Opisthorchis felineus or Siberian liver fluke is a trematode parasite (Opisthorchiidae) that infects the hepato-biliary system of humans and other mammals. Despite its public health significance, this wide-spread Eurasian species is one of the most poorly studied human liver flukes and nothing is known about its population genetic structure and demographic history. In this paper, we attempt to fill this gap for the first time and to explore the genetic diversity in O. felineus populations from Eastern Europe , Northern Asia (Siberia) and Central Asia (Northern Kazakhstan). Analysis of marker DNA fragments from O. felineus mitochondrial cytochrome c oxidase subunit 1 and 3  and nuclear rDNA internal transcribed spacer 1 (ITS1) sequences revealed that genetic diversity is very low across the large geographic range of this species. Microevolutionary processes in populations of trematodes may well be influenced by their peculiar biology. Nevertheless, we suggest that lack of population genetics structure observed in O. felineus can be primarily explained by the Pleistocene glacial events and subsequent sudden population growth from a very limited group of founders. Rapid range expansion of O. felineus through Asian and European territories after severe bottleneck points to a high dispersal potential of this trematode species. O. felineus, O. viverrini and Clonorchis sinensis are three species from the genus Opisthorchis  that are known to cause serious human diseases affecting bile ducts and the gall bladder. Opisthorchis infection is recognized as the major risk factor of cholangiocarcinoma O. felineus, O. viverrini and C. sinensis, respectively Opisthorchis are obligate endoparasites with a complex three-host life cycle (http://www.dpd.cdc.gov/dpdx/HTML/Opisthorchiasis.htm). The first intermediate hosts of O. felineus are sister-species of freshwater snails Bithynia  All species within the genus Opisthorchis, O. felineus has the largest distribution range, which does not overlap with the ranges of O. viverrini  C. sinensis  O. felineus spans over the territory of the Ob-Irtysh River basin (West Siberia), the Volga-Kama River system, as well as the Don and the Dnieper watersheds (Eastern Europe). The Yenisei River basin delimits the eastern border of the areal O. felineus has been reported in Belarus O. felineus in the final hosts in most European countries O. felineus have only been described for Italy and Germany Among human-infecting members of the genus Schistosoma mansoniS. japonicumParagonimus westermaniFasciola hepaticaF. magnumEchinostoma sp. internal transcribed spacer sequences, ITS, evolve much faster than genes encoding ribosomal RNA ITS have been employed to resolve phylogenetic problems in closely related species and they are the markers of choice in taxonomic studies of digeneans Over the past few years, many efforts focused on studying the population genetic structure and heterogeneity of important trematode species including O. viverrini and C. sinensis, have been the focus of active research over the past several years. In particular, this research involved studies of genetic variation in O. viverrini populations. Based on the sequences of several mitochondrial DNA markers (NADH dehydrogenase subunit 1 gene (nad1) and cox1), genetic variation among different geographical isolates of O. viverrini from Thailand, Cambodia and Lao PDR has been detected O. viverrini. A high level of genetic diversity was demonstrated for O. viverrini sampled in 5 localities O. viverrini from different geographical areas in Thailand and Lao PDR, high diversity between the isolates from different localities was observed. Thus, the authors speculated that O. viverrini did not represent a single species but rather comprised two cryptic species C. sinensis has been published to date. Nevertheless, the genetic diversity study of C. sinensis population from the Russian Far East showed two levels of intraspecific variation for ITS1 sequences Opisthorchiasis and clonorchiasis are diseases with pronounced impact on public health in the countries of South-Eastern and Eastern Asia. Two parasitic species that cause these diseases, O. felineus is widely spread across Europe and Northern Asia, its intraspecific structure still remains poorly explored. To our knowledge, no analysis of its population genetic diversity has been done. Beer and German previously reported O. felineus to be heterogeneous within its distribution range O. felineus were proposed to exist: European, Siberian and Kazakh Even though cox1, 573 bp-long fragment from cox3 and a 481 bp-long fragment of a nuclear marker ITS1 to gain insight into genetic diversity of O. felineus over its major distribution range. We also adopted this approach to address the question of its intraspecific population structure.In the present work, we used a 260 bp-long marker fragment from the mitochondrial gene O. felineus metacercariae were collected in different geographical localities in Eastern Europe , Northern Asia (the Ob-Irtysh and the Yenisei basins) and Central Asia (the Nura-Sarysu basin) (Leuciscus idus), roaches (Rutilus rutilus), daces  and rudds . We isolated DNA directly from 149 metacercariae. Species identity of the samples was established by comparing the sequencing reads with the reference sequences available . Thus, 124 samples were classified as O. felineus and so they were used in this work. The remaining samples were from P. truncatum and M. bilis (Opisthorchiidae family). Also, adult worms (97 samples) were grown in the lab by infecting golden hamsters (Mesocricetus auratus) with metacercariae. One month after infection, adult worms were isolated from hamster livers. Species identity was morphologically assessed under the light microscope. Each adult worm was cut in 2–3 parts, one of which was used for DNA isolation. The remaining material was used as a back-up and stored in 70% ethanol at +4°C.u basin) . MetacerO. felineus individuals from 16 localities were used in our analysis  took place were neither conservation areas nor private, nor otherwise protected, hence no fishing permits were required. The fish species collected are not considered endangered or rare, and fishing methods were in full compliance with the Federal Law N166-Ф3 of 20.12.2004 (ed. 18.07.2011) "Fishing and conservation of water bio-resources”.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Institute of Cytology and Genetics (Permit Number: 7 of 19.11.2011). Euthanasia was performed by decapitation, and all efforts were made to minimize suffering.Three markers showing different levels of polymorphism were selected for our analysis.Cox1 is a marker routinely used in population genetic studies. Cox3 is yet another michonodrial gene which is known to have increased frequency of SNPs relatively to cox1. O. felineus, C. sinensis and O. viverrini, demonstrated that cox3 is indeed more variable than cox1. This established cox3, similarly to nad1, as one of the highly variable mtDNA markers in liver flukes.ITS1 sequence is much more conserved. The specific choice of ITS1 rather than ITS2 sequence was based on the fact that it is the only region of rDNA which is variable enough to discriminate polymorphisms within species (even though this polymorphism is quite low) Compared to the mtDNA markers used, the nuclear O. felineus adults or metacercariae using standard phenol/chloroform extraction DNA was isolated from Opisthorchis genus. We aligned Genbank sequences for the four species of interest, and focused on the primer regions that were the most conserved.Vector NTI Advance 10 (Invitrogen) was used for primer design. These primers were universal for the Cox1 gene fragment was amplified using the primer pair cox1-F (5′-GGGTTTGGAATGATTAGTC) and cox1-R (5′-CACAGAGGCAGAAAGAACT), Tm –54°C; fragment of ITS1 was amplified with ITS1-F (5'-GTCGTAACAAGGTTTCCGTA) and ITS1-R (5'-ACACGAGCCGAGTGATCC), Tm –59°C; the cox3 fragment was amplified with cox3-F (5'-ATATTTATGGGGATTGTGAATT) and cox3-R (5'-AGCCCGAACTATAGACAACA), Tm –50°C.2, 1.5 U TrueStart Hot Start Taq DNA polymerase (Fermentas) and appropriate PCR buffer. Touch-down PCR was performed in a total volume of 20 mkl with the following thermocycling conditions: 37°C/5 min, 94°C/2 min, 10 cycles: 94°C/10 sec, Tm+10°C, decrement −1°C per cycle/20 sec, 72°C/40 sec, 20 cycles: 94°C/10 sec, Tm/20 sec, 72°C/40 sec followed with the final elongation step at 72°C for 5 minutes.Reactions contained 100 pg template DNA, 250 mkM dNTPs, 25 mkM dUTP, primers (200 nM each), 0.1 U UDG (Fermentas), 1.5 mM MgClAmplified DNA fragments were Sanger-sequenced using BigDye terminator cycle sequencing kit (Applied Biosystems), according to the manufacturer’s specifications. Sequencing primers were the same as those used for PCR. To assure the accuracy of sequencing, the amplified fragments were sequenced from both forward and reverse primers. Sequencing products were run on ABI 3130XL Genetic Analyzer (Applied Biosystems). In some individuals only one or two marker sequences could be retrieved.Chromatogram analysis and editing were done using Sequencing Analysis 5.2, Patch 2 (Applied Biosystems). All the sequences were deposited in Genbank, and their accession numbers are shown in cox1, cox3 and ITS1 were aligned separately using Vector NTI Advance 10 (Invitrogen). Intrapopulation variation was summarized using standard statistics: number of haplotypes (H), number of segregating sites (S), haplotype diversity (Hd) and nucleotide diversity (π), were all calculated using the program DnaSP 4.5 The sequences of cox1, 4 cox3 and 7 ITS1) had degenerate sites. In these cases, haplotypes were reconstructed using PHASE algorithms provided in DnaSP 4.5 Due to heteroplasmy mtDNA and partial homogenization and/or heterozygosity rDNA, several sequences  was performed, considering genetic distances between haplotypes and their frequencies among the defined populations, with statistical significance determined by 1000 permutations Neutrality tests were performed using Tajima’s D A distribution of pairwise genetic differences (mismatch distribution) analysis O. felineus populations studied here were combined into three geographically isolated groups: Northern Asian , Central Asian  and East European . This grouping was based on the data of German and Beer on the existence of three O. felineus subspecies in these territories All ITS1 (481 bp), and two mitochondrial markers, cox1 (260 bp) and cox3 (573 bp). The diversity indices calculated from the nucleotide data sets are summarized in We sequenced one nuclear marker, ITS1 and cox3 displayed the least and the highest genetic diversity, respectively. Surprisingly, the levels of nucleotide and haplotype diversity were very low across the whole territory studied (Hd = 0.28 (cox1), 0.61 (cox3) and 0.18 (ITS1); π = 0.0013 (cox1), 0.0019 (cox3) and 0.0004 (ITS1).In agreement with the expected degree of variability of the markers used, Mesocricetus auratus), we separately analyzed genetic diversity in the samples obtained directly from metacercariae and from all adult worms grown in the lab. No significant differences were found .In order to control for possible introduced bias while culturing metacercariae in the non-natural definitive host , 37 cox3 haplotypes (28 singlets) and 5 ITS1 haplotypes (3 singlets). Most of the identified haplotypes were distinct from the central haplotype by one substitution , or 2 substitutions (4– cox1 and 9– cox3). In one case the haplotype diverged by as much as 4 SNPs from the central variant for cox3. Notably, this exceptional haplogroup was not restricted to a particular geographic location. We also found 6 non-singlet region-specific haplotypes. For cox1 and cox3, there were 4 and 1 Northern Asia-specific haplotypes, respectively. One region-specific cox3 haplotype was found in the Eastern Europe. All these haplotypes were exceedingly rare, i.e. in a given region only 2–3 individuals shared such variant.Besides the common haplotypes, we identified 18 Cox1- and cox3-based analyses of molecular variance (AMOVA) failed to show considerable differences between separate populations and regions , −2.40 (cox3) and −1.55 (ITS1)), indicating an excess of rare nucleotide site variants compared to expected under a neutral model of evolution. Results of Fu's Fs test (which is based on the distribution of haplotypes) also show negative values (−29.70 (cox1), −48.83 (cox3) and −5.76 (ITS1)). This indicates an excess of rare haplotypes over what would be expected under neutrality. This could be due to population expansion, purifying selection or selective sweep. However, all the markers used here are known to be selectively neutral, therefore the effect observed should be best explained by population expansion.The results of Tajima's D test and Fu's FO. felineus population expansion, we calculated the distribution of pairwise nucleotide differences among individual haplotypes. The mismatch distribution for the species was clearly unimodal, and characterized with low ruggedness index r . Unimodal mismatch distributions are typical of expanding populations, whereas old populations of constant size are expected to be multimodal O. felineus.To test the hypothesis of cox1, 0.366 for cox3 and 0.191 for ITS1), which is the date of growth or decline measured in units of mutational time, and is useful to estimate the onset of expansion We also estimated τ : ITS rate being 0.8%/MY cox1–2.5%/MY Winter period ranges 5–8 months across Thus, this analysis suggests that our species of interest has recently gone through a severe population bottleneck followed with sudden expansion (21–25 kBP) from a small group of founders with a very limited gene pool.Nowadays it becomes increasingly important to study biodiversity of human parasites, given that many of such species are now observed to rapidly invade new territories and thereby pose an increasing epidemiological threat. Genetic diversity is one of the most important components contributing to species survival and ecosystem resilience. Knowledge of the genetic structure within and among populations of parasitic species combined with phylogeographic data can provide a deeper understanding of their ecological and evolutionary dynamics with practical implications for parasitosis control.O. felineus found in the largest river systems of Eastern Europe and Central and Northern Asia. We SNP-typed three DNA fragments from ITS1, cox1, and cox3, which have shown different substitution rates. The patterns of genetic structure and diversity obtained from these DNA markers were largely consistent.Nuclear and mtDNA markers are widely used to assess genetic relationships between individuals or groups of individuals. These markers are used to study the dynamics of genetic processes within populations, to describe the species population structure and to elucidate the phylogeographic history O. felineus across its very large range, we expected to see complex intraspecific structure. However, our analysis revealed that three regions tested  demonstrated very low values of genetic variation (Hd = 0.28 (cox1), 0.61 (cox3) and 0.18 (ITS1); π = 0.0013 (cox1), 0.0019 (cox3) and 0.0004 (ITS1). Nucleotide diversity was low because haplotypes typically differed by just a single nucleotide. Our estimates of genetic differentiation between the populations were based on the haplotypes frequencies and evolutionary distance between haplotypes. Fst<0.05 (0.008–0.021), as well as on AMOVA tests, were indicative of very low differences among populations. Haplotype networks displayed pronounced star-like structure with one major haplotype present in over 80% of cases for the relatively conserved cox1 and ITS1 and in over 60% of cases for the more variable cox3. The rest of the haplotypes were distinct from the major variant by one or two substitutions, and were predominantly singlets. Such peculiar pattern – low genetic diversity, star-like haplotype network with dominance of one major haplotype and many rare variants, combined with highly negative neutrality indices (Fu’s Fs and Tajima’s D reaching −29.70 and −2.10 (cox1), −48.83 and −2.40 (cox3) and −5.76 and −1.55 (ITS1) – are indicative of a recent severe bottleneck in the species population history, which was followed with rapid colonization and population expansion DNA markers similar to the ones used in our work have been extensively tested over the past ten years to establish the population structure and phylogeography of human-infecting trematodes from Europe Opisthorchis species. Selfers are expected to be less polymorphic than outcrossers O. felineus, differentiation both between and within populations was low.In addition to the recent severe bottleneck, the peculiar mating system in this species could also contribute to the decrease in genetic variability. Liver flukes are facultative hermaphrodites Besides being hermaphroditic, trematodes display obligate alternation between sexual and clonal reproduction during their complex life cycle. Although modeling has suggested certain changes in the population genetics of such parasites F. hepatica populations showed extensive genetic mtDNA diversity even in local studies (Ireland and the Netherlands) nad1 and cox1 revealed 4.1 and 2.3% sequence variation, respectively, in flukes sampled across Eastern Europe Opisthorchis species, O. viverrini populations displayed a striking Hd = 0.88 (nad1) just in the Lower Mekong River basin. Of the 19 haplotypes found in this population, there were at least three major haplotypes that were considerably distinct from each other ITS1 clades were found in an other Opisthorchis species, C. sinensis, in its Russian Far East population Studies of other Eurasian trematode species with similar reproductive cycle reported significantly higher genetic diversity O. felineus reproduction is not the main factor contributing to its extraordinary low genetic variability. Thus, we suggest that the low genetic diversity of O. felineus is best explained by some sort of environmental impact (such as severe climatic changes) which caused the effective population size to shrink. Whatever the case, as the population was undergoing expansion after the bottleneck, its low genetic variability should have largely relied on higher levels of genetic drift due to the stage of very active asexual reproduction in the life cycle of this species.Taken together, these data indicate that the peculiar mode of To gain deeper insight into the possible causes of low genetic variability in our species of interest, a phylogeographic approach can be very useful. Climatic oscillations of the Pleistocene are often invoked as major events that have shaped the geographic and genetic structure of current species via past range fragmentation, population contraction and recolonisation O. felineus is presently undergoing demographic expansion, which started relatively recently, approximately 21–25 kBP. We estimated that O. felineus split from the last common ancestor with C. sinensis about 3 MYA . Back then, Northern Asia had relatively mild climate, and in Siberia, Sino-Indian species of flora and fauna were widely distributed O. felineus across the draining territories. The major reason underlying the severe bottleneck that Siberian liver fluke population went through is harsh environment in the Quaternary combined with overall low infestation rate (<1%) Our mismatch distribution test clearly suggested that O. felineus refugium and to trace its migration routes. Unfortunately, the species’ low genetic diversity across the territory studied suggests the refugium was located elsewhere. Where would the most likely location of such refugium be? There is a line of fossil evidence suggesting that during the climate fluctuations and landscape changes in Pleistocene, Bithyniidae snails went essentially extinct in the present-day Ukraine and European part of Russia It would be interesting to pinpoint the location of O. felineus hosts include fresh-water and terrestrial species of animals, therefore this parasite could spread over the new territories by different ways. Obviously this has contributed to the rapid geographic expansion of the species. The land route of O. felineus colonization could occur upon completion of LGM via fish-eating animals, whose long-distance migrations upon post-glacial climate changes were reported O. viverrini demonstrated limited gene flow between the nearby regions F. hepatica, dating back to the Quaternary events is still very well-discernible O. felineus.Using standard DNA markers, we have shown very low levels of genetic differentiation between geographically distinct Europian and Asian populations of the Siberian liver fluke. In contrast to free-living species, there are far more factors that influence the genetic structure of parasites due to their complex life cycle and to the contribution of host biology. Trematodes have been previously reported to have less structured populations and lower genetic diversity as compared to snails, their first intermediate hosts O. felineus and re-colonization of the range from a single source population. Yet, earlier studies proposed that O. felineus is a composite of several lineages, subspecies or cryptic species inhabiting the territory being analyzed. These proposals were based on the noticeable morphological differences among the isolates collected from different localities O. felineus subspecies Absence of intra-species structure is in good agreement with short post-glaciation history of O. felineus which prevented us from singling out any cryptic species or subspecies. Clearly, this result conflicts with the morphology findings. Much like other trematode researchers, we favor molecular data over morphological classifications The markers used in our analysis are sensitive enough to track speciation process and to discriminate valid species S. mansoni, as little as 3 generations were enough to form host-parasite compatibility in lab experiments O. felineus.Limited compatibility of parasites and snails from different geographical localities can not be easily disregarded and implies some intraspecies diversity was necessary for successful host/parasite adaptation. Such local adaptation relies on the interplay of highly variable proteins of hosts and parasites O. felineus revealed its uniquely low genetic diversity across a geographically enormous range. We suggest that severe geoclimatic events during the Quaternary epoch led to virtually complete extinction of this species. Upon completion of the last glaciation, O. felineus underwent rapid expansion from a single ancestral population. In fact, this recovery of species could have occurred from a very small ancestral population, due to the ability of this parasite to reproduce clonally. Importantly, despite the recent bottleneck episode, O. felineus is presently a widely-distributed species. Therefore, high dispersal potential of this trematode species should be taken into account in parasite-control programs.The first population genetic analysis of Siberian liver fluke Table S1Comparison of diversity indices of samples isolated from adult worms and metacercariae.(DOC)Click here for additional data file.


Pachyhynobius shangchengensis (Hynobiidae) is a vulnerable species restricted to a patchy distribution associated with small mountain streams surrounded by forested slopes in the Mount Dabieshan region in southeastern China. However, molecular phylogeography and population genetic structure of P. shangchengensis remain poorly investigated. In this study, we explored the genetic structure and phylogeography of P. shangchengensis based on partial sequences of the mitochondrial DNA (mtDNA) cytochrome b and cytochrome c oxidase subunit I genes. Fifty-one haplotypes and four clades were found among 93 samples. Phylogenetic analyses revealed four deeply divergent and reciprocally monophyletic mtDNA lineages that approximately correspond to four geographic regions separated by complicated topography and long distances. The distinct geographic distributions of all lineages and the estimated divergence time suggest spatial and temporal separation coinciding with climatic changes during the Pleistocene. Analysis of molecular variance indicated that most of the observed genetic variation occurred among the four groups, implying long-term interruption of gene flow, and the possible separation of P. shangchengensis into four management units for conservation. The salamander  Molecular ecology primarily aims to understand the influence of abiotic factors, such as altitude, topography, and glacial history, on the spatial distribution of genetic variations . RecentlUsing phylogeographical tools to analyze the effects of landscape characteristics on species distributions over large spatial scales has provided remarkable insight into the spatial patterns of genetic diversity . Using hP. shangchengensis which lives in here should be affected by climatic changes. Besides landscape characteristic, climatic changes have also caused montane species to expand, change, or be in contact with each other along latitudinal or elevational gradients associated with Pleistocene glacial cycles –11. “EasP. shangchengensis (Hynobiidae) is an endemic species in china, its distributions restrict in the Mount Dabieshan regions, which was described by Fei et al. [Pachyhynobius shangchengensis has low vagility, its habitat is separated by valleys and low lands, and its distributions are getting smaller and smaller [Hynobius yunanicus based on specimens from Huangbaishan, Shangcheng County. Hynobius yunanicus differs from P. shangchengensis mainly in having little white spots on deep brown dorsal side, in lacking premaxillary fontanelle on the skull, and in lacking connection between maxillary and pterygoid. However, evidences from karyotypic and phylogenetic analysis rejected the validity of H. yunanicus [Hynobius yunanicus is a synonym of P. shangchengensis, and our sampling sites should include Huangbaishan.Shangcheng Stout Salamander i et al.  from Moui et al. ,18. Pach smaller . Chen et smaller  describeunanicus . TherefoP. shangchengensis, the sampling sites include Jingangtai (JGT), Tiechong (TCH), Tiantangzhai (TTZH), Huangbaishan (HBSH), Yingshanxian(YSHX), Yuexixian (YXX) and Huoshanxian (HSHX). The above sampling sites are isolated from each other by more than 20 kilometers  and cytochrome c oxidase subunit I (mtDNA COI) genes. We also determined whether climatic oscillations during glacial periods in the Quaternary affected the distribution of P. shangchengensis. In this study, we test whether the topography of the mountains affected the population genetic structure of the P. shangchengensis individuals were collected from seven locations on the Dabieshan Mountains, Southeast China  of Shaanxi Normal University and Chinese Academy of Sciences. A total of 93 st China . SamplesSamples were also permitted by following authorities: management committee of Jingangtai Nature Protection Area; management committee of Shangcheng Stout Salamander Nature Protection Area (Henan Shangcheng); management committee of Tiantangzhai National Forest Park; management committee of Yaoluoping Nature Protection Area.1, H1 and inner primers (nested primer) L2, H2 (COI) was amplified by PCR , with forward primer FZ and reverse primer RZ (COI) and KC162084-KC162162 (cyt b).Tissue samples were preserved in 95% ethanol and stored at -20 °C. Total genomic DNA was extracted through a standard phenol: chloroform method . A conti) L2, H2 . A contirimer RZ . The PCRhttp://darwin.uvigo.es). To examine whether the two analyzed regions (cyt b and COI) can be combined into a larger data matrix [The sequences were aligned with Clustal X1.83 . The alia matrix , we perfa matrix . The comHd) and nucleotide diversity (Pi) were determined through Arlequin 3.5.1.2 [The number of variable and parsimony-informative sites was determined using the program DnaSP 5.10.01 , and hap 3.5.1.2 .Hynobius chinensis and Hynobius guabangshanensis were used as the outgroup. The COI and cyt b sequences of H. chinensis were downloaded from GenBank with accession number HM036353, and the COI and cyt b sequences of H. guabangshanensis were downloaded from GenBank with accession number EF616473 (cyt b) and FJ913877 (COI).  The phylogenetic relationship among haplotypes was estimated through maximum likelihood (ML) analyses in PAUP*4.0b10 , as well We also used NETWORK 4.5.0.2  to draw  The population and phylogroup comparisons using pairwise difference and the partitions of genetic diversity within and among populations were analyzed through analysis of molecular variance (AMOVA)  using Ar The spatial genetic structure of haplotypes was analyzed through SAMOVA 1.0   was proposed by Weisrock et al. . This evFST and the geographical distances. Pairwise FST values between populations were estimated through Arlequin 3.5.1.2 [http://www.gpsvisualizer.com/calculators. Mantel tests  were con 3.5.1.2 , whereasFS test [D [D and Fu’s FS are sensitive to bottleneck effects or population expansion, causing these values to be more significantly negative [FS is particularly sensitive to recent population growth [We applied Neutrality tests through the program Arlequin 3.5.1.2  as an asFS test  and Taji test [D . Tajima’negative –49. Fu’sn growth . R) between observed and expected mismatches. The formula T=τ/(2ut) was used to estimate the time of the population expansions [t is the date of growth or decline , τ is the mode of mismatch distribution (evolutionary time), and u is the mutation rate per sequence and per generation [ Population expansion events were determined through mismatch analysis  using Arpansions  based onpansions , t is thneration . P. shangchengensis using the program BEAST 1.6.1 [P. shangchengensis as suggested by Weisrock et al. [ The Bayesian skyline plot (BSP) was used to estimate the demographic history of ST 1.6.1 . A piecek et al. . Tracer COI gene and 942 bp for the cyt b gene. A total of 195 polymorphic sites were found, of which 182 were parsimony-informative and 13 were singleton-variable. These polymorphic sites identiﬁed 51 haplotypes within 93 individuals from seven localities  was 1953, of which 1011 bp were sequenced for the calities . Each say low Pi . P. shangchengensis observed in the combined dataset formed four distinct clades  (93.92%), whereas differentiation among seven endemic populations  within groups only contributed 1.48% to the total population, and differentiation within seven endemic populations contributed 4.6% to the total population .K increased from 2 to 6, the FCT value was highest (FCT = 0.9421) when K = 4. Thus, the SAMOVA tests revealed the number of significant phylogeographic groups (K = 4) .For the spatial AMOVA, with p < 0.001), indicating the presence of isolation-by-distance. This finding suggests that the distribution of genetic variation is due to geographical separation. The Mantel test results provided evidence for large-scale geographical population structure in this species.Mantel test results showed significant correlation between the pairwise calculated genetic distance and pairwise calculated straight line geographical distance of the populations  between most local populations . Thus, aFs were negative, and some values have highly significant, except clade D possessed a positive value  of 1.54–2.74 Myr. The divergence time between clade C and clade A+B was calculated to have taken place in the mid-Pliocene 3.93 Myr (95% HPD: 3.06–4.83 Myr). The divergence time between clade D and clade A+B+C was calculated to have taken place in the early Pliocene 4.93 Myr (95% HPD: 4.06–5.88 Myr)  . P. shangchengensis. In our study, we can see that phylogenetic analyses support four major clades. The median-joining network yielded four unconnected subnetworks corresponding to the four clades in the phylogenetic tree, and there are no shared haplotypes between clades , between the main clades, the divergence time taken place in the Pliocene, and by Bayesian skyline plot (BSP) a sudden expansion occurred in the Late Pleistocene. Therefore, climatic fluctuations probably impacted the distribution, demography and diversification of the species. We infer that P. shangchengensis which lived in the low lands disappeared during the interglacial in the Quaternary, because it could not adapt to hot and dry climate. By contrast, Pachyhynobius shangchengensis, which lived in higher elevations, survived because these areas were suitable for survival. Thus, the populations of P. shangchengensis have been isolated in the fragmented mountain habitats in the last interglacial to the present. This pattern also existed in other montane organisms [Topography can drive divergence patterns , Pleistorganisms ,68. P. shangchengensis decreases each year because of human activities such as arbitrary arrest or killing [Pachyhynobius shangchengensis should be protected.    The two goals of any conservation program are to maintain the genetic diversity of species for long-term evolutionary success and ensure their survival ,69. The  killing ,19,61. TP. shangchengensis can be considered MUs because genotypes in the four populations are closely related but not shared. Any conservation policy should concentrate on protecting the distinct populations with similar MUs similar to the conservation efforts for the Tibetan gazelle in China [P. shangchengensis and avoid its extinction, we should take necessary measures, such as forbidding to kill any individual species in distribution areas of P. shangchengensis.Management units (MUs) are commonly used designations for threatened or endangered taxa ,71. The in China . We woulPachyhynobius shangchengensis has significant phylogeographic structure, the topography of the Dabieshan Mountains significantly affects the population genetic structure of it, and climatic oscillations during glacial periods in the Quaternary affected the distribution of this species. In conclusion,


Salmo trutta marmoratus, an endemic salmonid of northern Italy and the Balkan peninsula, was explored at the macro- and micro-scale level using a combination of mitochondrial DNA (mtDNA) and microsatellite data.The genetic structure of the marble trout Sequence variation in the mitochondrial control region showed the presence of nonindigenous haplotypes indicative of introgression from brown trout into marble trout. This was confirmed using microsatellite markers, which showed a higher introgression at nuclear level. Microsatellite loci revealed a strong genetic differentiation across the geographical range of marble trout, which suggests restricted gene flow both at the micro-geographic (within rivers) and macro-geographic (among river systems) scale. A pattern of Isolation-by-Distance was found, in which genetic samples were correlated with hydrographic distances. A general West-to-East partition of the microsatellite polymorphism was observed, which was supported by the geographic distribution of mitochondrial haplotypes.While introgression at both mitochondrial and nuclear level is unlikely to result from natural migration and might be the consequence of current restocking practices, the pattern of genetic substructuring found at microsatellites has been likely shaped by historical colonization patterns determined by the geological evolution of the hydrographic networks. Salmo trutta, which lead to the definition of five distinct evolutionary lineages: four of them, the Adriatic (AD), the Atlantic (AT), the Danube (DA) and the Mediterranean (ME) lineage correspond to the major drainage basins, whereas the fifth lineage is represented by the marble trout (MA). Examination of the evolutionary relationships among the five trout lineages suggested a scenario in which the most ancient phylogeographic separation resulted from allopatric isolation in the Pleistocene between the three major European drainages, Atlantic (AT lineage), Danube (DA lineage) and Mediterranean , followed by later divergence within the Mediterranean basin linked to the existence of glacial refugia that originated the AD, MA and ME lineages [Freshwater fish species show a greater average degree of genetic differentiation among locations than marine species, often resulting from the isolation of fish populations among drainages . The marlineages . More relineages . Complexlineages -11.Salmo marmoratus [Salmo trutta marmoratus within the Salmo trutta complex [The marble trout is characterized by a particular marbled colour pattern and different biological and ecological traits such as a very large body size (up to 1 m in length and 25 kg in weight) and the complete lack of black spots on the lateral side of the body. It has a restricted native geographic distribution within the Adriatic basin, found on the alpine part of the river Po in Northern Italy, former Yugoslavia and Albania . The taxrmoratus -16 or as complex ,18.The existence of marble trout has been seriously threatened by the introduction of nonindigenous brown trout throughout its natural range by means of massive restocking practices . HybridiSalmo trutta complex, while examination of the population genetic structure of marble trout has been mostly neglected. Using allozymes, Giuffra et al. [FST = 0.66 (p < 0.001) and all pairwise FST ranging from 0.31 to 0.88 [Up to now, molecular genetic studies on marble trout have focused on species identification using mitochondrial DNA haplotypes and assessing the rate of introgression among lineages in the a et al.  found no to 0.88 .The aim of this study is to perform the most extensive genetic survey to date of marble trout populations sampled throughout its geographic distribution, including tributaries of the Po and Soca  rivers, the two drainage systems in which the species is most prominently found Figure . A totalA total of seven mtDNA haplotypes were found in our dataset Table , three oComparison of mtDNA haplotype frequencies showed highly significant differences across sampling locations (p < 0.001). The genetic differentiation is essentially due to the differential distribution of haplotypes Ma-s1 and MA-s2: haplotype Ma-s2 appears to be fixed in the most western locations , decreases progressively in frequencies moving toward eastern locations , and is replaced by haplotype MA-s1 in the two Slovenian locations . The only exception was Ticino, the second most western sample, with 22% haplotype MA-s1 individuals  using STRUCTURE, a scenario with two clusters (K = 2) corresponding to the two taxa was the most likely. When using a 10% expected brown trout introgression as threshold, introgressed individuals were found at all locations except Predelica in Slovenia Table . IntrogrAR = 1.4).Values of genetic variability at microsatellite loci are summarized in Table e = 0.080) in comparison with the rest of samples (He = 0.164-0.388). Following Bonferroni correction, only one out of 45 tests departed significantly from HWE (locus Str85 at Piave). The software MICROCHECKER showed no evidence for scoring errors due to stuttering or large alle dropout. An homozygote excess was found at locus Str85, which might be attributable to the presence of null alleles, at Adige, Isonzo and Piave, but not at the rest of samples. No linkage disequilibrium was observed between any pair of loci after Bonferroni correction.In order to estimate the native patterns of population genetic structure and gene diversity, data was re-analyzed removing all individuals with >10% expected introgression. As a consequence, Adda  and Koritnica  were excluded from the analysis. The effect of removing introgressed individuals was a expected drop in genetic diversity Table . HeterozFST values  showed that our markers have enough statistical power to detect FST values > 0.005 (p = 1.000). This suggests that our markers can provide a reasonably accurate picture of the genetic population structure of marble trout.Since our study was carried out with a limited number of microsatellite markers, statistical power to detect genetic divergence was assessed with POWSIM prior to the genetic differentiation analysis. Simulations using our empirical microsatellite data, taking into account the sample size for each population, and a wide range of predefined FST comparisons were highly significant . Similarly, pairwise genetic distances between population pairs were all highly significant , which suggests an Isolation-by-Distance pattern , which might be due to the deep genetic break between Pellice and Ticino in the Po river and Isonzo and Predelica in the Soca river. In concordance with the MDS analysis, a hierarchical AMOVA partitioned significantly among groups only when considering four groupings corresponding to Western Italy (Pellice), Central Italy , Eastern Italy  and Slovenia (Predelica) (Table FST = 0.226), a significant Isolation-by-Distance pattern , and the same sequential distribution of samples following the West-to-East distribution of the sampling locations.When investigating the genetic structure of native populations , all pairwise FST = 0.68. Similan Figure . AMOVA aFST = 0.68. SimilaNe values ranging roughly between 2000 and 9000 individuals, with the exception of Predelica, for which a mean Ne value of 858 individuals was inferred  and macro-geographic (among river systems) scale.FST values between samples using microsatellite loci, with an average pairwise FST value of 0.79 , indicative of complete absence of genetic exchanges. They suggested the remnant marble trout populations from Slovenia to be completely isolated from each other and from the main river system as immigration from downstream is precluded by impassable barriers. Furthermore, in the populations analyzed by Fumagalli et al. [He = 0.04- 0.22) were remarkably low, pointing to possible past or present bottleneck episodes. By comparison, most populations in our study showed expected heterozygosities up to 0.388 and allelic richness ranging between 2.3 and 3.4, with the only exception being Predelica , a location in the Soca river in Slovenia shared with the study of Fumagalli et al. [Ne = 858). The fact that the rest of locations in our study showed (i) moderate to high genetic diversity, (ii) no signatures of a genetic bottleneck, (iii) and an inferred effective population size over 2000 individuals in all cases suggests that marble trout population in Northern Italy are not extremely isolated and small, unlike the secluded headwater streams of the Soca river.Genetic heterogeneity was found not only between geographically distant rivers but also within the same river basin. Samples collected no more than 15 km apart in tributaries of the same river, for instance Adige and Prissiano, resulted significantly differentiated, indicative of limited genetic exchange. However, genetic differentiation in our study was much lower in comparison with the study of Fumagalli et al.  on Slovei et al. , microsai et al. . ConcordAt the macro-geographic level microsatellite data reveal a highly significant genetic differentiation consistent with a pattern of Isolation-by-Distance. A significant correlation was found between genetic and hydrographic distances that suggests a reduction in gene flow among populations directly related to the increase of waterway distances among sampling locations. According to the Isolation-by-Distance effect, positioning of samples on the horizontal axis of the Multi-Dimentional Scaling plots Figure  clearly It is generally assumed that the intra-specific genetic structure largely reflects the timing and the pattern of habitat colonisation across the entire range distribution of the species. This holds true especially for freshwater species, for which colonisation patterns have been strongly determined by the geological evolution of the hydrographic networks in the past. The Wurmian glacial period (with a maximum peak 18000 years ago) is thought to have played a fundamental role in determining habitat expansion and contraction of most of native freshwater species in the North-Italian and North-western Balkan regions . During FST values were found when including all individuals in the analysis and when including only native (non-introgressed) individuals, some introgression could remain considering that we excluded introgressed individuals using a 10% criterion. In addition to past biogeographic events, the higher introgression observed in Central Italy could contribute to the deep genetic break between Pellice and Ticino, and between Isonzo and Predelica, and the differences within river basins. In regards to the differences in genetic diversity, introgression might have also contributed to a higher native genetic diversity in Central Italy in comparison with the more extreme locations , but the fact that a similar native genetic diversity is observed in locations with high  and low introgression (Adige) suggests that introgression alone cannot account for those differences.Besides historical patterns of isolation among drainages, the high rates of introgression may have affected the native patterns of population genetic structure. While a similar East-to-West partition of the microsatellite polymorphism following an Isolation-by-Distance pattern and comparable Preserving genetic diversity within a population or species sustains its evolutionary potential to cope with current and future environmental changes. In this sense, native species are specially vulnerable to human activities including introduction of nonindigenous species. Loss of genetic integrity as a consequence of introgression of exogenous gene pools is considered the most serious thread to native gene pools. Thus, detecting hybridization and introgression in wild populations is a primary concern in developing conservation and management strategies. The risk of genetic erosion caused by introgression cannot be ignored as it obscures native gene pools through alteration of genetic composition, reduction of fitness, diminution of population sizes, loss of genetic diversity and extinction of local populations.Salmo trutta marmoratus individuals were caught using electrofishing over the period 1995-1997. Collecting sites included most of the geographic distribution of the species, ranging from North-western Italy to Slovenia  and aligned using CLUSTALW [DNA was extracted from ethanol-preserved finclips following a resin-based protocol  as described in Walsh et al. . A 352 bCLUSTALW .Salmo trutta, Str15 [Salmo salar: Ssa85 [2, 0.3 μl 10 mM forward and reverse primers, 0.04 μl Taq polymerase  and water up to 8 μl. PCR products were run on a 725 VISTRA automatic sequencer (Molecular Dynamics) and analyzed using FRAGMENTOR (Molecular Dynamics).All samples were scored for five microsatellite loci: four previously described in brown trout a, Str15 , Str85, a, Str15 ; and oner: Ssa85 . PCR pro4 iterations followed by 105 additional Markov Chain Monte Carlo (MCMC) iterations were performed as recommended by Pritchard et al. [Analysis of mtDNA data was performed using ARLEQUIN v. 3.5 , includid et al. .Ho) and expected (He) heterozygosities per locus using GENETIX version 4.05 [AR) using FSTAT [At microsatellite loci, within-sample genetic diversity statistics were assessed by observed .Prior to the population structure analysis, the statistical power of the markers employed in our study for investigating genetic differentiation was assessed with the program POWSIM . We testF-Statistics [FST/(1- FST)) vs. geographic distance (shortest waterway distance between pairs of samples).Population structure was studied using non-hierarchical and hierarchical atistics  calculatatistics . Signifiatistics . Pairwisatistics  and a muNe (lower limit 100 and upper limit 100 000), mean μ (lower limit 1 × 10-5 and upper limit 1 × 10-3) and locus μ (lower limit 1 × 10-5 and upper limit 1 × 10-2). Microsatellite repeat motif and allele range were specified for each locus. The stepwise mutation model was used and 500,000 replicate runs were performed to generate the reference tables.The software DIYABC  was usede) at polymorphic loci. Thus, in a recently bottlenecked population, the observed gene diversity is higher than the expected equilibrium gene diversity (Heq) which is computed from the observed number of alleles (k), under the assumption of a constant-size (equilibrium) population [crit estimated after 10,000 simulations and assuming the population to be at equilibrium. In all simulations, three different values of θ were used . A range of mutation models were examined and conservative values were used for ps (frequency of one-step mutations) and Δg (average size of non one-step mutations), ps = 0.90 and Δg = 3.5.Finally, we tested for a genetic bottleneck episode in two different ways. First, we used the software BOTTLENECK , based opulation . We usedpulation , which cpulation . The propulation . Second,pulation ). Hence,The authors declare that they have no competing interests.JMP analyzed the data and drafted the manuscript. ANL and MS conducted the molecular analyses, contributed to the data analysis and writing of the manuscript. TP designed and coordinated the study. All authors read and approved the final manuscript.


In recent years, a number of technological advancements started to modify the long standing appearance and functionalities of traditional optical microscopes used in Pathology and other medical fields. In fact, at present many new tools for microscopical visualization exist that are based on digital imaging, robotization, and remote communication. Such tools are typically adopted in activities ranging from education to telediagnosis to remote consultation.Present paper describes the features of a basic digital microscope that has been tested to verify its performance for occasional remote consultation inside an international project between Italy and Slovenija, funded by Interreg initiative of the European Regional Development Fund.The system is composed by a pair of digital microscopes  associated to a high resolution videoconferencing systems . The systems are connected through the Internet. Sixty histology and cytology cases have been collaboratively diagnosed between two Pathology Institutes to verify the diagnostic performance of the system, regarding the image quality point of view as well as time needed for diagnosis. The system has also been tested for compatibility with standard videoconferencing software.No discrepancies between local and remote diagnoses have been identified, with diagnosis time reasonably close to typical microscope observation times. Time needed for most operations is not far from that needed on a traditional microscope, except for startup.The system can be considered usable as a standard microscope, and also for occasional remote consultations. The traditional optical microscope is made for individual examination of samples through the eyepiece, and for this application it is very ergonomic and efficient . HoweverIn the last years, the availability of digital imaging systems drove to novel ways of sharing images for the above purposes . In factIn addition to that, digital images also gave momentum to research fields like image analysis and telepathology .For relatively long time since the availability of digital imaging systems, microscope manufacturers did not change the basic shape and functions of microscopes, concentrating instead on providing high quality photo and video cameras, viewing stations, and software. Other systems were directed to telepathology in its various meanings, i.e. for remotely exchanging images with some expert consultant, either in real time or store-and-forward way .Only recently devices that could be defined as digital microscopes started to appear, aimed at routine examination of specimens . The firAnother system of the same kind of more recent development is the digital microscope Leica DMD108 . It is a basic digital microscope with manual interaction (versus the robotized one on the Nikon Coolscope), aimed at routine work. With an additional videoconferencing module (Tandberg), it can become a tool for occasional telepathology consultations or distant teaching sessions.In the present paper we present a technological assessment exercise on the Leica DMD108 digital microscope, aimed at establishing whether the image quality provided is adequate for collaborative viewing of pathology images.Assessment has been carried out between two workstations, one in the Institute of Pathology at the University of Udine, Italy and the other in the Institute of Pathology at the Hospital of Izola, Slovenia.Each workstation was composed by a Leica DMD108  digital microscope connected by means of an hardware integration module to a high definition videoconferencing system . The videoconferencing system was in turn connected to the Internet.Aims of our assessment regarding the overall system were:- to understand whether it can be considered a substitute for a microscope,- to evaluate diagnostic quality of images, and finally- to verify the reliability of communications.For the first aim, we examined the behaviour of the digital microscope by measuring time needed for main operations and features of the embedded software.To test the system for collaborative discussion of cases, as well as to evaluate diagnostic quality a remote encounter programme was established, with week-based frequency, to examine 60 cases of interest for both institutes. We also evaluated optical and digital resolution of images on both static and dynamically delivered images.During encounters, discussion time for each case was recorded as well as discussion outcome and errors and problems occurred during the communication session (to verify communication reliability).Cases were mostly equally provided by the two institutes of Pathology involved in the study. Involved personnel was first briefly trained by three Authors of the present paper , during two encounters in the Izola and in the Udine Hospital.The DMD108 digital microscope has manual stage and focus movements like in a traditional microscope, and robotized objective revolver. In both installations, objectives included the following magnifications: 5x, 10x, 20x, 40x, 63x.Real time image acquisition and display occured at 1280x960 pixels, 24 bit depth. For the videoconference mode, images were scaled down to 1024x768, to allow real time transmission with reasonable bandwidths.Apart from looking directly, it is possible to capture still images at a resolution of 3 MPixels (2048x1536). In this case, frame position on the slide was recorded with the image, in order to be able to return to the same place . Images can be stored on a USB drive, or on some file server on the network. When connected to a network, images can also be sent through email directly from the system interface.Display is not an integral part of the microscope, so any monitor can be attached to the system. In our setup, two 19” 4:3 monitors were connected to the digital microscope, which show the same image when used locally, while in videoconference mode one is on the local microscope field, the other on the remote video. Also a keyboard and a mouse were attached to the microscope, to allow for input of textual descriptions. Figure Another feature not traditionally available with microscopes is a macro overview of the slide, obtained by manually placing the slide in a different area of the stage.Finally, it is also possible to directly trace lines, circles, comments on the image, as well as to measure lengths and areas, and also a voice recorder is available to record audio comments associated to the pictures.Both systems were connected to the Internet, with specific ports open to allow for the videoconference. The Institute of Pathology at the University of Udine is hosted in an academic environment, and the public Internet, without closed ports, was already available. To avoid use of the hospital network, the hospital of Izola adopted a radio bridge to connect to an Internet provider.An operating system provides a graphical user interface to the functions of the microscope. For example, different specimen stainings may be viewed in an optimized way by means of profiles that adapt colour curves to the specific staining to be displayed. It also provides some typical imaging services, like storage of still images on an USB drive or an external server, and delivery of images through email.We first evaluated system features regarding the quality of acquired and transmitted images. For this aim, we measured the size of fields acquired with the microscope.Table As in most digital imaging applications in pathology, image resolution matches optical resolution only at higher magnifications, which however are the most frequently used to establish a final diagnosis.Start-up time was about 1’05”. Shut-down time was 8 seconds. During every start-up it is necessary to calibrate the stage position by moving it in a corner. Time needed for any basic microscope operation  did not differ from a traditional microscope. The computer interface for digital imaging operations was not based on commonly known operating systems, but was easy to use without previous knowledge.Communications occurred flawlessly in all but one discussion session, which was not carried out because of lack of connection. An analysis of the situation recognized that the radio bridge used by the Izola Hospital to connect to Internet was suffering of problems due to bad weather.A total of 60 cases have been discussed between Udine and Izola, of which 30 cytologic samples, 27 histologic samples, 3 intraoperative samples. A pathologist and a biologist were present in Udine; a pathologist was present in Izola.Discussion lasted 1’17” on average . Only among the very first cases, duration has been more than two minutes for some cases.Outcome of discussion was an agreement on the diagnosis on 56 out of 60 cases. However, also the other discussions were concluded with an agreement: no disease in one case, need for further immunohistochemistry analyses in 2 cases, and insufficient experience on both sides for 2 other cases.The tested digital microscope attempts at substituting a regular optical microscope, while providing also basic digital imaging facilities.In our tests, the digital microscope behaviour has been very close to the one of a traditional microscope. Start-up time was of course higher than time needed to start a basic optical microscope, but not far from time needed to start a research microscope with robotized movements. Slightly more disturbing is the need for stage calibration at every start-up, but this can be avoided by leaving the system in standby mode.Most common operations  were very similar in activation modality and execution time to the same operations carried out on a traditional microscope. According to pathologists, quality of images as seen on video is good at magnifications greater than 10x, and video refresh is sufficiently quick to see any change in focus and in captured field in real time. However, no quantification has been made to objectively evaluate these subjective assessments.So, for the sake of pure microscopic examination, the present digital microscope can be considered a good substitute for a traditional microscope.However, some concern exists regarding all digital imaging applied to microscopes, and compared, the ergonomics of the traditional microscope has two advantages:• the view field is wider, due to the circular shape that cannot be digitally reproduced with an equivalent area; this may translate in more time needed for examination, like already shown for digital slides ;• the binocular vision helps in focusing attention on the slide, by making the external world invisible during examination.While the former concern cannot be really overcome, regarding the latter it is probably matter of habits and attitudes, that will change when the “videogame generation”, more used to computer screens, will be in charge of microscope examinations .On the other side, the system seemed easy to use, and only a short training period, during which part of discussion time was spent in understanding the device functioning, has been needed before reaching a good knowledge of the tool.However, for the pathologist that only needs to look at slides alone, with only occasional need for digital images, there is no strict recommendation of leaving a traditional microscope for a digital one like this, because traditional functions are performed more or less in the same way.The pathologist with need for collaboratively discussing cases  instead has different needs that can be easily fulfilled with the examined digital microscope. Current approaches to collaborative vision include the multi-headed microscope, which presents two or more eyepieces, or a video camera able to show images on a television-style monitor on the side of the microscope. The former represents the traditional way of teaching, but –except for the double eyepiece version- it is very cumbersome. The latter provides slightly sub-standard images, because resolution is often 600 lines according to television standards.The digital functions provided by the microscope are to be compared with a traditional microscope enriched by a microscope photo camera. In this comparison, any digital microscope including the studied shows some clear advantages, including:• integrated environment: no need for an external computer to be activated every time a picture is needed;• easier focusing: not always aftermarket microscope cameras are installed in a perfect parafocality, so the sample cannot be focused directly at the eyepiece. Microscope cameras almost always allow for real time viewing on a computer or on an external monitor at a resolution much lower than the final one, thus making difficult to focus at the maximum level of detail.The only microscope feature that was not functioning appropriately, at least in our device, was polarized light. It is an infrequently needed feature that however, when needed, cannot at present be reliably obtained with this microscope.Whenever a pathologist needs to routinely acquire digital images, a digital microscope may become an efficient way of accomplish the task, in particular when coupled to an institutional server to store images.Finally, we also preliminarily evaluated the digital microscope behaviour as a remote discussion tool. For this, the system was coupled with a videoconferencing system chosen by the microscope manufacturer and thus well integrated with it. To allow transmission through videoconferencing, image resolution has been reduced to 1024x768 pixels.However, no specific problems have been recognised regarding image quality. Also ease of use of the combined microscope plus videoconference system has not been an obstacle to communications, so that after the first discussion sessions there has not been necessary to provide technical support.As with other telepathology techniques , remote The test was carried out in the framework of an international project between Italy and Slovenija, funded by Interreg Interreg initiative of the European Regional Development Fund. This allowed to experiment with a heterogeneous access to the Internet, that always but in one occasion behaved consistently. It should however be noted that implementing the system inside a protected hospital network may be not so easy due to the presence of firewalls and closed ports . A closeFinally, occasional collaboration can be also done with remote users of videoconference systems, because the enclosed system is compliant with H.263 standard. Such systems could even be realised only by software, although the ones we tested were not able to negotiate the maximum resolution provided by the source videoconference system.The Leica DMD108 digital microscope has been assessed to verify its system functionality in routine telepathology work and in a collaborative case discussion scenario. The system can be considered usable as standard microscope, i.e. as a substitute for a traditional microscope, and also for occasional remote consultations.The authors declare that they have no competing interests.VDM and CAB designed the evaluation experiment; VDM wrote the first draft of the paper. NB and SS managed all technical details. DI, VS and FR did the remote collaborative diagnoses and discussed results. All authors read, revised and approved the article.


Transgenerational sources of biological variation have been at the center of evolutionary studies ever since Darwin and Wallace identified natural selection. This is because evolution can only operate on traits whose variation is transmitted, i.e. traits that are heritable. The discovery of genetic inheritance has led to a semantic shift, resulting in the tendency to consider that only genes are inherited across generations. Today, however, concepts of heredity are being broadened again to integrate the accruing evidence of non-genetic inheritance, and many evolutionary biologists are calling for the inclusion of non-genetic inheritance into an inclusive evolutionary synthesis. Here, we focus on social heredity and its role in the inheritance of behavioral traits. We discuss quantitative genetics methods that might allow us to disentangle genetic and non-genetic transmission in natural populations with known pedigrees. We then propose an experimental design based on cross-fostering among animal cultures, environments and families that has the potential to partition inherited phenotypic variation into socially  and genetically inherited components. This approach builds towards a new conceptual framework based on the use of an extended version of the animal model of quantitative genetics to integrate genetic and cultural components of behavioral inheritance. Ever since Darwin and Wallace identified natural selection, a central objective of evolutionary biology has been to evaluate the role of inheritance mechanisms on the evolution of phenotypic diversity. Darwin Linaria vulgaris) which was described more than 250 years ago by Linnaeus Periplaneta americana) to flee light and move towards darkness is so strong and invariable in nature that it suggests genetic encoding. However, a study reported that this tendency can be reversed by social influences, suggesting that its determinism is plastic and that part of this tendency can be acquired socially, opening the way to social inheritance Genetic and non-genetic inheritance are intricately interconnected and easily confounded Thus, the long prevailing assumption that the inheritance of phenotypic variation rests exclusively on genetic variation is no longer tenable Here, we focus on the vertical component of cultural inheritance, which can be used to calculate what we have termed the ‘inclusive heritability’ of a trait et al. Several methods exist that provide us with indirect tests to determine the inheritance of social information, for example the ‘Option-bias method’ Cyanistes caeruleus) and great tits (Parus major), two species with contrasting foraging niches, Slagsvold and Wiebe A method for decoupling genetic and non-genetic effects is the manipulation of progeny, such as cross-foster experiments, which entail exchanging offspring between families A promising opportunity to measure inclusive heritability and to evaluate the relative importance of genetic and non-genetic inheritance is the pedigree based “animal model” framework used in animal breeding and long-term studies of wild animal populations  environmental effects on phenotypic variation The animal model approach is a pedigree-based mixed linear model of variance that is one of the most advanced methods in quantitative genetics for studying populations with known pedigrees  as long as its variation is not confounded by genetic or environmental variation. Cross-fostering experiments should allow us to statistically test the significance of the effect of such a random variable and to estimate its effect size. As a result, the impact of cultural changes on phenotypic variation would be known. Adding the effect of an individual's birth place in an animal model makes it possible to measure the effect on phenotypic variance of the common environment shared by a family, part of which may be cultural. Although such an effect can be used to measure common brood and long-term environmental effects, it is not suitable for quantifying the extent to which the inheritance of a behavioral trait is socially mediated, i.e., to estimate cultural heritability, because cultural variation occurs among populations and not among families. In humans for instance, statistical settings based on twins are used similarly to cross-fostering experiments to estimate the heritability of traits such as cognitive capacity ij, the coefficient of coancestry between individuals i and j. Θij is the probability that one allele chosen randomly in individual i is identical by descent to an allele chosen randomly in individual j. On the basis of Θij, we can then calculate the additive genetic covariance between i and j, which is calculated by doubling Θij: ijA = 2Θij. Because Aij can be calculated for all pairs of individuals in the pedigree, it is possible to build the additive genetic relationship matrix A for the entire population. Solving an animal model consists of obtaining the additive genetic variance σ2A from the covariation between the matrix A and phenotypic variation h2 is the part of a trait's phenotypic variation that is determined by additive genetic variation: h2 =  σ2A/σ2P. The additive variance component σ2A is therefore the key parameter for the empirical estimation of heritability.The key to understanding why the animal model framework is suitable to estimate and partition inclusive heritability lies in the use of the pedigree. The pedigree of the population recapitulates the genealogy among all individuals in the population  when seeking extrapair, or to lay parasitic eggs, and return. Another problem stems from the potential inaccuracy of pedigrees because most often observational data is blind to extrapair paternity, adoption or brood parasitism. Several studies have dealt with this issue For instance, genetic and cultural pedigrees can be confounded in the process of offspring learning their first language, which is mainly transmitted by parents. Estimates of the genetic inheritance of language calculated from data on sedentary individuals would misleadingly incorporate the part of language variation that is inherited vertically, but non-genetically, by social learning. The same reasoning holds for song learning in birds, whales and dolphins Pundamilia pundamilia and P. nyererei. They concluded that female mating preferences were heritable and discussed the supposed genetic system involved in the inheritance of this behavior. However, given that parents provide care to their offspring, the experiment did not exclude the possibility of cultural transmission through sexual imprinting early in life. A second study that cross-fostered fry between the same sister species resulted in the reversal of each species' preference for its own kind Despite the fact that heritability estimates of behavioral traits may include substantial overlapping information from genetic and cultural pedigrees Sialia mexicanaSimilarly, a study of a wild population of western bluebirds  culturally inherited Accounting for epigenetic transmissibility and environmental inductions can also allow the estimation of the epigenetic component of inclusive heritability The double pedigree experimental design uses partial cross-fostering manipulations between identified behavioral groups to uncouple the genetic and cultural sources of phenotypic variation of behavioral traits . This isPartial cross-fostering uncouples sources of genetic and cultural variance by fostering half of newly born clutches, litters or broods between genetic and cultural environments . Because social influences can occur very early in development 2σP is the total phenotypic variance, 2σA is the additive genetic variance, 2σC is the additive cultural variance, 2σM is the maternal effect variance, 2σCB is the common brood effect variance, 2σENVT is the environmental variance and 2σR is the residual variance. In this model, partitioning inclusive heritability between the cultural and genetic components is possible: 2σA/2σP would estimate the narrow sense genetic heritability and 2σC/2σP the cultural component of inclusive heritability. Such experiments could be performed in animals with vocal dialects, particularly in species that are easy to manipulate, such as hole-nesting birds. This design also avoids the developmental problems resulting from social deprivation because by being fostered in another cultural environment, rather than being deprived of social stimulation, cross-fostered young should develop normal social learning capacities.Various techniques may be used to avoid confounding common environment or parental effects with the effect of culture. For instance, by using a species with multiple offspring in a single reproductive event, it may be possible to foster several sibs in different families of the same and different cultural groups. This could be performed over several generations so that the variables capturing the variability of cultures, common brood (by foster family) and parental effects  would be independent given that genetically related individuals are spread across those factors. Our purpose is to separate shared culture from genetic resemblance. As a result of the design proposed in The resulting data should be analyzed in a model incorporating both genetic and social pedigrees simultaneously. In this multivariate model, sources of phenotypic variation would thus be partitioned between their genetic and cultural components and their interaction. The genetic component is estimated by the covariance between trait variation and genetic relatedness, while the cultural component would be estimated by the covariation between trait variation and cultural relatedness as defined above. The cross-fostering experiment allows partitioning of phenotypic variation between these two sources of trait variation by uncoupling the  overlapping pedigrees.A. The information derived from the cultural pedigree (included in the model as a random variable) may take various forms: 1) the identity of the cultures between individuals that were cross-fostered, which is an individual characteristic, 2) the pairwise sum of social contacts in the form of a matrix C, and 3) the vertical component of socially transmitted information built on the basis of the transgenerational segregation of different cultures that would account for the complex nature of culture as defined by the analyst ).In this model the information derived from the genetic pedigree takes the form of the matrix of genetic relatedness The value of these models is not restricted to detecting cultural inheritance. It is also in the opportunity to compare the phenotypic variation explained by genetic relatedness, cultural transmission and the inclusive genetic and cultural determinism. This can be done by comparing the goodness of fit of those models with parameters such as the Akaike Information Criterion (AIC) for maximum likelihood tests or the Deviance Information Criterion (DIC) for a Bayesian Markov Chain Monte Carlo approach et al. C that recapitulates the information gained from experimentally manipulated cultural pedigrees in place of Slate et al.'s genomic data The approach developed by Slate G matrix, which defines how microevolutionary parameters  change over time and across environments CVC matrix’ . We will then be able to compare the role and stability of these genetic and cultural links across time and environments. For instance, language inheritance and mate choice copying experiments illustrate how such cross-foster experiments constitute a powerful tool that could be used to estimate the inclusive heritability of these traits and disentangle their cultural and genetic determinism  will become more complex with the addition of a third dimension, that of cultural relationships . Such prerminism  and 2. WEvolutionary biologists seem to periodically rediscover that, by concentrating on the genetic component of inheritance, we are missing the phenotypic, environmentally induced component of inclusive heritability. This fundamental statement was first formulated four decades ago by Anthony D. Bradshaw


Dipteryx alata Vogel) is an endemic species from the Cerrado biome, but widely distributed within the biome and abundant in several of its habitats [in natura and are source of raw material for small and middle-sized food industries [in situ and ex situ conservation efforts. Data for such analyses can be provided by new molecular markers, such as microsatellites, whereas spatial analyses and optimization procedures can be applied to analyze these data and thus improve conservation efforts. Here we investigate the genetic variability within the germplasm bank of D. alata based on microsatellite data and compared it with a large sample of 25 widely distributed natural populations of the species. We then used a simulated annealing algorithm to find the smallest number of these natural populations that should be sampled to conserve the full genetic variability of the species.The “Baru” tree , mean number of alleles per loci (A), expected heterozigosity under H-W equilibrium (He) and observed heterosigozity (Ho). Population structure and divergence among natural populations were analyzed using A simulated annealing algorithm, implemented in the software SITES  was usedD. alata for the microsatellite loci analyzed here was low compared with other forest species in the same region . Out ofThe simulated annealing revealed that only 4 local populations, located in Mato Grosso and Mato Grosso do Sul , there will be genetic erosion reducing total genetic variability in this species. So, an effort to sample and grow up individuals or progenies from the 4 populations, preferentially coupled with in situ conservation programs in these natural populations from Mato Grosso region, must be urgently done. Our optimization analyses reveals that only a few samples are necessary to achieve a more complete representation for the species, and this is important to reduce sampling and maintenance costs without losing efficiency in terms of establishing conservation and future breeding programs for the species.