lineages characterized by higher genetic diversity. To test for this relationship we performed regression analyses of nucleotide diversity  form a separate clade, in which the Balkan endemic A. graeca is in a sister position to the eastern widespread species, A. colchica . Anguis graeca is mostly confined to the Hellenides in the southern Balkans where it is distributed from the northern Peloponnese, along the Pindus Mts. and the Albanian Mts. to the southernmost Dinaric region (southern Montenegro) and western Macedonian-Thracian Massif (northeastern Rep. Macedonia). Anguis cephallonica was found in the Peloponnese and Kephallonia Island .A. fragilis and A. colchica was detected in eastern and southeastern Serbia and central-western Bulgaria . Three zones of contact were further detected between A. fragilis and A. graeca in southernmost Montenegro , northwestern Rep. Macedonia (sites 63 and 158), and in the tri-border area of Serbia, Bulgaria and Rep. Macedonia . Sympatric occurrence of A. graeca and A. cephallonica was confirmed from northern Peloponnese .Our detailed sampling also revealed several areas where haplotypes of different species could be found in distances from ca. 15 to 80 km, indicating the existence of contact zones  sequences, which yielded a total of 100 haplotypes. Nucleotide diversity was higher in the two Balkan endemics, A. graeca (π = 1.17 ± 0.11 %) and A. cephallonica (π = 0.81 ± 0.21 %), than in the Balkan populations of the two northerly distributed taxa, A. colchica  and A. fragilis , and the southern one . Haplotypes from the ICE haplogroup were also detected in Central Europe and southern Great Britain . The ICAnguis colchica, a deep intraspecific divergence  and the southern Caspian region ; see also [Incerta clade (16 haplotypes within 43 specimens) is widespread along the Carpathians and the Stara Planina Mts., with relatively high genetic variation and diversified into three main well-supported lineages: (i) Stara-Planina lineage in the region of the Stara Planina Mts. and the northern foothills, reaching the Serbian Carpathians ; (ii) Banatian lineage detected in the Banat (southwestern Carpathians); and (iii) Carpathian lineage present in most of the Carpathians with a further sub-structure forming at least four haplogroups; Carpathian I–IV  than the less widespread A. cephallonica. The genetic structure of A. graeca is complex, characterized by many haplogroups but without deep divergences ; the second in western Greece, Corfu Island and southern Albania (graeca V); and the third one in Rep. Macedonia (graeca XI).Of the two Balkan endemics, ces Fig. . Only thA. cephallonica, has a similarly complex phylogeographical structure with 13 haplotypes detected among 16 specimens  population growth was detected in the Carpathian lineage  of the lineages/haplogroups plotted against the Q3, median above Q3, and modus above Q3 of the terrain ruggedness index (TRI), and an area size inhabited by these lineages/haplogroups, were statistically significant (Table beta) regression coefficients were highly significant both when samples from the Apuseni Mts. were included among the Carpathian samples as well as when they were treated separately.Multiple linear regressions of nucleotide diversity , A. coleca Fig. . Historiene Fig. . Anguis istocene , 45. It A. cephallonica), Hellenides (A. graeca), southern Carpathians (A. colchica), and northwestern Dinarides (A. fragilis)  Fig. . Existenronensis . This paronensis , 47–49 aronensis , 51. Theronensis ) or in nronensis .A. fragilis and several haplogroups of the Carpathian lineage of A. colchica colonized broad areas of temperate Europe from their northern extra-Mediterranean refugia. On the contrary, A. cephallonica, A. graeca, the Pontic and Stara-Planina lineages of A. colchica, and the South Balkan haplogroup of A. fragilis did not disperse much from their southern Mediterranean refugia and their distribution has remained more localized south of the Danube River  a star-like pattern of the haplotype network and low genetic variation of the ICE haplogroup and (ii) the broad area presumably colonized from a single source population [A. fragilis suggests not only quick expansion to the north, but also a gradual north-to-south/west-to-east expansion during the Pleistocene, which is very rare in terrestrial animals .Lissotriton vulgaris, [Bombina variegata, [A. colchica . These lareolus .A. colchica shows relatively high mtDNA polymorphism. Close phylogenetic relationships of the southeast Bulgarian and Anatolian populations (own unpublished data) indicate that the Pontic lineage might have colonized the Black Sea region of the Balkans during the Pleistocene when the peninsula was accessible from northern Anatolia via terrestrial route [Despite its limited distribution in the Balkans, the Pontic clade of al route , 73.A. cephallonica, and the region west of the Pindus Mts. (with high haplotype diversity of A. graeca) have favourable geography with deep long valleys providing stable climatic conditions. Consequently it is known for high endemism of numerous plants, invertebrates, and vertebrates [A. graeca during the Plio-Pleistocene and allowed divergence of its lineages. In contrast, the overall flat Skadar region enabled colonization of southern parts of the region of present-day Montenegro and forming a narrow zone of sympatric occurrence with A. fragilis. The existence of several distinct haplogroups in A. graeca indicates that this species has a longer and complex evolutionary history. Overall high intraspecific genetic diversity with up to 3.6 % in p-distances . The siifestyle . Such coifestyle .Anguis species and hidden diversity of their populations in the Balkans. These species have mostly parapatric distributions that correspond with major mountain ranges. We showed that biogeography of the genus in the Balkans is concordant with the refugia-within-refugia model previously proposed for other main European Peninsulas. The role of Mediterranean as well as extra-Mediterranean refugia was detected in the evolutionary history of slow worms with varying ages and degrees of post-glacial recolonization. Beside climatic historical events, we consider the complex topography of the Balkans as one of the most important factors in shaping recent genetic diversity of slow worms. Topographic heterogeneity seems to be a good predictor of both genetic and species diversity, in general. The pattern observed on slow-worm refugia in the Balkan Peninsula thus illustrates and highlights the fact that many global biodiversity hotspots and endemism centres are located in montane regions [Our study uncovered mitochondrial DNA variation and distribution of four  regions –82. As i regions , 83–86, New sequences have been deposited in GenBank (accession numbers KX020147–KX020322) and other input data are provided in Additional file Not applicable.Not applicable.


Bos indicus) is declining (8.94 % in last decade) and needs immediate scientific management. Genetic characterization is the first step in the development of proper management strategies for preserving genetic diversity and preventing undesirable loss of alleles. Thus, in this study we investigated genetic diversity and relationship among eleven Indian cattle breeds using 21 microsatellite markers and mitochondrial D loop sequence.Indian agriculture is an economic symbiosis of crop and livestock production with cattle as the foundation. Sadly, the population of indigenous cattle  contains supplementary material, which is available to authorized users. Bos indicus) evolved over centuries under low levels of selection followed in traditional animal husbandry. As a result, Indian cattle adapted to harsh native environment, resistance to tropical diseases and external parasites and sustenance on low quality roughages and grasses. A large and divergent range of agro-ecological zones in India have helped to develop number of cattle populations. The state of world’s animal genetic resources, SoW-AnGR listed a total of 60 local, eight regional trans-boundary and seven international trans-boundary cattle breeds from India [India is home to the largest cattle population (13.1 % of world’s cattle population) in the world which constitutes 37.3 % of its total livestock [om India . Among tGenetic characterization of breeds allows evaluation of genetic variability, a fundamental element in working out breeding strategies and genetic conservation plans. Molecular markers have revolutionized our ability to characterize genetic variation and rationalize genetic selection . MarkersConsidering the importance of cattle in Indian agriculture, few efforts have been made to evaluate the genetic diversity and relationship in Indian cattle using microsatellite markers –12. HoweThe objectives of this study were to use microsatellite markers and mitochondrial DNA control region polymorphisms to characterize the within-breed genetic diversity, to establish breed relationships and to assess their population structure. The use of molecular information supplied by nuclear and mtDNA markers is aimed to provide a rational basis for suitable strategies of management and conservation.No animal experiments were performed in this study, and, therefore, approval from the ethics committee was not required. Blood samples were collected with the help of veterinary doctors from respective State Animal Husbandry Department. In total, 508 animals from 11 different cattle breeds  were sampled from Northern, Central and Southern India  and 1 min at 72 °C and a final extension of 5 min at 72 °C. Amplified fragments were separated by capillary electrophoresis using an ABI PRISM 3100 automatic sequencer  and allele sizing was accomplished by using the internal size standard GeneScan™-500LIZ™. Fluorescently labeled fragments were detected and sized using GeneMapper software . Stutter related scoring error, often seen in dinucleotide repeats, was absent and alleles could be scored unambiguously.DNA samples were amplified by PCR in correspondence with the selected panel of 21 bovine specific loci. The loci were chosen, according to ISAG/FAO recommendation aiming to analyze high polymorphic markers spread all over the genome and with the ability to co-amplify in PCR reactions . The fluF-statistics (FIS (f), FST (θ) and FIT (F) and Nei’s [GENALEX 6.2 software  was usednd Nei’s  standardnd Nei’s . Bootstrnd Nei’s . The sofnd Nei’s  was usednd Nei’s . Simulatnd Nei’s , delta Knd Nei’s . Principnd Nei’s  and Prin2, 50pmol primer, 0.5 U Taq polymerase  and Taq buffer. Negative controls (lacking template DNA) were included in all reactions, and produced no products. The PCR reaction cycle was accomplished by denaturation for 6 min at 94 °C, 30 cycles of 94 °C for 45 s, 60 °C for 30 s, 72 °C for 60 s, and finally extension at 72 °C for 6 min, before cooling to 4 °C for 10 min. The size of amplification product was checked by loading 5 μL PCR product ontoa 1.8 % agarose gel containing 0.5 μL/mL ethidium bromide. The product was purified usinga QIA quick PCR purification kit . Purified product was labeledusing the BigDye Terminator 3.1 Cycle sequencing kit  and sequenced directly using an ABI3100 Prism automatic DNA sequencer followingmanufacturer instructions. The primers used for sequencing were the same as those used in the PCR. Both strands of PCR product were completely sequenced. All finalsequences were determined from both strands for verification.The non-coding D-loop region was amplified by PCR, using primer pair , as described by Suzuki et al. . The D-ld) of breeds were calculated by Arlequin 3.1 [http://www.fluxus-engineering.com/sharenet.htm), resolving the reticulations through a maximum parsimony criterion [The DNA sequences were edited manually using EDITSEQ (DNASTAR) and the MegAlign program (DNASTAR) was used for multiple alignments. Sites representing a gap in any of the aligned sequences were excluded from the analysis. We compared 60 D-loop haplotypes of a 230-bp hypervariable region-I (HVR-I) fragment of mtDNA control region obtained from 170 cattle from India. Mean number of pairwise differences and nucleotide diversity (π) within cattle breeds, nucleotide divergence between breeds and haplotype diversity (Hquin 3.1 . The Neiquin 3.1 . Networkriterion .Genetic status and diversity of indigenous cattle populations of India was established using nuclear (microsatellite markers) and mitochondrial polymorphisms. All microsatellite markers used in this study were successfully amplified in five multiplex sets designed with consideration for annealing temperature, product size and specific dye label in all the populations Table . The genA total of 359 alleles were detected with ILSTS34 presenting the highest number of alleles per locus (37) while CSSM08 was least  polymorphic. The average observed number of alleles per locus ranged from 6.571 ± 0.732 in Hariana to 10.619 ± 0.824 in Shahabadi cattle with the mean allele number across all the loci of 8.784 ± 0.25 (Table IS evidenced heterozygote deficiency which was highest in Ongole (22.1 %) and lowest in Ponwar (1.4 %).Estimates of observed heterozygosity including all loci and populations (0.653 ± 0.01) confirmed the remarkable level of diversity in the Indian cattle. Among populations, observed heterozygosity ranged from 0.459 ± 0.07 to 0.724 ± 0.036 with the lowest value found in Ongole cattle and the highest in Kenkatha cattle (Table d) was high in all the populations, ranging from 0.80526 (Hariana) to 0.96429 (Ponwar).A fragment of 230 bp hypervariable region-I (HVR-I) of the non-coding mtDNA control region was unambiguously explored resulting in identification of 223 variable sites. Consequently, 60 haplotypes were identified with haplotypic diversity of 0.90240 (Table IT) amounted to 17.5 % (P <0.001). An overall significant deficit of heterozygotes (FIS) of 4.9 % occurred in the analyzed loci because of inbreeding within populations. The multi-locus FST values of breed differentiation indicated that 13.3 % of the total genetic variation was due to unique allelic differences between the breeds, with the remaining 86.7 % corresponding to differences among individuals within the breed across the 21 markers. All loci contributed to the differentiation with the highest values found for ETH225 (32.4 %). The pair-wise FST values of breeds . However, a minor peak at 22 and 24 mismatches was also observed  and nucleotide diversity (π) is also congruent with previous results of Indian cattle [Estimates of observed heterozygosity including all loci and population (0.653 ± 0.014) confirm the remarkable level of diversity in the studied populations. Higher genetic variation in Indian cattle must have contributed to its adaptability as genetic variation is necessary to allow organisms to adapt to ever changing environments with some of this variation stemming from introduction of new alleles by the random and natural process of mutation. Overall heterozygosity estimates were comparable with Tharparkar cattle (0.64) , Orissa n cattle , 39. Hign cattle  using geIS) was observed for eight of the 12 breeds investigated being highest in Ongole (0.221). On the contrary, Kenkatha, Kherigarh and Gangatiri presented slight heterozygote excess in the population  which was expressed in heterozygosity pattern too (Table IS estimate for remaining populations indicates either the presence of inbreeding and /or Wahlund effect (presence of population substructure within breed). Since blood samples were collected from different villages, presence of a hidden substructure cannot be ruled out. Paucity of pure bulls as well as management seems to be the main reasons for heterozygote deficiency in these cattle. Moreover exotic/crossbred semen (Jersey and Holstein Friesian) is available in the breeding tracts whereas, local bull semen is usually unavailable to the owners. Together these two factors are resulting in the reduction of true to the breed type animals. In case of draft breeds, most of the males are used for carrying loads and agricultural operations. These males are castrated around the age of one year leading to their genetic death. With the modernization of agriculture and sub-division of land holdings, bullock power in Indian agriculture is losing its importance. Thus, with the diminishing demand for bullock power, the farmers are not adequately motivated to conserve these draft breeds.Significant heterozygote deficit  and Central and Northern Indian cattle have distinctive genotypes, both in nuclear . These results reflect that within-breed genetic variation is more (86.7 %) than between-breed (13.3 %) and this variation could be a valuable tool for genetic improvement and conservation of cattle populations of India. Genetic differentiation of similar magnitude has been reported in some other indigenous cattle [ST value has been reported among cattle breeds of Orissa and hill cattle of Kumaun (0.044) from India [Bos taurus) viz. North European breeds FST = 0.107 [ST =0.112 [ST = 0.090 [The studied populations showed a moderate and significant genetic differentiation  [Bos indicus) [Bos primigenius) have been clearly identified from Banahalli, Karnataka (India) [These different lines of evidence suggest that some degree of genomic divergence has occurred between Ongole and other cattle breeds of India. The genomes of modern cattle basically reflect the history of animal movements by migratory farmers out of the ancient centers of the cattle domestication. At the time of Neolithic transition, zebu cattle were considered to be the most abundant and important domestic livestock species in Southern Asia. Indus Valley is the major centre of domestication for Indian cattle (indicus) , 44. Howindicus) , 45. In indicus) . Moreoveindicus) . Further (India) , 47.The inferences obtained from nuclear (STRs) and mitochondrial (D-loop) markers are consistent and in agreement with geographical distribution and historical backgrounds. Both proved the clear genetic differentiation between southern and other Indian cattle breeds. However, clustering solutions of mitochondrial and nuclear DNA showed extensive sharing of diversity and absence of genetic substructure between the breeds and populations of a single geographic area. Further studies involving genome-wide approaches are apparently needed for further elucidation of differentiation.This study involves detailed analysis of the genetic diversity and differentiation of Indian cattle from different regions. It is vital to report that indigenous cattle populations of India retain high levels of genetic diversity based on the results from analysis of two genetic markers (microsatellites and mtDNA control region). Inbreeding was detected in some breeds, suggesting the need for appropriate measures to be taken to avoid the negative effects. The results presented here can be used to assist all stakeholders as breeds with wide range of genetic diversity are required in the future for generating transgressive variation for quantitative loci mapping and developing new genotypes for particular management systems and market needs.


Poecilimon veluchianus, a bushcricket endemic to central Greece, show a parapatric distribution and are partially reproductively isolated. Therefore, P. veluchianus is suitable to investigate an ongoing speciation process. We based our analysis on sequences of the internal transcribed spacer (ITS) and the mitochondrial control region (CR). The population genetic analysis based on the nuclear marker ITS revealed a barrier to gene flow within the range of Poecilimon veluchianus, which corresponds well to the described subspecies. In contrast to the results based on the nuclear ITS marker, the mitochondrial CR marker does not clearly support the separation into two subspecies with restricted gene flow and a clear contact zone. Furthermore, we could identify isolation by distance (IBD) as one important mechanism responsible for the observed genetic structure (based on the ITS marker). The population genetic analysis based on the nuclear marker ITS also suggests the existence of hybrids in the wild. Furthermore, the simultaneous lack of strong prezygotic barriers and the presence of postzygotic mating barriers, observed in previous laboratory experiments, suggest that a secondary contact after an allopatric phase is more likely than parapatric speciation.Different mechanisms such as selection or genetic drift permitted e.g. by geographical isolation can lead to differentiation of populations and could cause subsequent speciation. The two subspecies of  Speciation is still a hot topic in evolutionary biology, and different mechanisms and geographic modes of speciation have been proposed to cause this event –4. SelecPoecilimon veluchianus, a flightless bushcricket, which is endemic to central Greece, as a promising system to investigate forces that could drive speciation. The two subspecies, P. v. veluchianus and P. v. minor [We chose v. minor , are desv. minor ,10. Sizev. minor , whereasv. minor ,11.P. veluchianus rarely occurs. As speciation might occur over large time scales, it is important to consider past fragmentations that might have served as potential barriers to gene flow. During ice ages in the Quaternary, some 2.4 million years ago, central Greece was not fragmented [As elevation might influence the distribution of the two subspecies , the Itiagmented  and mighHowever, previous laboratory experiments have shown that, despite the females do not distinguish between songs of the two subspecies ,the matiP. veluchianus using sequences of the mitochondrial control region (CR marker) and the internal transcribed spacers (ITS marker 1 and 2). Due to the lack of premating barriers [To test for possible genetic differences between the proposed subspecies ,10,13, wbarriers , hybridibarriers . To testOur investigations revealed one main barrier rather similar to the contact zone formerly hypothesized due to phenotypic differences. IBD has great influence on the genetic structure observed, but secondary contact seems to be rather likely than ongoing speciation in parapatry.P. veluchianus is a flightless bushcricket with two parapatrically distributed subspecies that are endemic to central Greece. In three different years, 2010–2012, we collected in total 283 individuals at 28 sites. P. v. veluchianus was collected at 16 sites and P. v. minor at 12 sites  and a modified TI-N18 primer (CTCTATCAARRTAAYCCTTT) [TAGAGGAAGTAAAAGTCG and GCTTAAATTCAGCGG) described in Weekers et al.(2001) [The mitochondrial CR marker was amplified using primer SR-J14610 (l.2001) . These m01 . Thesl.(2001) .2O added to a final volume of 30 μl. According to Zhao et al. (2011) [To amplify the mitochondrial CR marker the following PCR protocol was used: 1.2 μl genomic DNA, 0.1 μl innuTaq Hot-A DNA Polymerase , 2.5 μl Hot Start Buffer complete, 1.2 μl of a 10 μM solution of each primer, 0.6 μl of a 2 mM dNTP-Mix and ddH. (2011) , the PCR2O added to a final volume of 20 μl. According to Weekers et al. (2001) [To amplify fragments of the ITS marker the following PCR protocol was used: 0.8 μl genomic DNA, 0.07 μl innuTaq Hot-A DNA Polymerase , 1.7 μl Hot Start Buffer complete, 0.8 μl of a 10 μM solution of each primer, 0.4 μl of a 2 mM dNTP-Mix and ddH. (2001)  the PCR After purifying, following the ExoSAP-IT PCR clean-up protocol (USB Corporation), PCR products were directly cycle-sequenced using the amplification primers and then analyzed on an ABI 3730 capillary sequencer (Applied Biosystems) at the sequencing facility of the CeBiTec (Bioinformatics Resource Facility) of Bielefeld University.P. v. veluchianus = 164 sequences, P. v. minor = 119 sequences). The assembly of forward and reverse sequences was performed in MEGA 5.2 [In the resulting analysis datasets for both markers (ITS and CR) contained sequences of the same 283 individuals .The genetic structure and diversity of the datasets were investigated with the program DNAsp5 . At firsTo find out whether the subspecies are supported by the Fst-values we used the software Barrier v. 2.2  and its To determine the percentage of genetic variation explained by the affiliation into subspecies a distance based redundancy analysis (dbRDA) ,30 was pTo further investigate the genetic structure of the studied species and subspecies we performed an AMOVA. The AMOVA was calculated by using the program Arlequin 3.5.1.2 based on the haplotype information, provided by DNAsp5 and the affiliation of the sample sites to the both subspecies and additionally the new affiliation into the subspecies as calculated by Barrier for the ITS marker. Significance test was performed with 1023 permutations.We tested for IBD by performing a dbRDA on three different arrangements  of the p-distance-matrix of the individuals and the associated geographical coordinates as variables. We also tested both sexes separately on the whole dataset without any partitioning to test for sex biased dispersal. Again, significances of the dbRDA were established with 9999 permutations in DISTLM v. 5.To test whether the analyzed sequences show departure from neutral evolution, we calculated Tajimas D and Fu’s and Li’s F* and D* as implemented in DNAsp5. We performed both tests as Fu’s and Li’s F* and D* are supposed to be more sensitive to population expansions and genetic hitchhiking than Tajimas D . Both teThe aligned sequences of the CR dataset contained 794 bp and the ITS dataset consisted of 706 bp. For the CR dataset the model test resulted in GTR+G+I  with the lowest AIC value. This model was used for calculation of the p-distances in MEGA 5.2. As this model is not provided in Arlequin 3.5.1.2, for this program we chose the model with the next best AIC value of the selectable models, Tamura-Nei (G = 0.184).The model with the lowest AIC value for the ITS dataset was T92+G+I  in the model test. This model was used for calculation of the p-distances in MEGA 5.2. For Arlequin 3.5.1.2 the model Kimura2P+G  had the lowest AIC value and was therefore used in the Arelquin analyses.The mitochondrial CR marker as well as the nuclear ITS marker showed high genetic variation . For botP. v. minor from all remaining sampling sites (data not shown). This reconstructed barrier is not corresponding to an obvious geographic barrier and is not adding further information on the question about the subspecies or the genetic pattern concerning the two subspecies. Moreover, the mitochondrial CR marker did not support two different subspecies with restricted gene flow.To ascertain whether the assignment of the individuals to subspecies is supported by the Fst-values for both markers, the first four barriers to gene flow were calculated using the software Barrier v. 2.2. The first calculated barrier for the ITS marker with their bootstrapping values for every decision is shown in P. v. veluchianus, was rather unexpected. The calculated barrier indicates that these P. v. veluchianus populations are more similar to the adjacent P. v. minor populations and hence, did not correspond to the proposed contact zone. Many of the decisions made by Barrier are supported by bootstrap values higher than 70%. In the north-east the contact zone between sample site 14 and 21 is only supported by a bootstrap value of 35. In the south two possible ways for the continuing barrier to gene flow were calculated. The barrier between sample site 04 and 09 is supported by a bootstrap value of 54. The other possible way of the contact zone, including sample site 04 to P. v. minor, is supported by bootstrap values lower than 25. Low bootstrap values may suggest unsampled locations [The barriers to gene flow determined are not exactly in accordance to the hypothesized contact zone, but barrier 1 calculated for the ITS marker supports it almost as proposed by Eweleit & Reinhold (2014) . Just thocations , the preocations . BetweenBased on the bootstrap support values we assume the most possible contact zone as shown in For further analysis we will focus on the ITS marker, to focus on the contact zone between the two subspecies calculated by barrier. The CR marker is not diagnostic in this respect, as it shows too much variability to resolve on the subspecies level. The Genetic structure and diversity for the ITS marker, according to the new subspecies is shown in 2 = 0.092). The partitioning of individuals into the corresponding subspecies with respect to the newly calculated contact zone (barrier 1) for the ITS marker was also significant and is adding some more explanation of the genetic variation .The result of the dbRDA supports the calculated barrier . The affThe amount of explained variation of the genetic pattern in the ITS marker, analyzed by an AMOVA, is different for the diverse genetic levels  by using the geographical coordinates as variable. Within the two subspecies the influence of IBD is almost the same as for the overall dataset .The test for IBD as a force for separation revealed 15.62% of the genetic variation was explained , indicating that sex has no strong influence on the observed genetic structure. Furthermore, this contradicts the expectation that females, as the songless sex, should be more actively walking around and hence could have a higher impact on the population structure.To test for the influence of sex on the genetic pattern we also performed the dbRDA based on the p-distances and the sex of each individual . The resP. v. minor  and also for the new arrangement of sample sites into the subspecies P. v. minor along barrier 1 . Hence, the individuals in these areas seem to be under some sort of selection and the DNA- sequences do not evolve randomly. Population growth or population subdivision may also be a possible explanation as these genetic signatures are hardly to distinguish from selection effects [To investigate whether the sequences show signs of non-random effects or evolve neutrally, we calculated Tajima’s D and the Fu’s and Li’s F* and D* . For thePoecilimon veluchianus. This contact zone is diagonally extending from the north-east of central Greece to the south-west  as calculated by the AMOVA  Files. NWe could identify IBD as a mechanism generating structure within the species as the geographical coordinates significantly explained 15.6% of the genetic variability (based on the ITS dataset). When analyzed within subspecies another big part of the genetic variation (14.9% for Pvv and 12.8% for Pvm) was explained by the coordinates , which mP. veluchianus to be reduced. Reinhold (1994) [P. v. veluchianus mother. Therefore, we expect different amounts of shared haplotypes between the subspecies for both subspecies. Instead both subspecies feature a similar amount of haplotypes shared between subspecies . When locations close to the new contact zone were considered, only two haplotypes are shared across the contact zone.Based on laboratory experiments we expect gene flow between the two subspecies of d (1994)  found a P. veluchianus, males are the sound producing sex and waiting for females to approach them for mating [As for r mating , we woulP. veluchianus. As both values were significant for the species, we concluded the ITS sequences evolved non-randomly . Responiewed in  and thusP. v. veluchianus mother grow bigger than pure P. v. minor individuals and hybrids with a P. v. minor mother stay smaller than pure P. v. veluchianus individuals [The frequency of haplotypes which are shared between the two subspecies along the new contact zone is quite high and most prominent at a transect  [P. veluchianus is part of a ring species. To further investigate this hypothesis, samples of closely related species of Poecilimon are necessary. Our findings are also able to be interpreted with non-ecological speciation were no geographical barrier is needed, but sexual selection plays a major role. This way of speciation is consistent with the neutral biodiversity theory (NBT), based on Hubbel (2001) [P. veluchianus  ,43–45, ad (1993) ,46, the d (1993)  is stilld (1993) ,49. Therl (2001)  (reviewel (2001) ). It is uchianus , also th hybrids  and mighP. veluchianus and the more detailed characterization of the suggested hybrid zone of the two subspecies.An extension of the former mate-choice tests and the cross-mating-experiments ,13 accorPoecilimon veluchianus, and thus supports a possible hybrid or contact zone between the two subspecies. The existence of hybrids cannot be finally clarified but might be indicated by the high frequencies of shared haplotypes between both subspecies which are quite prominent along the contact zone. In contrast the mitochondrial CR marker does not clearly support the separation of P. veluchianus into two subspecies with restricted gene flow and a clear contact zone. The factors responsible for the observed discrepancy between both markers, potentially be due to saturation of the mitochondrial marker cannot be finally answered. In general IBD has a great influence on the observed genetic pattern with a more prominent influence in females than in males indicating a sex biased dispersal capacity. Both subspecies show signs of non-random evolution, which either could indicate natural selection or that the species experienced a recent bottleneck and is now in a phase of range expansion. Testing both subspecies separately for departure of neutral evolution, the results are only significant for P. v. minor. This might indicate that P. v. minor is either more affected or that only this subspecies experienced the bottleneck. The frequency of haplotypes which are shared between the two subspecies along the contact zone is quite high but surprisingly a difference in shared haplotypes between both subspecies, as expected due to the quasi sterility of F1-hybrid-males with a P. v. veluchianus mother, was not observed. Together with our findings, the previously published lack of strong prezygotic barriers combined with the presence of postzygotic mating barriers suggest that a secondary contact after an allopatric phase is rather likely for the two subspecies of P. veluchianus.The population genetic analysis, based on the nuclear ITS marker revealed one main barrier to gene flow within the range of S1 FigBetween population p-distances are coloured in green, in the upper left side of the matrix. Within population p-distances coloured in orange, diagonal line of matrix. The net number of nucleotide differences between pairs of populations are coloured in blue, in the lower right side of the matrix.(TIF)Click here for additional data file.S2 FigBetween population p-distances are coloured in green, in the upper left side of the matrix. Within population p-distances coloured in orange, diagonal line of matrix. The net number of nucleotide differences between pairs of populations are coloured in blue, in the lower right side of the matrix.(TIF)Click here for additional data file.S1 File(FAS)Click here for additional data file.S2 File(FAS)Click here for additional data file.S3 File(XLS)Click here for additional data file.S4 File(XLS)Click here for additional data file.S5 File(XLS)Click here for additional data file.S6 File(XLS)Click here for additional data file.S7 File(XLS)Click here for additional data file.


RCN1, therefore, acted as a link between Glc and the BR-signalling pathway. Polar auxin transport worked further downstream to BR in controlling Glc-induced root deviation response. Glc also affected other root directional responses such as root waving and coiling leading to altered root architecture. High light intensity mimicked the Glc-induced changes in root architecture that were highly reduced in Glc-signalling mutants. Thus, under natural environmental conditions, changing light flux in the environment may lead to enhanced Glc production/response and is a way to manipulate root architecture for optimized development via integrating several extrinsic and intrinsic signalling cues.Directional growth of roots is a complex process that is modulated by various environmental signals. This work shows that presence of glucose (Glc) in the medium also extensively modulated seedling root growth direction. Glc modulation of root growth direction was dramatically enhanced by simultaneous brassinosteroid (BR) application. Glc enhanced BR receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) endocytosis from plasma membrane to early endosomes. Glc-induced root deviation was highly enhanced in a PP2A-defective mutant,  The ability for plant organs to guide their growth at a specified angle from the gravity vector (gravitropism) ensures that the shoot is positioned to maximize its light-harvesting capabilities and that the roots are positioned downward so as to maximize the uptake of water and nutrients. The ability to readjust their growth direction by sensing any deflection relative to the direction of gravity involves gravity perception and signal transduction, followed by differential growth .Arabidopsis roots  is important modulator of plant growth; BRs promote organ growth by affecting cell elongation and cell division. The effects of BR on root gravitropic response have been studied. Exogenous BL application has been shown to increase the gravitropic curvature in primary roots . Following lines were obtained from the original published source as: pBRI1::BRI1::GFP . For experiments testing the effects of different sugars/hormones/inhibitors on root directional growth, 5-d-old light-grown seedlings were transferred to the treatment medium and kept vertically under culture room conditions. For experiments exploring the root directional growth on increasing light intensity, light intensities ≈2000 Lux (≈30 μmol m–2 s–1) and ≈7000 Lux (≈100 μmol m–2 s–1) were used. Root coiling and waving responses were studied as described previously (–2 M stock solutions and FM4-64 (Invitrogen) was prepared as a 2mM stock solution in dimethyl sulphoxide.Seeds were surface sterilized and imbibed at 4 °C for 48h. The imbibed seeds were germinated and grown vertically on Petri dishes containing 1/2 MS supplemented with 1% sucrose (Suc) and solidified with 0.8% agar. Seed germination was carried out in climate-controlled growth rooms under a 16/8 light/dark cycle (22±2 °C and light intensity 60 μmol mhttp://rsb.info.nih.gov/ij/). Root elongation was measured using ImageJ. Root coiling and waving response was studied as described previously . For imaging GFP, the 488nm line of the Argon laser was used for excitation and emission was detected at 520nm. For imaging FM4-64, 543nm line of the helium/neon laser was used for excitation and emission was detected at 590–620nm. All fluorescence intensity measurements were performed using LCS Lite software version 2.61 (Leica Microsystems). GFP/FM4-64 experiments were performed by using sequential scanning. To observe the actin filament organization in root epidermal cells, 5-d-old light-grown, 35S::GFP-ABD2-GFP-expressing seedlings were transferred on treatment medium for 24h. The laser, pinhole, and gain settings of the confocal microscope were kept identical for different treatments. Images were assembled using Photoshop (Adobe Systems). At least three biological replicates, with each replicate having 10 seedlings, were performed for all the experiments.Five-d-old light-grown Col-0 (WT) seedlings were transferred to Glc-free or 3% (w/v) Glc-containing 1/2 MS medium solidified with 0.8% agar in a climate-controlled growth room for 3 d under a 16/8 light/dark cycle. The root epidermal cell profile visualization and amyloplast staining was performed as previously described for etiolated hypocotyls in Arabidopsis thaliana Col-0 seeds, 5 d after germination on 1/2 MS medium supplemented with 0.8% agar and 1% Suc, were transferred to Glc-free and 3% Glc-containing medium for 48h. Root tissue from each sample was harvested in liquid N2 and processed for RNA isolation. Total RNA was extracted and QC was performed as described (Supplementary Table S1 (available at JXB online).For quantitative real-time PCR analysis, P value was 0.001 except where stated otherwise. All endpoint analyses were taken 72h after transfer to treatment medium otherwise specified, although plates were observed for up to 10 d.All experiments reported in this work were performed at least three times yielding similar results. Each experiment was considered as an independent biological replicate. All data measures are averages from two independent biological replicates each with at least 20 seedlings otherwise specified. Error bars represent SE. Statistical significance for all the experiments was evaluated using Excel (Microsoft). For all experiments, statistical differences between both control/treatment and WT/mutant pairs were analysed using Student’s t-test evaluation with paired two-tailed distribution. A cut off for Supplementary Fig. S1A). Study of root elongation during the course of deviation suggests that changes in root length do not correspond to extent of root deviation. The roots of 5% Glc-grown seedlings were the smallest, yet they showed the highest degree of deviation as compared to seedling roots growing on 0, 1, and 3% Glc (WT Col-0) seeds were germinated and grown vertically on increasing concentrations of Glc-containing 1/2 MS medium solidified with 0.8% agar in the light. The primary root grew vertically down in Glc-free medium, whereas on increasing concentrations of Glc, the primary root showed deviation from straight/vertical growth in a dose-dependent manner . Root deol-0 seedd 3% Glc .Supplementary Fig. S1B). The maximum gravitropic curvature angle recorded for 5% Glc-treated seedling roots was ~50° at 72h after reorientation in contrast to the ~20° angle recorded for seedlings placed in Glc-free medium . The root elongation kinetics during the course of gravistimulation suggests that rate of root elongation is independent of extent of root gravitropism in media containing different concentrations of Glc .Root gravitropism kinetics of gravistimulated seedlings in presence of Glc was also affected. Five-d-old WT (Col-0) seedlings grown on 1/2 MS media were transferred to different concentrations of Glc-containing media and subjected to gravity reorientation assay by giving a 90° gravistimulation. The root gravitropic response was quantified as a measurement of angle of curvature at various time points after the seedlings were reoriented. Higher Glc concentration  caused significant delay in gravitropic bending of roots at almost all time points tested (O-Methyl-d-glucopyranose (3-OMG), which is transported to cell but not sensed as a sugar, has been used as Glc analogue to study sugar signalling .In order to find if root deviation is Glc specific or other sugars could also induce similar response, the effect of various sugar analogues was observed. Five-d-old WT (Col-0) seedlings grown on 1/2 MS media were transferred to different sugars and root deviation from vertical was checked after 72h. The seedlings could show pronounced root deviation from vertical on Suc- and Glc-containing media whereas other slowly metabolizable sugars displayed the response at a much lesser extent . The mai2-KI solution. There was no disruption of amyloplasts in columella cells in fact, an increase in amyloplasts was observed at higher concentration of Glc  suggesting that Glc-mediated gravitropic defect may not involve amyloplast degradation as a way to induce root deviation.Amyloplasts play a significant role in gravity sensing and are essential for full gravitropic response (Arabidopsis includes AtHXK1 (HEXOKINASE1)-dependent and -independent components. In the AtHXK1-dependent pathway, HXK1 acts as a Glc sensor . Root growth measurement showed no correlation between the effects of Glc on root deviation angle and root elongation in HXK1-independent Glc-signalling mutants . These results suggest that Glc involves both HXK-dependent and -independent signal transduction pathways to modulate root directional growth.The Glc-signalling pathways in gin2-1 and rgs1-1. The gin2 mutant exhibited significant decrease in gravitropic-bending kinetics  while rgs1-1 showed slightly increased gravitropic-bending kinetics as WT . These results further substantiated the involvement of both HXK-dependent and -independent signal transduction in Glc modulation of root directional responses.Gravitropic bending kinetics was also assayed in gravitationally stimulated seedlings of the Glc-signalling mutants Supplementary Fig. S4B). All these results in combination suggest that Glc may involve downstream BR signalling in controlling Glc-induced root growth deviation.Plant hormones such as auxin, cytokinin, jasmonic acid, ABA, and BR have been shown to play important roles in affecting root directionality . Presencbrassinosteroid insensitive1-6 (bri1-6) mutant, which lacks the reception ability towards BR, showed no root growth deviation in presence of Glc . Roots of the BR-hyperresponsive mutant brassinazole resistant1-1D (bzr1-1D) displayed an exaggerated Glc-mediated root deviation response as compared to WT , further confirming that BR signalling positively mediates Glc-induced root deviation. Root growth measurement showed no correlation between root deviation and root elongation .To find out the involvement of different BR-signalling components in Glc-induced root deviation response, the deviation of BR-signalling mutants was checked in presence of Glc. Compared to WT, the DWF4, CPD, ROT3, and BR6OX2 were studied in presence of Glc . Glc not only increased the overall BRI1 abundance in cell, but also significantly increased the ratio between intracellular versus plasma-membrane-localized BRI1 signal . The protein phosphatase inhibitors okadaic acid and cantharidin caused a pronounced increase in Glc-induced root deviation as compared to control . Okadaic acid and cantharidin treatment could also enhance BRI1 endocytosis similar to Glc supplementation . Previous studies have indicated the involvement of variety of protein phosphatase 1 (PP1) and 2A (PP2A) in plant sugar signalling (ROOTS CURL IN NAPHTHYL PHTHALAMIC ACID1 (RCN1), which encodes one of the three members of regulatory subunit A and functions as a positive regulator of PP2A holoenzyme . Root growth measurement showed no correlation between root deviation and root elongation . Since BRI1 protein turnover in Arabidopsis has been shown to be regulated by activity of PP2A .To regulate signalling output, receptor internalization and its subsequent targeted fate such as recycling or degradation is very crucial. We also tried to test whether other inhibitors related to endocytosis/degradation can effect Glc-induced root deviation. Physiological treatments with the proteolysis inhibitor MG132 , the protein-trafficking inhibitor BFA, and the protein biosynthesis inhibitor CHX, were used in presence of Glc to find the extent of the response. While CHX treatment inhibited overall growth of the seedlings, MG132 and BFA did not induce much difference as compared to WT (ethylene insensitive root 1 (eir1) and auxin resistant 1 (aux1-7) have been found to be involved in BR-mediated root gravitropism  mutants showed exaggerated Glc-induced root growth deviation as compared to WT, suggesting that Glc may interact/modulate auxin transport machinery to cause Glc-induced root direction response  could also substantially enhance the Glc-induced root direction reset , further supporting that Glc may modulate auxin transport machinery to cause Glc-induced root deviation. Auxin transport changes may work downstream even to BR response, since NPA was able to induce root deviation even in bri1-6 , which could not show Glc-induced root growth deviation. Root growth measurement showed no correlation between root deviation and root elongation . All these observations suggest that differential auxin transport may lie downstream to Glc and BR in controlling root growth deviation.Our group has previously reported that Glc-induced root growth deviation involves various auxin-signalling components . Exogenous Glc treatment also caused changes in the cell profile of the root epidermis, as seedlings grown in Glc-free 1/2 MS medium displayed a straight arrangement of epidermal cells across the root, while twisting of epidermal cells in a spiral manner was observed in Glc-treated roots . Changes in actin filament organization in Arabidopsis roots upon Glc treatment was also studied using 35S::GFP-ABD2-GFP lines and it was found that Glc treatment caused alteration of actin filaments organization .Cytoskeleton has been widely studied with respect to gravitropism, and reorganization of cytoskeleton has been shown to positively regulate gravitropism during the signal transduction stage and gravitropic bending. Glc has been reported to interfere with actin filament organization . BRI1 inSupplementary Fig. S7A); the wave frequency was reduced with seedling roots forming larger waves . Altogether, Glc-induced changes in root directional responses led to an increased area spanned by the seedling root, hence enabling better anchorage to the medium/soil and support for the enhanced corresponding shoot and root biomass . To question whether a light signal is actually transduced by Glc, the effect of light on Glc-signalling mutants was studied in terms of root deviation. While gin2 could not show light-induced root deviation from vertical, rgs1-1, thf1-1, and gpa1-3 showed a significantly reduced sensitivity towards light-induced root deviation as compared to WT , suggesting that Glc can mimic effect of light in controlling root architecture. Together, these results suggest that light may use Glc-signalling components to modulate root architecture under natural environmental conditions.The availability of Glc under natural environment conditions depends on the presence of light, and varying light conditions may modulate root architecture by using Glc as an intermediate. Light levels have been used in past to modulate endogenous sugar levels, which correlate positively with primary root growth and the density of lateral roots in bidopsis . To tested to WT . Higher Arabidopsis (pgm (phosphoglucomutase) and eal1 , have also been reported to be less gravitropic (Arabidopsis starch excess 1 (sex1) mutant, having greater amount of starch relative to the wild type, have also been shown to possess almost similar gravitropic sensitivity . These results in combination suggest that Glc, either directly or via modulating BR and auxin response, can alter the cytoskeleton and cellular patterning the most downstream steps responsible for inducing Glc-induced root deviation.Cytoskeleton reorganization has been widely studied with respect to control of gravitropic responses . Glc enhanced BR signalling to induce root deviation response by increasing BR receptor BRI1 internalization . Increased BRI1 endocytosis upon Glc treatment may be a results of reduced PP2A activity, as phosphatase inhibitors okadaic acid as well as cantharidin also caused increased BRI1 endocytosis and root deviation similar to Glc treatment (rcn1-1 also showed exaggerated Glc-induced root deviation that could be restored by application of BRZ (bri1-6, which was otherwise insensitive to Glc-induced root deviation (eir1-1, aux1-7, and mdr1-1 displayed highly exaggerated Glc-induced root deviation response (A testable model based upon the aforementioned findings and previously published reports is presented in reatment , B. rcn1n of BRZ . RCN1, teviation . Also, tresponse . IncreasRoot growth direction can be correlated with architectural stability to provide increased anchorage and water and nutrient absorption. Increasing Glc concentration not only induced root deviation from vertical but also altered root waving and coiling, eventually leading to a root architecture that was able to better support the aboveground parts. Glc treatment also increased the area spanned by seedling root for better exploration of the soil for water and nutrient. All these features in combination would lead to a root architecture leading to better fitness in a natural environment condition. Root architecture has been reported to be involved in regulating seedling fitness and survival (Under natural environmental conditions, Glc as such is not available to the plant root. Plants can produce Suc via photosynthesis in the presence of light, which is eventually hydrolysed to Glc and fructose, the only source of Glc to plant root. Thus, availability of Glc under natural environment conditions depends on the presence of light, since high light intensity boosts photosynthesis, sugar production, and Glc signalling in a physiological context in plants . PhotosyJXB online.Supplementary data are available at Supplementary Fig S1. Glc-induced root deviation from vertical is not correlated with root growth inhibition.Supplementary Fig. S2. Role of Glc metabolism and signalling in root growth and direction.Supplementary Fig. S3. Both HXK -dependent and -independent components of Glc signalling are involved in root deviation from vertical response.Supplementary Fig. S4. Involvement of BR-signalling elements in regulating root directional growth.Supplementary Fig. S5. Involvement of protein phosphorylation and de-phosphorylation in regulating root directional response.Supplementary Fig. S6. Changes in polar auxin transport and actin cytoskeleton organization regulate Glc-induced root directional response.Supplementary Fig. S7. High light intensity could mimic Glc effects on root waving and coiling responses.Supplementary Table S1. List of primers used for real-time PCR analysis.


Vulpes vulpes deletrix Bangs 1898) poses interesting questions about genetic distinctiveness and the post-glacial colonization history of the island’s depauperate mammalian fauna. Here, we characterized genetic variability at the major histocompatibility complex (MHC) class II DR β1 domain (DRB1) locus in 28 red foxes from six sampling localities island-wide and compared it with mitochondrial control region (CR) diversity and DRB1 diversity in other canids. Our goals were to describe novel DRB1 alleles in a new canid population and to make inferences about the role of selection in maintaining their diversity. As in numerous studies of vertebrates, we found an order-of-magnitude higher nucleotide diversity at the DRB1 locus compared with the CR and significantly positive nonsynonymous-to-synonymous substitution ratios, indicative of selection in the distant past. Although the evidence is weaker, the Ewens-Watterson test of neutrality and the geographical distribution of variation compared with the CR suggest a role for selection over the evolutionary timescale of populations. We report the first genetic data from the DRB1 locus in the red fox and establish baseline information regarding immunogenetic variation in this island canid population which should inform continued investigations of population demography, adaptive genetic diversity, and wildlife disease in red foxes and related species.As the only native insular Newfoundland canid between the extinction of the wolf in the 1930s and the recent arrival of coyotes, the red fox ( The major histocompatibility complex (MHC) contains among the most polymorphic genes in the vertebrate genome  comprises some 40 subspecies and occupies a broad range of habitats from tundra to urban (Larivière and Pasitschniak-Arts V. vulpes deletrix Bangs 1898), described as pale and straw-colored relative to mainland counterparts, has been considered distinct enough from neighboring mainland populations to warrant subspecies status (Dodds Angiostrongylus vasorum), the causative agent of verminous pneumonia and coagulopathy , thus providing a comparative model for understanding adaptive genetic variation in non-threatened populations, as advocated by Kohn et al. , one of the most monomorphic sexually reproducing animal populations known, revealed remarkably high levels of MHC variation  and the progressively more distantly related South American foxes (genus Dusicyon), red fox-like canids (genus Vulpes), and grey and island foxes (genus Urocyon). Suites of DRB1 alleles have been described in several wild canid species including island foxes  covering the geographic range of insular Newfoundland  according to the manufacturer’s instructions for tissue samples.Taq DNA polymerase (Qiagen Inc.), and 25–200 ng DNA template. The amplification profile for DRB1 consisted of an initial denaturation for 5 min at 95 °C, followed by 14 cycles of 95 °C for 30 s, 62 °C (decreasing by 0.5 °C per cycle) for 30 s, and 72 °C for 1 min, followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, and a final 10-min elongation at 72 °C. PCR products were purified using the QIAquick PCR Purification kit (Qiagen Inc.).Exon 2 of the DRB1 locus (302 bp) was amplified with DRBIN1-F (5′-CCGTCCCCACAGCACATTTC-3′)  following the manufacturer’s instructions. Inserts were directly amplified from five to ten colonies for each amplicon using the M13 primers recommended by Qiagen, with reaction mixtures prepared as above but using a small portion of the bacterial colony as a template, and an amplification profile consisting of initial denaturation for 5 min at 95 °C, followed by 40 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min, and a final extension at 72 °C for 10 min. PCR products were purified as above.All PCR products were sequenced in both directions using BigDye Terminator 2.0 chemistry and electrophoresed on the ABI Prism 3130 DNA Analyzer  using Sequencing Analysis version 5.0 software. Sequence reads were assembled and edited with Sequencher version 4.9 .Initial examination of MHC DRB1 exon 2 sequences identified as many as eight distinct sequences per individual fox. However, molecular cloning produces errors such as recombinant alleles, singleton mutations, and non-target DNA incorporation into the final sequence  and dA  relationships among red fox DRB1 alleles and two sets of alleles representing other canids  accessed from GenBank were determined via neighbor-joining phylogenies of maximum composite likelihood distances and tested by 10,000 bootstrap replicates, using MEGA, as a measure of trans-species polymorphism.The following tests were performed as suggested by Garrigan and Hedrick . To testV. vulpes), from the island of Newfoundland, Canada. Our goals were (1) to make inferences about the role of balancing selection over short, medium, and longer term evolutionary scales in maintaining patterns of allelic diversity at this locus in Newfoundland red foxes and (2) to introduce data regarding diversity at a functional immune system locus as a baseline for future studies in this wildlife population. We present evidence in favor of balancing selection over the medium and especially longer evolutionary timescales in this species and compare patterns of diversity in Newfoundland foxes with those observed for other canids.Herein, we describe and characterize class II MHC DRB1 alleles in red foxes  and each fox was found to contain at most two alleles, consistent with a single locus. Allele and genotype frequencies in each of the six Newfoundland red fox localities are given in Table Among the eight alleles, 38 nucleotide sites were variable Fig. . The vasπ = 0.030–0.061; Table π = 0.0017–0.0062; Langille et al. HE = 0.56–0.82 and very similar to the haplotypic diversity of h = 0.54–0.69 for the CR . Patterns of variability at the DRB1 locus among Newfoundland red foxes locations are uncorrelated to variability at the CR .Nucleotide diversity for the DRB1 locus (P ≫ 0.05), indicating no evidence of selection in the short term. Although the small sample size investigated here may compromise power for this test, the observed and expected heterozygosities are exactly the same, so there is certainly no indication that a departure from Hardy-Weinberg equilibrium is present.Expected and observed measures of heterozygosity for each location and overall are presented in Table P = 0.051) evidence for departure from the expected allele frequency under neutrality for the 56 alleles at the DRB1 locus. Indeed when a simulation is done by doubling the sample size but keeping the allele distribution the same, significance is observed (P = 0.019). In contrast, the CR frequency distribution was quite consistent with neutrality , suggesting that the result in the DRB1 locus is not due to the effects of demography. Moreover, demographic tests of expansion for the CR imply no recent expansion of this population  or pairwise differences . Pairwise estimates of FST or dA among individual localities were significant only for the WEC population compared with the NEC and Central (CEN) localities , and most pairwise values were positive  or dA . Lower FST values for the DRB1 compared with the CR may suggest uniform selection pressure across subpopulations  in a set of V. vulpes and V. lagopus sequences  were found to be under positive selection in the inclusive data , consistent with continuing selective pressure since species divergence, and we observe a new amino acid variant in red foxes at a site under positive selection among Vulpes.The excess of nonsynonymous to synonymous substitutions that exists between alleles is among the most long-standing and powerful lines of evidence in favor of balancing selection at the MHC and is well documented in the peptide-binding regions of the DRB1 in a wide array of vertebrates  in the Channel Islands, with a colonization history timescale of 800–16,000 years ago, and the Arctic fox (V. lagopus) in the Commander Islands, isolated from the mainland since the Pleistocene. Among the Channel Islands, expected heterozygosity ranges from 0 to 0.36 for a set of populations of effective populations sizes of 163–984 and levels of neutral microsatellite genetic variation on the order of 0–0.36. Notably, the San Nicolas Island population  showed both the lowest microsatellite and the highest DRB diversity, attributed by the authors to a severe bottleneck accompanied by strong selection . Notably, the dN measures are virtually identical among taxa, and the differences in ratios are explained by dS differences. This suggests that over the long term, there may be similar levels of selective pressure in each of these taxa. Second, while species or genera tend to group together loosely phylogenetically, the inclusion of all genera illuminates the tendency of alleles to be shared past species and even genus barriers, although this is not well supported by bootstrap analysis, probably due to the length of the sequence analyzed. We also note that unlike in Canis, deeper divergences within the Vulpes only phylogeny are not well supported by bootstrap values. This may be due to longer divergences times among species in the two genera.Investigation of DRB1 alleles from The signature of positive and balancing selection in red foxes in Newfoundland is consistent with the known functional significance of the genes of the MHC and particularly the DRB1 in numerous studies across vertebrates. Species which exhibit low or no detectable polymorphism in MHC genes might suffer from increased susceptibility to disease, whether enzootic or epizootic Hedrick, . Here, wAs suggested by Castillo et al.  for racc


Mycteroperca rosacea). We tested the hypothesis that source–sink larval metapopulation dynamics describing the direction and frequency of larval dispersal according to an oceanographic model can help to explain empirical genetic data. We described modeled metapopulation dynamics using graph theory and employed empirical sequence data from a subset of 11 sites at two mitochondrial genes to verify the model predictions based on patterns of genetic diversity within sites and genetic structure between sites. We employed a population graph describing a network of genetic relationships among sites and contrasted it against modeled networks. While our results failed to explain genetic diversity within sites, they confirmed that ocean models summarized via graph and adjacency distances over modeled networks can explain seemingly chaotic patterns of genetic structure between sites. Empirical and modeled networks showed significant similarities in the clustering coefficients of each site and adjacency matrices between sites. Most of the connectivity patterns observed towards downstream sites (Sonora coast) were strictly asymmetric, while those between upstream sites (Baja and the Midriffs) were symmetric. The best-supported gene flow model and analyses of modularity of the modeled networks confirmed a pulse of larvae from the Baja Peninsula, across the Midriff Island region and towards the Sonoran coastline that acts like a larval sink, in agreement with the cyclonic gyre (anti-clockwise) present at the peak of spawning (May–June). Our approach provided a mechanistic explanation of the location of fishing zones: most of the largest areas where fishing takes place seem to be sustained simultaneously by high levels of local retention, contribution of larvae from upstream sites and oceanographic patterns that concentrate larval density from all over the region. The general asymmetry in marine connectivity observed highlights that benefits from reserves are biased towards particular directions, that no-take areas need to be located upstream of targeted fishing zones, and that some fishing localities might not directly benefit from avoiding fishing within reserves located adjacent to their communities. We discuss the implications of marine connectivity for the current network of marine protected areas and no-take zones, and identify ways of improving it.Understanding patterns of larval dispersal is key in determining whether no-take marine reserves are self-sustaining, what will be protected inside reserves and where the benefits of reserves will be observed. We followed a multidisciplinary approach that merged detailed descriptions of fishing zones and spawning time at 17 sites distributed in the Midriff Island region of the Gulf of California with a biophysical oceanographic model that simulated larval transport at Pelagic Larval Duration (PLD) 14, 21 and 28 days for the most common and targeted predatory reef fish, (leopard grouper  Knowledge of patterns of larval dispersal is essential to implement fully-protected marine reserves (no-take zones), a tool frequently used to enhance the conservation of biodiversity and the recovery of fisheries . ReserveMarine connectivity within a single species is influenced by multiple biological and physical factors including: spawning time and location, pelagic larval duration (PLD), their interaction with ocean current speed and direction, as well as the distribution of suitable habitat for settlement . The bathymetry of this region constitutes one of the most notable features of the GC, which is formed by a series of deep basins  and sills, and which restrict the circulation between the northern and central GC . The comMycteroperca rosacea, Streets 1877) is the most common and heavily targeted grouper by small-scale and recreational fisheries in the GC  endemic to the GC bioregion. It ranges from Bahía Magdalena in the Pacific coast of the Baja California Peninsula south to Bahía Banderas in Nayarit, Mexico, including all rocky-reefs within the interior of GC (or of GC . Ecologior of GC . Spawninor of GC . Histoloor of GC .M. rosacea (April–June) in 2008, 2009, and 2010. Evidence of the formation of spawning aggregations were based on standard protocols .We determined the duration of the spawning season using a combination of two methods. First, we examined the histological preparations of all gonad samples to identify the percentage of females capable of spawning or actively spawning during each month of sampling. Females categorized as ‘spawning capable’ included those with ovaries dominated by oocytes in advanced stages of vitellogenesis , whereas those categorized as ‘actively spawning’ contained ovaries with oocytes in the migratory nucleus, hydrated, or ovulated stage or post-ovulatory follicles were present. Data were pooled by month to estimate the monthly proportion of spawning females over a calendar year. Collection dates of females classified as actively spawning based on histology were used as indicators to determine exact dates of spawning. A second estimate of the spawning season was obtained by calculating the mean monthly gonadosomatic index  of female M. rosacea was captured through a series of interviews implemented across 17 fishing communities in the northern GC  the north end of Angel de la Guarda Island; (2) on the south, western and northern edge of Tiburon Island, and on sites in mainland Sonora north of Tiburon, around Las Cuevitas, Puerto Libertad and Puerto Lobos (yellow areas http://www.ngdc.noaa.gov/mgg/shorelines/shorelines.html) and the marine protected areas in Mexico (www.conanp.gob.mx), respectively. We used the spatial union function to integrate the different boundaries and define the spatial units, under ArcGIS 10.1 (ESRI) with the Spatial Analyst Extension and Model Builder tools. The size of the spatial units varied from 13 to 812 km2 . We assumed that larvae are advected as passive particles and do not migrate vertically downward to deep depths, as in other studies   to calcues e.g., , given t5 m deep .Hourly latitude and longitude data for each modeled particle were imported into MatLab (MATHWORKS). We estimated connectivity at different time intervals: 336 h (14 days), 504 h (21 days) and 672 h (28 days) respectively after the release dates. These PLDs were selected based on the average time of flexion in groupers that corresponds to the onset of larval behavior (14 days)  and the Marine connectivity patterns were displayed using graph theory and a spatial network approach  with theWe used the software GENETICSTUDIO  to calcuM. rosacea from 11 sites included in our modeling exercise around the Midriff Islands region (b using primers Gludgl 5′-TGAYTTGAARAACCAYCGTTG-3′ and CB3H 5′-GGCAAATAGGAARTATCATTC-3′ (M. rosacea (MYCROS Forward: 5′-TTCTCCCACTACCCTGATTC-3′ and MYCROS Reverse: 5′-TACGTAGGCTTGGATCATTG-3′) to amplify a 726 bp fragment of mitochondrial marker ATPase. Thermocycler parameters were as follows: initial hold at 94 °C/5 min, 35 cycles of 94 °C/30 s, 54 °C/30 s, 72 °C/30 s, with a final extension of 72 °C/7 min. After purification of PCR products following ABI manufacturer’s protocols , we sequenced clean PCR products on an ABI 3730xl automated sequencer .We collected tissue samples from the pectoral fins of s region . Sampless region . We amplCATTC-3′ . Thermocπ) and haplotype diversity (h). We corrected haplotype diversity using CONTRIB  the evolutionary relationship among haplotypes can provide additional resolution to explain where an haplotype is found among distinct populations . We also calculated the standardized index STF by the maximum value STF can achieve while maintaining the within-population diversity. Maximum values were calculated by recoding the alleles in such a way that every population only contained alleles unique to that population, following STF and STϕ which are fixation indexes, Phylogenetic relationships among sequences were inferred from a haplotype network based on pairwise differences between haplotypes generated using Arlequin  and R soent}FST′  by dividulations .We constructed a population graph from the distribution of haplotype frequencies with the software GENETICSTUDIO. A population graph is an analysis that allows genetic structure to define a graph-theoretic topology by capturing the high dimensional genetic covariance among all nodes considered simultaneously. An edge is placed between nodes that are conditionally dependent of each other, based on the remaining data in the model. With the empirical network, we calculated the same five node-based network metrics in NODEXL, and the adjacency and graph distance matrices  STF and (log) STϕ, (log) STF and (log) STϕ, STF and P value 0.05 with the graphically sharpened method  from sampling localities in the Baja Peninsula, the Midriff Islands, and the Sonoran coast. One model assessed the hypothesis of unidirectional gene flow from the Baja Peninsula across the Midriff Islands and towards the Sonoran coast, while the other model tested gene flow in the reverse direction. The latter two models reflect seasonal differences in directionality of a cyclonic and anticlyclonic gyres in the northern GC. Using a Bezier approximation, we chose the most appropriate model for our dataset by taking the natural log of the ratio of the marginal likelihoods (Bayes factors) for each model . Analyses were done with GENETICSTUDIO, where we considered each group in a sub-graph one at a time, separated from the other two.We identified 17 sites associated with 17 distinct spatial units for the release of virtual larvae in the simulation model based on direct and indirect evidence of the presence of spawning aggregations for leopard grouper . AlthougM. rosacea were collected from commercial fishers, with an average of 14 samples collected each month (range = 8–27). Based on microscopic examinations of gonadal tissue samples, females in the spawning capable phase were collected from March through June, and actively spawning females were collected mainly during May and June. The GSI of adult females showed elevated levels from April to June, with a peak during May  and two days in 2010 .A total of 162 samples of female ring May . When thFrom all simulated particles released , coastal areas that are suitable for larval recruitment captured 55.13% (PLD 14 days), 48.07% (PLD 21 days) and 42.03% (PLD 28 days). Remaining particles did not reach any coastal habitat by the end of the PLD. Simulations of ocean currents produced the highest concentrations of larvae in: (1) Canal de Ballenas, (2) north end of Angel de la Guarda Island, (3) north end of Tiburon Island, and (4) in coastal areas located to towards the north after PLD 28 days , the northern end of Tiburon Island , followed by the southern coast of Tiburon Island , San Esteban Island (range: 0.22–0.23) and Puerto Libertad (range: 0.38–0.16).Our analyses illustrated that several of the connectivity metrics covary , highlighting a few sites that were important despite variation in PLD . For exab and a 726 bp fragment of ATPase for 260 individuals. We identified a total of 79 haplotypes (GenBank accesion numbers KJ004770–KJ004925), with adjacent haplotypes in the haplotype network separated by 1–4 bp , but patterns were not significant after correcting for multiple tests using FDR . No other comparisons were significant. The linear regression between modeled and empirical values of betweenness centrality and clustering coefficient at each PLD showed only the clustering coefficient estimates were correlated at PLD 21 days . After correcting for multiple tests, only patterns at PLD 28 days were significant .Node-base analyses via fit of a linear model for explaining haplotype and nucleotide diversity using in-degree, out-degree and betweenness centrality at each PLD as explanatory variables revealed that nucleotide diversity was lower at locations showing higher out-degree according to the model at PLD 14 days . The STϕ matrix and the (log) graph distance matrix were also significantly correlated at PLD 28 days .Matrix-based analyses showed a lack of correlation between pairwise distance . First, χ2 = 186.876, d.f. = 152, P = 0.0286) and an AMOVA test . The first segment consisted of Baja + Midriffs and the second segment included Sonora. The empirical network did not show any evidence of modularity.Regional genetic subdivision was observed when sampling sites were clustered into the Baja Peninsula, Midriff Islands and Sonoran coast groups, as supported by a chi-squared test , along with large levels of local retention according to the model  of larvae from upstream sites.Although the Baja, Midfriffs and Sonora groups were significantly differentiated, the best supported gene flow model (based on genetic subdivisions) agreed with the general cyclonic (anti-clockwise) direction of the gyre in the northern GC during the spawning period of Sargassum sp.  and 11 sites where genetic samples were also collected (blue polygons).Click here for additional data file.10.7717/peerj.511/supp-2Figure S2Tides in the HAMSOM oceanographic model for the northern Gulf of California, showing the four neap and four spring tides during May and June selected for the release of virtual larvae. Red lines separate distinct months.Click here for additional data file.10.7717/peerj.511/supp-3Table S1STF possible while maintaining the within-population diversity above diagonal and pairwise Pairwise maximum Click here for additional data file.10.7717/peerj.511/supp-4Table S2STF) values and various explanatory variables, including geographic distance and three distinct matrices calculated from the modeled networks, including adjacency, graph distance and log graph distance. P values below 0.05 are shown in bold. * Indicate statistical significance after correcting for multiple tests .Mantel and partial Mantel tests between an empirical matrix of genetic structure .Mantel and partial Mantel tests between an empirical matrix of genetic structure  for PLD 14, 21 and 28 days. Scale in kilometers.Click here for additional data file.10.7717/peerj.511/supp-8File S1Detailed results from three independent runs with Migrate-n software for unrestricted gene flow among Baja, the Midriff Islands and Sonora.Click here for additional data file.10.7717/peerj.511/supp-9File S2Detailed results from three independent runs with Migrate-n software for a model of gene flow from Baja across the Midriff Islands towards Sonora.Click here for additional data file.10.7717/peerj.511/supp-10File S3Detailed results from three independent runs with Migrate-n software for a model of gene flow from Sonora across the Midriff Islands towards Baja.Click here for additional data file.