The known plant viruses mostly infect angiosperm hosts and have RNA or small DNA genomes. The only other lineage of green plants with a relatively well-studied virome, unicellular chlorophyte algae, is mostly infected by viruses with large DNA genomes. Thus RNA viruses and small DNA viruses seem to completely displace large DNA virus genomes in late branching angiosperms. To understand better the expansion of RNA viruses in the taxonomic span between algae and angiosperms, we analyzed the transcriptomes of 66 non-angiosperm plants characterized by the 1000 Plants Genomes Project.Marchantiophyta), hornworts (Anthocerotophyta), lycophytes, a horsetail Equisetum, and gymnosperms. Polymerase genes in algae were most closely related to homologs from double-stranded RNA viruses leading latent or persistent lifestyles. Land plants, in addition, contained polymerases close to the homologs from single-stranded RNA viruses of angiosperms, capable of productive infection and systemic spread. For several polymerases, a cognate capsid protein was found in the same library. Another virus hallmark gene family, encoding the 30 K movement proteins, was found in lycophytes and monilophytes but not in mosses or algae.We found homologs of virus RNA-dependent RNA polymerases in 28 non-angiosperm plant species, including algae, mosses, liverworts  contains supplementary material, which is available to authorized users. The study of plant viruses since the early days has been dominated by the analysis of pathogens infecting crop plants. Isolation, characterization, and genome sequencing of plant viruses was later expanded to those viruses that have weeds or wild relatives of cultivated plants as their hosts. Viruses and virus-like replicons in non-agricultural habitats were in the meantime being discovered serendipitously, for example when symptoms suggestive of an infection were observed in nature, but it was only recently that technological developments – large-scale sequencing of cloned DNA at first, and next-generation sequencing in the last decade – allowed virologists to screen more systematically for the presence of known and novel viruses in plant populations of choice .These efforts have provided us with a relatively good understanding of the general landscape of genomic diversity in viruses of angiosperm plants. This landscape appears to be completely devoid of viruses with large and/or linear DNA genomes, but includes DNA viruses with small, circular, single-stranded genomes; pararetroviruses that encapsidate double-stranded DNA and express their genome through an RNA intermediate; and a large variety of viruses with RNA genomes, comprising viruses whose virions encapsidate positive (+) or negative (–) sense single-stranded RNAs (ssRNA), double-stranded RNAs (dsRNA), or ambisense RNA. Sequence similarity analysis and phylogenetic studies have established profound, ancient relationships between each of these virus groups with viruses that infect fungi and animals; more recently, such studies have drawn the evolutionary links between these virus lineages and viruses infecting unicellular eukaryotes, bacteria, and archaea .Chlorophyta 16] and iIn this work, we used computational sequence analysis to detect several virus hallmark genes  in a colViral RNA-dependent RNA polymerases  are a distinct group within the superfamily of nucleotidyltransferases with the catalytic right-hand palm domain. This superfamily includes several other kinds of DNA and RNA polymerases of viral and cellular origins, as well as more distantly related nucleotidyltransferases and nucleotidyl cyclases . Viral RVirus-like RdRP-encoding genes are not found in the DNA genomes of cellular organisms. The closest cellular homologs of RdRPs are the right-hand RNA-dependent DNA polymerases, or reverse transcriptases  involved in propagation of retroviruses and retroelements, as well as their domesticated derivatives, such as telomerase and RNA splicing factor Prp8 in eukaryotes. Notwithstanding the sister relationship between RdRPs and RTs, these two groups of replication enzymes are clearly distinguishable in sequence similarity searches and with phylogenetic inference. Furthermore, viral RdRPs are not related to the cellular RdRPs involved in RNA interference, which possess a distinct configuration of conserved sequence elements and adopt a different double-psi barrel fold . All thiChlamydomonas acidophila and the streptophyte Penium margaritaceum. The combined results of these searches are represented in Table We selected RdRP amino acid sequences representing every RNA virus family recognized by the International Committee for the Taxonomy of Viruses (ICTV), as well as the majority of the unclassified RNA viruses that are included in the ICTV database. These 80 protein queries were used to interrogate the joint nucleotide database of 66 green plant transcriptomes with the TBLASTN routine of the BLAST suite of programs . The setUlvophyceae Ulvophyceae, where they have been noticed before [Chlorophyceae and the prasinophyte Pyramimonas parkeae. Viruses were also found in streptophyte algae, including the first sightings in the representatives of classes Coleochaetophyceae and Zygnemophyceae. Among the land plants, RNA virus-like replicons are seen in two hornwort and four liverwort species, and also in several true mosses, from Sphagnopsida (Sphagnum), to more derived Bryopsida . Furthermore, virus RNA polymerase-encoding replicons are seen in three species of lycophytes, in a horsetail Equisetum, and in several gymnosperms. All these putative viral RdRPs are novel – their sequences are not found in the nucleotide or protein databases at NCBI, except for the three ESTs mentioned above.In total, RdRP homologs were discovered in the transcriptome libraries from 28 species, spanning the vast “virus desert” between green algae and angiosperms. In particular, RNA virus-like replicons are found in several classes of the chlorophyte algae – not only d before , 25, butThe lengths of the sequence fragments varied from 31 to 875 amino acid positions alignable with the closest database homolog (median 133), and the range of pairwise similarity to the nearest database homolog was from 19 % to 81 % (median 42 %). Despite this variation, all matches reported in Table We collected all cDNA fragments that showed statistically significant matches to virus RdRP protein sequences, and for each such fragment recorded the identity of its best match in the reverse BLASTX scan of the protein non-redundant (NR) database at NCBI. Out of 28 species in which we detected matches to any viral RdRP, 19 libraries encoded an RdRP most similar to a homolog from a virus with a dsRNA genome, five had virus replicons most similar to (–)ssRNA viruses, and 10 libraries contained encoded RdRP most closely related to a (+)ssRNA virus homolog Table .E-values for the best matches against true viral sequences, although wide (range 0 to 4.83 · 10−1) was highly skewed towards a low value, with a median of 1.8 · 10−33. In contrast, the median of the distribution of E-values for the best match from a plant was 32 orders of magnitude higher, that is, 3.79 · 10−1 (range 1.08 · 10−61 to 9.61). Interestingly, all matches with E-values < 10−10 in this second search against “plant proteins only” corresponded either to proteins already classified as virus-like RdRPs  or to unannotated proteins clearly related more closely to virus RdRPs than to proteins encoded by plant genomes. Moreover, such “plant” proteins typically came from cDNA sequencing projects, often explicitly targeted to recover virus RNA sequences, and did not appear to be represented in the genomic databases for the plant species they have been assigned to, suggesting that the vast majority of them are virus proteins annotated as host proteins. A paired-samples t test showed a highly significant difference between the two distributions of similarities (P = 5.7 · 10−6), and the statistic became even more extreme if all such putative RdRPs were removed from the list.To further validate the virus origin of these contigs, we performed several tests. In the first test, we asked whether the similarity in a BLASTX search with default parameters of these 94 contigs against true viral sequences  was significantly higher than the similarity to the best match annotated as non-viral plant sequences . The distribution of B. cinicola), and the remaining five corresponded to uncharacterized proteins. Even allowing that all these six proteins are not confirmed to be of viral origin, the distribution of top matches is clearly dominated by viral sequences .In another test, we used the 94 contigs to interrogate the entire NCBI nucleotide NT database in a TBLASTX search (with default parameters), and recorded the identity of the best matches for all the contigs. In 88 cases, the top match corresponded to a bona fide viral sequence, one case corresponded to a likely misannotated virus RdRP . Therefore, despite the partial character of the matches, it seems unlikely that these RdRP-related sequences reside on aberrant transcripts from the integrated and pseudogenized DNA copies of virus genomes. Such fragments of integrated cDNA copies of non-retroid RNA viruses in any case may be rare in plants, as has been documented, for example, for grape genome . Thus, iPartitiviridae, a large family that includes viruses infecting plants, fungi, and protozoa. Partitiviruses typically possess two dsRNA genome segments, one encoding the RdRP and another encoding the capsid protein. Partitiviruses are transmitted by cell division, via plant seeds or pollen, fungal spores, or by hyphae anastomoses, but appear to lack the capacity to move from cell to cell via plasmodesmata, or by vasculature in plants. The vast majority of partitivirus infections are asymptomatic [As seen from Table ptomatic .A taxonomy revision of partitiviruses, delineating four genera, has been proposed recently by the ICTV study group . At a loIn order to place more precisely at least some of the partial RdRP sequences detected in our study onto the phylogeny of dsRNA viruses, we inferred a phylogenetic tree, utilizing the partitivirus RdRP dataset from Nibert et al.  and a muWe used the maximum-likelihood phylogenetic inference approach, implemented on the PhyML algorithm , to infeEquisetum, which appear to be a basal cluster close to Clade 2 ssRNA, and a (–)ssRNA virus . Phylogenetic inference was performed on the alignment of 219 sequences that contained 259 sites RNA genomes, were too short to provide clear phylogenetic assignment, though partial sequences from the green moss Rhynchostegium serrulatum almost certainly belong to the same novel clade as its close homologs from H. ciliata ssRNA genomes in arthropods has been completed recently, which revealed a substantial biodiversity, including at least one new family-level clade . We tookGnetum montanum, which contained a nearly complete RdRP domain, to genus Marafivirus  ssRNA viruses: partial sequences were found in different clades of land plants, with the identity of the nearest database matches suggesting a variety of virus families and genera. We were able to assign a partial sequence from a gymnosperm es) Fig. .Fig. 3MaGnetum montanum GTHK-0070069, also contained a nearly-complete open reading frame in the same strand, encoding a homolog of the marafivirus capsid protein. Prompted by this finding, we asked whether any of the libraries had sequences encoding for virus capsid proteins, another class of virus hallmark genes possessing spatial folds that are rare in cellular proteins [Nothoceros vincentianus, capsids of unassigned partitiviruses in the lycophyte Huperzia squarrosa and the gymnosperm Prumnopitys andina, and an ophiovirus-like capsid protein in the lycophyte Pseudolycopodiella caroliniana ssRNA capillovirus and of a (–)ssRNA ophiovirus. This extends the host range of the 30 K protein-encoding plant viruses to lycopods.Another class of plant virus hallmark proteins are movement proteins of the 30 K superfamily . This sundorffii . In thisPyramimonas, Penium, Cylindrocystis, and Chaetosphaeridium, whereas in land plants all statistically significant matches appear to belong to transcripts that are paralogous, but not orthologous, to NCLDV genes, as judged by reverse searches (data not shown). An earlier report of NCLDV DNA fragments integrated into the genome of the green moss Physcomitrella has concluded that similar fragments also were present in another related species of moss but were not transcribed in either of the two hosts [S. moellendorffii [Wickerhamomyces ciferrii than to the NCLDV homologs ,Marchantiophyta), in lycophytes, in a horsetail Equisetum, and in a broader range of green algae than before. Some of the live plants used as the source of RNA in the 1KP effort are likely not to be axenic, so it is possible that some of the virus replicons seen in our study in fact come not from the plant cells, but from tightly associated fungi or other eukaryotes that may be a part of the plant holobiont. This possibility requires further examination.In this study, we have provided the evidence of diverse RNA plant virus-like replicons in non-angiosperm land plants and in their algal relatives, that is, throughout the plant kingdom. The sequence libraries analyzed in our work have not been targeted towards selective recovery of virus RNA; the sampling of plant species by the 1KP project was phylogenetically broad but not particularly dense, likely affecting the accuracy of tree reconstruction; virus genes with low similarity to the hallmark sequences were most likely missed in our searches; and most matches to virus genes that we detected were partial and resided on short cDNAs. Despite these concerns, and notwithstanding that the fern libraries drew a near-blank, we identified – as far as we know, for the first time – sequences homologous to various hallmark genes of RNA viruses in true mosses, hornworts Various dsRNA viruses, all of which belong to the picorna-like tribe, have been found in four out of five supergroups of protists; it has been suggested that they evolved from their ancestors with ssRNA genomes in the “Big Bang” of picornavirus radiation in primitive eukaryotes . DespiteThe common ancestor of mosses and vascular plants may have existed about 475 MYA, in the Ordovician . InteresBazzania trilobata, which is likely to carry a 3′ tRNA-like structure instead of the polyA, is one exception from this trend. Interestingly, the same trend appeared in a pilot bioinformatics study of virus-derived small RNAs [Yet another bias in the virus sequences shown in Table all RNAs , which uall RNAs . Thus, tOur analysis also revealed the existence of virus movement proteins from the 30 K superfamily in a lycopod virome. In contrast, RNA libraries from true mosses, hornworts, and liverworts showed a wide variety of RdRP gene products but did not contain their cognate, or indeed any, 30 K-superfamily proteins. Further analysis of RNA viromes from mosses and their relatives will show whether the 30 K proteins have established themselves throughout the plant viruses, or only among those viruses that infect hosts with the dominant sporophyte phase.We found that colonization of land and subsequent growth in anatomical complexity in plants coincided with the acquisition or expansion of novel sets of RNA viruses with different strategies of infection and reproduction. Evidence of various hallmark genes of RNA viruses have been found in mosses, hornworts, liverworts, lycophytes, horsetail, and in a broader range of green algae than before. Analysis of the phylogenetic affinities of the putative dsRNA and ssRNA replicons suggests that viruses associated with the evolutionarily more ancient groups, such as chlorophyte and streptophyte algae, tend to form deeper clades in the phylogenetic trees. Conversely, RNA viruses from land plants more often fall within the families or genera that have already been established by analysis of viruses infecting angiosperms and fungi.Sequence database searches were mostly performed in July 2015. The 80 protein queries were RdRP sequences representing every RNA virus family recognized by ICTV, as well as the majority of the RNA viruses that are included in the ICTV database but unclassified  were collected and verified by reverse BLASTP and BLASTX searches against the NCBI NR database. The protein alignments produced by reverse BLAST were inspected for the presence of conserved sequence motifs. In these reverse searches, > 91 % of the selected transcriptome library sequences showed statistically significant matches to the homologs from viral RdRP. The remaining sequences were either closely similar to an unrelated database protein sequence or without homologs in the database; those were not examined further. One sequence from the Penium margaritaceum library (scaffold AEKF-0068309) was identical to a fragment of RdRP of Tobacco vein banding mosaic virus, a potyvirus, at both the nucleotide and amino acid levels; this is the only instance of a likely library contamination that we saw in this study, and it was also removed from further analysis.All moderately significant matches to RdRP proteins in the 66-species subset of the 1KP transcriptome database (BLAST Sonchus yellow net virus to build a profile Hidden Markov Model (profile-HMM) and to interrogate the profile-HMMs generated from the CDD; the automatically generated, highly scoring alignment between rhabdovirus and bunyavirus RdRP models was used as a guide to trim both ends of the long alignments of RdRP-containing multidomain proteins of mononegavirales and bunyavirales, and the two alignments were aligned together using the -profile option of the MUSCLE program. Orthomyxovirus replication enzymes were excluded from the analysis because they were much more distantly related to the plant virus sequences than the other virus clades (data not shown). The new sequences described in this study were first aligned to the protein family to which they showed the highest similarity in BLAST searches, and this information was used to remove the distal portions of the master alignment not shared with the partial sequences from the transcriptome library. The alignment of the marafivirus RdRP domain was also obtained using the MUSCLE program and checked against the alignment of multidomain replicases  that was submitted to ICTV in 2006 as part of the proposal that led to establishing the order Tymovirales in 2009.For phylogenetic inference, the partitivirus dataset from Nibert et al.  was downPhylogenetic inference was done using the PhyML server  with a LSelaginella moellendorffii gene product, the 298-amino-acid EFJ10074, which includes the complete sequence of EFJ26844 with only three mismatches. The closest 250 BLASTP matches to EFJ10074 were collected, and each group of sequences from one genus that had multiple homologs with more than 95 % identity in the same genus was replaced by one representative sequence; this mostly concerned yeast Candida and angiosperms from the core eudicots. The alignment and tree were constructed as described above.To reanalyze putative NCLDV sequences thought to be integrated in the lycopod genome, we could not use the data from t he original study , which


TotheEditor—In this issue of the Journal and in a recent study, Pawar et al reported results of serologic tests performed on a high-risk group of 446 poultry workers and 162 individuals from the general population in Maharashtra and Jamshedpur states, India [P < .0005, Pillai 0.1256), with the H9 titer being the highest. However, when we investigate other birth year cutoffs (±20 years), we find the same trend, suggesting that this effect is more related to an increase in age rather than to a specific exposure event.As noted by the authors, H9 titers were highest among all antibodies to avian influenza virus strains in our general population sample . Other pP < .001), but this curve provided no additional qualitative explanations of the data than the second best fit model, a simple quadratic regression , antibody titers to avian influenza A virus antigens increased with age. The optimal fitted regression curve among nonlinear models was a fifth-order polynomial .Figure 2 = 89.9; P < .001). With a more conservative (and probably more appropriate) titer cutoff of 1:80, this percentage is 41% , suggesting that age distribution needs to be carefully taken into account when designing seroepidemiologic studies of avian influenza virus in humans. This result is robust for site effects in Vietnam, strengthening the hypothesis that this phenomenon is not related to poultry exposure.As noted in our article, the microarray assay used in this analysis is more sensitive than traditional HI and MN tests when comparing titers for the homologous antigens. A problem in doing studies to evaluate zoonotic exposure is that the exact antigenic composition of the infecting virus may be unknown. Therefore, HI/MN tests may yield false-negative results, a problem that is less evident with the microarray, which measures antibodies to the head of hemagglutinin and is, therefore, more broadly reactive within subtype. The differences we see in response between H9 and H5 avian strains (and to a lesser extent between H7 and H5 strains) are robust even when a much higher titer cutoff is used . With a cutoff titer of 1:20, 76% of individuals aged ≥50 years test positive for H9 antibodies . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.


Visual objects are effortlessly recognized from their outlines, largely irrespective of viewpoint. Previous studies have drawn different conclusions regarding the importance to shape recognition of specific shape features such as convexities and concavities. However, most studies employed familiar objects, or shapes without curves, and did not measure shape recognition across changes in scale and position. We present a novel set of random shapes with well-defined convexities, concavities and inflections (intermediate points), segmented to isolate each feature type. Observers matched the segmented reference shapes to one of two subsequently presented whole-contour shapes (target or distractor) that were re-scaled and re-positioned. For very short segment lengths, performance was significantly higher for convexities than for concavities or intermediate points and for convexities remained constant with increasing segment length. For concavities and intermediate points, performance improved with increasing segment length, reaching convexity performance only for long segments. No significant differences between concavities and intermediates were found. These results show for the first time that closed curvilinear shapes are encoded using the positions of convexities, rather than concavities or intermediate regions. A shape-template model with no free parameters gave an excellent account of the data. The visual system is exposed daily to a vast array of shapes and objects, both natural and artificial. Object recognition, categorization and classification, across a wide range of circumstances, is effortlessThere is a long history of theoretical and experimental investigations concerning the importance of specific shape features in shape recognition and object perception. Alhazen (965–1040 CE) suggested that points of convexities and concavities are crucial for the perception of shapes ; convexities and concavities are the parts that curve outwards and inwards with respect to a reference point, such as the center of the object. Over 60 years ago, Attneave (1954) emphasized the significant role of regions of high curvature in shape perception and argued that:“…information is concentrated along contours at those points on a contour at which its direction changes most rapidly…”and he additionally proposed:“Common objects may be represented with great economy, and fairly striking fidelity, by copying the points at which their contours change direction maximally, and then connecting these points appropriately with a straightedge.”et al. (2001). In their study, subjects were presented with silhouettes of smoothly curved sweet potatoes and were tasked to position ten points along each shape’s outline which they considered to be the most informative. Results showed that subjects chose regions of high curvature of both signs, i.e., convexities and concavitiesTo underpin his hypothesis he took an image of a sleeping cat, extracted the points of maximum curvature (convexities and concavities) and connected them with straight lines, thus omitting intermediate shape information. Despite the absence of this intermediate contour information, the sleeping cat was still recognizableet al. (2003) demonstrated that sensitivity is impaired when small gaps are introduced at points along the contour . Occluding points of maximum convex curvature led to the highest threshold elevations, suggesting that radial frequency (RF) patterns are detected using points of local maximum curvatureet al. (2010) employed a compound adaptation paradigm21et al. (2010) concluded that all parts of an RF contribute equally to shape coding.Other studies have drawn different conclusions with regards to the importance of convexities, concavities and intermediate points. Kennedy and Domander 1985) used fragmented familiar shapes  and showed that the areas of least curvature  are the most important for recognition, questioning Attneave’s earlier proposal. In contrast, Biederman (1987) removed zones of maximum curvature (vertices) or intermediate points (midsegments) from images of familiar objects and showed that removing the convex regions led to a larger impairment than removing either the concave or intermediate regions, suggesting a predominant role for convexity. Results on the detection of changes in shapes5 used fr91213et al. 03 demonsSome investigators have also tried to illuminate the topic from a theoretical point of view. Formalized on the basis of Shannon’s mathematical definition of information24In summary, previous results on the role of specific shape features are inconclusive and appear to be dependent on both the task and the stimulus design  showed that only for compound shape 1–2–5 was performance significantly different between segment length 1 and 7 (p < 0.01) and segment length 2 and 7 (p < 0.001). This analysis shows that performance is largely independent of segment length. The average percent correct responses across all segment lengths was: 2–3–4: 81.6% (SEM ± 4.0%); 1–2–5: 80.3% (SEM ± 2.2%); 2–3–6: 88.6% (SEM ± 4.0%) and 3–5–8: 82.6% (SEM ± 2.1%).shapes and segment length was conducted. There was no significant effect of shape  or segment length . Additionally no significant interaction was found . In summary, the ability to recognize the shape from convexities is independent of segment length and type of compound shape.To determine whether performance for convexities was dependent on the type of compound shape, a two-way repeated measures ANOVA with factors In contrast to convexities, performance for concavities and intermediate segments improves gradually from close-to-chance for the shortest segment lengths to that of convexities at the longest segment length. The results for the shortest, dot-sized segments for all tested shapes and shape features are given in ANOVA with factors of shape feature (concavity & intermediate), shapes  and segment length (7 different lengths). This analysis revealed no significant differences between concavities and intermediates  and shapes . However, performance increases significantly with increasing segment length . No significant interactions were found between factors .As the Table shows, for the dot-sized segment lengths, performance is on average around chance level for concavities (58.1 ± 2.8%) and intermediates (57.7% ± 2.5%) but much higher for convexities (80.7 ± 1.9%). As et al. (2012) conducted a series of experiments on symmetry perception and suggested that an advantage for convexities in symmetry detection may be caused by the observers’ tendency to attend to convexitiesBertamini In summary, performance for concavities and intermediate segments increases with increasing segment length. No significant differences exist between concavities and intermediates. Performance is also independent of shape type.turning angle function, a function designed to compare two polygonsθTem, θTgt and θDis, which refer to the angles subtended by the gradients for the template shape, target shape and distractor shape respectively, were calculated as follows:We propose a model with no free parameters in which the visual system creates a ‘shape primitive’ in the form of a polygon formed by combining the endpoints of the segments in the reference shape. The polygon constitutes a template shape that is compared to both the whole-contour target and distractor shapes. The decision rule is to select the shape, either target or distractor, most similar to the template. The model is illustrated in λ of the pie-wedge sector was set to 5°, which resulted in 72 values around the circumference of each shape (360°/5° = 72). θ) between the template shape (Tem) and whole-contour target shape (Tgt) and distractor shape (Dis) (∆θTem vs. Tgt & ∆θTem vs. Dis).The central angle This procedure was simulated for 1500 randomly generated reference shapes and the corresponding target and distractor shapes, for each compound shape, i.e. RF combination  and for each of the seven different segments lengths placed at convexities, concavities and intermediates. The Min Rule was applied; that is a smaller outcome translates to a closer similarity between the template and either distractor or target shape. This procedure was utilized for each segment length. The results from the simulations are percent correct of the model observer. The results were averaged across compound shapes and are illustrated by the solid lines in R2 for convexities (R2 = 0.89), concavities (R2 = 0.91) and intermediates (R2 = 0.92), demonstrating very good performance of the model. It is worth reiterating that the proposed model has no free parameters.The shaded areas represent ± SEM of the data averaged across compound shapes. The goodness of fit between the model and the data was evaluated by calculating the coefficient of determination Our results show that for dot-sized segment lengths performance is significantly better for convexities than for either concavities or intermediate points. For convexities, performance remains constant as a function of segment length, for concavities and intermediate segments performance increases with increasing segment length. These results suggest that points of local curvature convexities are the critical features for recognizing the class of shapes tested here. This is in agreement with Biederman (1987), who demonstrated that the ability to recognize familiar shapes is significantly impaired when convex features (vertices) were removed. Our results add to the existing literature on convexities by showing that the predominant role of convexities in shape recognition also applies to non-familiar complex shapes. Furthermore, for the specific shapes used here, the ability to recognize the shape from convexities is independent of segment length or compound shape type and is scale-invariant.Dot-sized points at the position of convex curvature maxima are sufficient to extract crucial information for the observer to match it to a whole-contour shape. It is a surprising and novel result that performance is independent of the complexity of the shape, as performance is similar for smoothly curved shapes possessing a low number of curvature maxima and minima (2–3–4) and shapes with many, salient curvatures (e.g. 3–5–8). Our results suggest that the information contained in between convexities is largely redundant. Performance for concavities and intermediate points on the other hand is around chance level for dot-sized segments, improves gradually with increasing segment length and reaches the same level as convexities for longer segment lengths. No significant differences were found between concavities and intermediates.Independent of whether from a mathematical point of view more information is contained in curvature convexities or concavities, which remains a controversial issue242526272829We speculate that the human visual system has developed a strategy that uses convex curvature maxima to encode whole shapes instead of using other features, i.e. concavities and intermediate points. Given that we found for our stimuli that only dot-sized points positioned at convexities were required to recognize a shape, we further propose a novel and simple model of whole-shape recognition in which the visual system creates polygon-like template shapes, by joining the endpoints of the convexities. This is analogous to a rubber band stretched around these points, i.e., forming a convex hull.Any conceivable closed shape must contain the same number or more convexities than other shape features, such as concavities. Moreover, convexities always represent the outer boundary, i.e. the hull, of closed shapes. That is, if one connects convex curvature maxima in any closed shape, the resulting polygon envelops most of the shape and especially its concavities and intermediate regions. However, polygons created by connecting either concavities or intermediate points do not do this, as convexities tend to extend beyond the polygon’s confines.Many models of object recognition3132333537394042et al. (2011) showed that neurons in extrastriate area V4 are selectively responsive to stimuli with acute high curvatures. In addition, they analyzed images of familiar objects from the HEMERA Photo-object database (Hemera Technologies Inc.) with respect to the distribution of curvatures and showed that smooth low curvatures are much more common than acute curvatures (an order of magnitude). They suggest that the sparse coding of objects is reflected in the bias of V4 neurons towards less frequent object features, i.e., acute curvature maxima, and a more pronounced representation of uncommon, but salient (diagnostic) shape features, which contain most information about object identity. The authors suggest that sparse coding for uncommon, yet informative or diagnostic features could be a general encoding strategy of high-level visual processing4445Carlson et al.’s (2011) hypothesis that shapes might be encoded from specific salient shape features. Our experimental and modeling results suggest that convex curvatures in particular, rather than concavities and intermediate regions, are utilized to perform this recognition task. The information from shape features other than convexities seems to be less important for the recognition task employed here. However, these features might become more important for other types of stimuli or perceptual tasks. Otherwise, the visual system would not be able to distinguish shapes that only differ in concavities or intermediate regions.The superior and shape-independent performance for convex shape features observed here is supportive of Carlson The kind of segmented stimuli used here are reminiscent of partly occluded shapes. Previous studies on curve completion (interpolation) Three non-experienced observers who were also naïve as to the purpose of the study participated in this study. All had normal or corrected-to-normal visual acuity. Informed consent was obtained from each observer. All experiments were approved by the McGill University Ethics committee and were conducted in accordance with the original Declaration of Helsinki.2), under the control of an Apple Mac Pro (3.33 GHz). Observers viewed the stimuli at a distance of 120 cm. At this viewing distance one pixel subtended 0.018˚ of visual angle. Experiments were performed in a dimly illuminated room. Routines from the Psychophysics Toolbox were employed to present the stimuli48The shapes were generated using MATLAB  and presented on a gamma-corrected Iiyama Vision Master Pro 513 CRT monitor running with a resolution of 1024 × 768 pixels and a frame rate of 85 Hz  patterns. Combinations of different RF patterns have been employed previously to describe complex shapes, such as human heads5051r (radius) and θ refer to the polar coordinates of the contour and rmean the radius of the modulated circle, which was set to 100 Pixels . The modulation amplitude A was set to 0.1. The variables ω1, ω2 and ω3 define the radial frequencies and φ1, φ2 and φ2 the phases (orientations) of each RF component, respectively. Four different RF combinations  were tested with frequencies of: 2–3–4, 1–2–5, 2–3–6 and 3–5–8. We refer to these as compound shapes. The highest radial frequency (ω) determines the maximum number of cycles in the compound shape. The phase (φ) of each RF component was randomly varied on each trial, such that observers never saw the same shape twice. Thus observers were presented with a wide range of shapes, varying from shapes with smooth curved boundaries  to shapes with very salient convexities and concavities . The shapes were black on a mid-grey background and had a width of 4 pixels .Where In order to vary the segment length of the feature of interest, the maximum and minimum radii along the circumference of the shape were first determined to find the points of convexities and concavities, respectively. Intermediate points were defined as the angular midpoint between these convexities and concavities. Hence, the shapes contain twice as many intermediates as convexities and concavities. In order to allow a valid comparison, we presented only every second intermediate segment on a given trial. The position of these intermediate segments was randomly changed each trial (see The noise-shapes (distractor) were composed of the same RF components as the segmented reference shapes, but with different pseudo-randomly determined phases. Example stimuli are shown in Using a Match-to-Sample task, observers were asked to match a segmented reference shape to one of two whole-contour shapes; a distractor shape and a target-shape. The segmented reference shape was presented in the center of the screen. The distractor and target-shapes were presented simultaneously and their locations were randomly determined on a trial-by-trial basis (left or right side of the display). A vertical and a horizontal jitter (±60 pixels) was also applied. Furthermore, the target and distractor shapes were each scaled in size by a random amount in the range: −1/2 to +1/4 relative to the reference shape. This scaling minimized the possibility of observers simply judging the distance between shape features but was presumed not to impair performance significantly due to the scale-invariant nature of shape recognition (see Introduction). An experimental trial consisted of a 400 ms presentation of the segmented reference shape followed by a 300 ms blank interval (mid-grey blank screen), followed by a 400 ms presentation of the two whole-contour stimuli. A blank screen followed, during which observers were free to make their choice - choose the whole-contour shape that matched the segmented reference shape – and register their response by choosing left or right on a numeric keypad. The key press initiated the next trial. Auditory feedback was provided for incorrect responses. The experimental paradigm is illustrated in A blocked design was employed. Each combination of compound shape  and shape feature  was tested in a separate block. The seven different segment lengths were each tested 30 times, resulting in a total number of 210 trials per block. Subjects typically repeated each block three times.How to cite this article: Schmidtmann, G. et al. Shape recognition: convexities, concavities and things in between. Sci. Rep. 5, 17142; doi: 10.1038/srep17142 (2015).


Phocoena phocoena), despite being widespread in temperate coastal waters of the Northern Hemisphere. Therefore, we examined harbor porpoises that stranded alive in the Netherlands, Belgium, and Germany between 2000 and 2014 for herpesvirus infection and associated disease. Porpoises that died or had to be euthanized were autopsied, and samples were collected for virological and pathological analyses. We found one known herpesvirus —a gammaherpesvirus—and two novel herpesviruses (PPHV-2 and PPHV-3)—both alphaherpesviruses—in these porpoises. A genital plaque, in which PPHV-1 was detected, occurred in 1% (1/117) of porpoises. The plaque was characterized by epithelial hyperplasia and intranuclear inclusion bodies that contained herpesvirus-like particles, and that stained positive by a PPHV-1-specific in situ hybridization test. PPHV-2 occurred in the brain of 2% (1/74) of porpoises. This infection was associated with lymphocytic encephalitis, characterized by neuronal necrosis and intranuclear inclusion bodies containing herpesvirus-like particles. PPHV-3 had a prevalence of 5% (4/74) in brain tissue, 5% (2/43) in blowhole swabs, and 2% (1/43) in genital swabs, but was not associated with disease. Phylogenetically, PPHV-1 was identical to a previously reported herpesvirus from a harbor porpoise, PPHV-2 showed closest identity with two herpesviruses from dolphins, and PPHV-3 showed closest identity with a cervid herpesvirus. In conclusion, harbor porpoises may be infected with at least three different herpesviruses, one of which can cause clinically severe neurological disease.Herpesvirus infection causes disease of variable severity in many species, including cetaceans. However, little is known about herpesvirus infection in harbor porpoises ( Phocoena phocoena)  vs 2% [1/74]) and genital slit (2% [1/43] vs 0% [0/43]), but is not associated with disease. PPHV-1 was only found in association with genital plaque, which had a prevalence of 1% (1/117) in live-stranded harbor porpoises. These results indicate that herpesvirus infections in harbor porpoises are more common than previously known and may be associated with severe, potentially fatal disease.As part of a long-term study of morbidity and mortality factors of live-stranded harbor porpoises in the Netherlands, we here describe two cases of herpesvirus infection: a gammaherpesvirus  and subsequently released into the wild. Since 2004, this activity has been organized in the form of an independent foundation, SOS Dolfijn, which operates at the same site. SOS Dolfijn has two 50 mBetween 2009 and 2014, the genital slits, blowholes and oral cavities of rescued harbor porpoises were sampled upon admission by use of a dry cotton swab for virological diagnosis. Swabs were stored in virus transport medium .Admission and rehabilitation of live-stranded harbor porpoises at SOS Dolfijn was authorized by the government of the Netherlands (application number FF/75/2012/036). Samples used in the present study were collected from harbor porpoises for diagnostic purposes by qualified personnel of the SOS Dolphin Foundation under veterinary supervision. SOS Dolfijn provided permission to the Department of Viroscience, Erasmus Medical Center to use the samples for the present study. No samples were collected from animals for research purposes.In recent years, survival of rehabilitated harbor porpoises at SOS Dolfijn has been close to 50%. Since 2000, those harbor porpoises that die or have to be euthanized, based on poor prognosis, have been autopsied at the Department of Viroscience  as part of a long-term program to understand morbidity and mortality factors of the North Sea harbor porpoise population.Autopsies were performed according to a standard protocol . The folFormalin-fixed, paraffin-embedded samples of brain tissue and genital mucosa were deparaffinized and embedded in epoxy resin. Thin sections were prepared, stained with 6% saturated uranyl acetate and lead citrate, and examined with a Philips Morgagni 268D electron microscope .For detection of gammaherpesvirus RNA, 5-μm-thick tissue sections were stained with a commercial in situ hybridization (ISH) technique as described previously . The proThe following porpoise tissues were sampled for polymerase chain reaction (PCR) Table : brain  were processed for sequence-independent DNA virus screening as described previously , 23. In Using the 454-sequencing reads obtained from porpoise #6, open reading frame 56 (ORF56)-specific primers were designed based on ORF56 Ovine herpesvirus 2 to obtain partially overlapping PCR amplicons using AmpliTaq Gold DNA polymerase (Roche). The overlapping PCR fragments were sequenced, resulting in a 1246 bp fragment of the ORF56 homologue of the herpesvirus from porpoise #6 (GenBank accession number KU200258).Phocoena spinnipinnis) [A papillomavirus-specific PCR was performed on the clitoral plaque sample from porpoise #6 using primers MY11/MY09  and GP5+ipinnis) .Ten percent homogenates of selected tissue samples that had been stored at −70 °C were inoculated on the following cell types: primary harbor porpoise kidney cells, Madin-Darby bovine kidney cells, Madin-Darby canine kidney cells, and Crandell feline kidney cells. Cultures were washed twice with Dulbecco’s modified Eagle’s medium supplemented with antibiotics and 10% fetal calf serum and incubated at 37 °C. Cultures were checked daily for cytopathic changes for a maximum of 10 days. At least three passages were made before cultures were considered negative. At the end of each passage, all cultures were tested for the presence of herpesviral DNA by PCR as outlined above.Porpoise #2 was a juvenile female harbor porpoise  found alive on 11 July 2005 at Middelskerk  (Table Stenurus minor), multifocal proliferative arteritis of the pulmonary arteries associated with nematode infection , and diffuse pulmonary oedema associated with euthanasia. By PCR, herpesvirus DNA (PPHV-2) was detected in the brain sample of this porpoise. The viral DNA polymerase 201 bp amplicon showed strongest identity with an alphaherpesvirus of a bottlenose dolphin (Tursiops truncatus) from Germany (92%) and a striped dolphin  that died during the dolphin morbillivirus outbreak in Spain in 2011 (87%) . No macroscopic changes were seen in the brain, but histologically in the cerebrum, there was a locally extensive area of increased density of nuclei in the gray matter. In this area, blood vessels had lymphocytic cuffs about three cells thick and randomly scattered neutrophils in the neuropil. In multiple neurons, the cytoplasm was eosinophilic and the nuclei had large amphophilic inclusion bodies and marginated chromatin Figure . By elec) Figure . Other tn = 74) for herpesvirus infection and encephalitis. The animals identified included 1 male abortion, nine male neonates, 25 male juveniles, seven male adults, six female neonates, 16 female juveniles and 11 female adults. Brain samples were tested for herpesvirus DNA by PCR and examined for histopathological changes by light microscopy. If brain tissues were found positive for herpesvirus DNA, other frozen tissues from the same porpoise were also tested. We found four additional porpoises  with herpesvirus DNA in the brain (Table Cervus canadensis) , aggregation of macrophages around neurons (satellitosis), and aggregation of mononuclear cells around blood vessels (perivascular cuffing). Brain tissues of two #1, #3) of these six porpoises also were positive for herpesvirus DNA  found alive on 24 November 2003 at Noordwijk aan Zee (The Netherlands). After long-term rehabilation at SOS Dolfijn the porpoise developed kidney failure. This was most evident from blood serum values: urea increased from 18 to 30 mmol/L, creatinine from <27 to 81 μmol/L and sodium from 158.5 to 196.4 mmol/L. The porpoise was euthanized on 2 May 2005 because of poor prognosis.At autopsy, there was a plaque on the mucosa of the clitoris. It consisted of a round yellow-white soft nodule  with an irregular surface. By histology, this plaque consisted of moderately thickened epithelium, characterized by hyperplasia and folding. In the upper half of the epithelium, many epithelial cells had large, round to oval, amphophilic INIB surrounded by a clear halo. The superficial connective tissue subjacent to the epithelium contained more capillaries than normal Figure . By elecPhocoena spinnipinnis papillomavirus (accession number: AJ23837.1) for a 327 bp fragment to 83% with Miniopterus schreibersii papillomavirus (GenBank accession number: JQ692938.1) for a 41 bp stretch of a 232 bp fragment. The clitoral plaque sample tested negative in the papillomavirus PCR.By next-generation sequencing of the skeletal muscle sample, which had the strongest herpesvirus DNA polymerase PCR band of all PCR-positive tissues, multiple reads of herpesvirus were obtained and used to design specific primers and obtain multiple overlapping PCR fragments. This resulted in a 1246 bp fragment homologous to gammaherpesvirus ORF56. By ISH using a probe designed based on this fragment, gammaherpesvirus RNA was detected in epithelial cells of the clitoral plaque  for herpesvirus infection and genital plaques. These porpoises were one male neonate, 18 male juveniles, two male adults, three female neonates, 16 female juveniles and three female adults. Swabs were collected from genital slits, blowholes and oral cavities and tested for herpesvirus DNA by PCR. Of these 43 porpoises, 16 were later autopsied. Furthermore, we screened autopsy reports of the live-stranded harbor porpoises that were autopsied between 2000 and 2014 (n = 74). We found two additional porpoises (#7 and #8) with swabs positive for herpesvirus DNA (Table Cervus canadensis)  and in another striped dolphin. PPHV-2, the striped dolphin herpesvirus and the bottlenose dolphin herpesvirus belong to the subfamily of Alphaherpesvirinae, suggesting that these viruses have a tropism for the nervous system and are potentially pathogenic in cetaceans. More extensive sampling of the brain to account for localized infection, and the application of molecular methods  in conjunction with histological methods  for pathogen detection are warranted to elucidate the causes of encephalitis, including herpesvirus infection, in harbor porpoises and other cetacean species.Encephalitis is commonly diagnosed in harbor porpoises, but its cause is rarely established. Encephalitis was a significant diagnosis in 9% (7/74) of porpoises in this study, 3% 4/133) of porpoises stranded along the German coast , and 11%y et al.  diagnose3 of porpThe next herpesvirus we detected in these harbor porpoises, PPHV-1, was associated with a plaque in the clitoral mucosa of an adult female harbor porpoise  [Grampus griseus) [Gammaherpesvirinae. In a zoo collection of bottlenose dolphins, infection was endemic and seroconversion occurred around the age of onset of sexual behavior [Genital lesions associated with herpesvirus infection have been found in four other cetacean species: a male Blainville’s beaked whale (rostris) , a femalgriseus) , a male griseus)  and bothgriseus) , 31, 32.griseus) , the botgriseus) , the mucThere is ongoing debate about the roles of herpesvirus infection and papillomavirus infection as the cause of genital lesions in cetaceans . In thatThe last herpesvirus we detected in harbor porpoises, PPHV-3, was not related to disease. Despite detection in brain, pulmonary lymph node, genital slit and blowhole samples of several porpoises , indicating that this morbidity factor is rare in harbor porpoises that strand alive on the Dutch and adjacent coasts. However, a caveat is that only one brain sample  was collected for virological analysis as part of our standard autopsy protocol. Given that herpesvirus infections may be limited to certain areas of the brain , we may Tursiops truncatus herpesvirus type 1 infection in bottlenose dolphins in a zoo collection—19% (4/21) for juvenile and adult males, 33% (5/15) for juvenile and adult females [The low prevalence of herpesvirus-associated genital plaques in live-stranded harbor porpoises—0% (0/52) in juvenile and adult males, 2% (1/46) in juvenile and adult females—contrasts with the much higher prevalence in bottlenose dolphins in a zoo collection—23% (3/13) in juvenile and adult males, 27% (4/15) in juvenile and adult females . Similar females . PossiblTursiops truncatus herpesvirus in bottlenose dolphin sera [To obtain a better idea of the prevalence of infection with these herpesviruses in autopsied harbor porpoises, it would be important to determine the site of latent infection and to sample those sites. For example, herpes simplex virus 1 in human beings establishes a latent infection in the root ganglia of the trigeminal nerve . The devhin sera .


Hybridization may drive rare taxa to extinction through genetic swamping, where the rare form is replaced by hybrids, or by demographic swamping, where population growth rates are reduced due to the wasteful production of maladaptive hybrids. Conversely, hybridization may rescue the viability of small, inbred populations. Understanding the factors that contribute to destructive versus constructive outcomes of hybridization is key to managing conservation concerns. Here, we survey the literature for studies of hybridization and extinction to identify the ecological, evolutionary, and genetic factors that critically affect extinction risk through hybridization. We find that while extinction risk is highly situation dependent, genetic swamping is much more frequent than demographic swamping. In addition, human involvement is associated with increased risk and high reproductive isolation with reduced risk. Although climate change is predicted to increase the risk of hybridization‐induced extinction, we find little empirical support for this prediction. Similarly, theoretical and experimental studies imply that genetic rescue through hybridization may be equally or more probable than demographic swamping, but our literature survey failed to support this claim. We conclude that halting the introduction of hybridization‐prone exotics and restoring mature and diverse habitats that are resistant to hybrid establishment should be management priorities. It has long been recognized that hybridization, defined here as mating between genetically distinguishable populations, can have a variety of evolutionary outcomes Stebbins . These i, outbreeding depression), and hybridization is common, population growth rates of one or both parental lineages may decline below replacement rates due to wasted reproductive effort, leading to extinction , that paper was excluded (84 cases). Then, the two sets of categorizations were compared and corrected for consistency by three final ‘editors’ who collectively decided how to treat ambiguous cases. Finally, we combined multiple publications about the same species in the same location for a total of 143 independent cases (Table S1). We are aware that this survey is not comprehensive, as some relevant studies were not published in the Web of Science core collection or were not detected by our keyword search.Our survey was based on a Web of Science (Thomson Reuters) search for the keywords ‘hybridi*ation’ and ‘extinction’, including research articles published between January 1975 and May 2015. We determined that 357 papers were broadly relevant to this study based on information presented in the abstract. Of these, we excluded 66 reviews and 37 publications that focused on theoretical models. Each empirical publication was read by two people who independently scored the study for 17 categories , genetic swamping was the more common cause (87% of cases) and local (56%) rather than global extirpation was more frequently predicted . This trend appears to be driven by fish (85%) and birds (79%). There were too few cases to make conclusions about invertebrates.It is possible that our conclusions will hold for some taxa, but not others. In our survey, plant hybrids were most commonly studied, followed by fishes, birds, and mammals Fig. . ExtinctDespite our broadly inclusive definition of hybridization, most studies focused on the outcomes of interspecific (83%) rather than intraspecific hybridization Fig. . HoweverP = 0.007). Among the cases where human involvement promoted hybridization, 55% involved husbandry or agriculture, 54% involved invasive species, and 36% involved habitat disturbance. Note that these factors are not independent; any one case could be affected by all three. Climate change, another consequence of human activities, was infrequently associated with hybridization in the current survey , 72% of studies with human involvement reported an extinction threat while only 46% of studies without human involvement reported a threat (P = 0.6). Nonetheless, this trend might partly account for the reduced extinction risk in plants versus vertebrates, since studies of the former are more likely to report the presence of one or more reproductive barriers (57% in plants versus 33% in vertebrates). Unfortunately, samples sizes were too limited to test the relative importance of prezygotic versus postzygotic barriers to extinction risk.Extinction risk through hybridization is lower when there are reproductive barriers (54%) and higher in the absence of reproductive barriers (67%), but this is not a significant difference . This makes sense because females typically invest more resources into reproduction than males. Likewise, when nuclear introgression is into the ‘common’ species, there is less risk of extinction (8%) than when introgression is into the ‘rare’ species (67%) (P = 0.019). This finding accords well with expectations from the process of extinction through genetic swamping.All types of hybridization/introgression asymmetry are associated with extinction threat, but the details matter. In cases of F1 asymmetry, when the ‘common’ species tends to be the mother, there is little risk of extinction (14%). When the ‘rare’ species tends to be the mother, the risk is high . Conversely, simulations predicted that the native species can persist when heterozygote fitness is high, but this counterintuitive finding is an artifact of species membership being determined by a single bi‐allelic locus. In this situation, a large fraction of offspring from hybrid matings are considered to be pure parental genotypes, and the taxon that is initially more frequent will be less likely to go extinct.The first attempts at modeling hybridization–extinction dynamics had a genetic focus. Huxel  considerThis limitation was accounted for in subsequent genetic models. Ferdy and Austerlitz  simulate, Huxel Compared to genetic models, ecological models place a larger emphasis on life‐history traits. Wolf et al. , for exaResults from Wolf et al.  also enaOther predictions of Wolf et al.'s  simulatiTheorists have also investigated the frequency of hybridization‐mediated extinction relative to hybridization outcomes that maintain or increase biodiversity, although comparatively less effort has so far been devoted to this question. In Wolf et al.'s  simulatiA more inclusive species definition was used by Buerkle et al. . In theiHybrid speciation was the least frequent outcome (2.1% of simulations), observed under conditions of no habitat separation but when F1 fertility and ecological selection were high, as well as with habitat separation when F1 fertility was high but when ecological selection was weak. The second outcome, extinction, was also infrequent (13.9% of simulations). It occurred only when F1 fertility was at its highest (90%), and there was no habitat separation. With habitat separation, extinction again occurred only at the highest F1 fertility level, although in this case moderate to strong ecological selection decreased the likelihood of extinction. Of the observed cases of extinction, all involved adaptive trait introgression: The common species acquired the locally adapted alleles from the rare species. Finally, Buerkle et al.  estimateWhile the theory of extinction by hybridization has made important contributions to our conceptual understanding of this process, it has also made it clear that no single model can provide predictions that are universally applicable. This is because model assumptions, which are dependent on the biology of the hybridizing taxa, can have important bearing on the predicted outcome. The degree of spatial clustering is one such assumption. In the model of Wolf et al. , for whiHabitat disturbance section below).Keeping these limitations in mind, several more general predictions can be formulated based on results obtained from theoretical work. For one, given that the relative proportions of the interacting taxa in contact zones are consistently ranked high by modeling studies, extinction by hybridization is likely to be common in scenarios of species introduction characterized by heightened propagule pressure . Below we describe some of the empirical literature that links human activities with extinction risk through hybridization, as well as some the factors underlying this linkage.Hybridization has long been known to be associated with human activities (Anderson and Stebbins Oncorhynchus mykiss) and charr , which are eroding the genetic identities of native congeners  in America  in Hawaii  and subsequent hybridization. In addition, captive breeding programs such as that involving endemic Cuban crocodiles , where the number of stocking events in Canadian lakes was shown to be positively correlated with levels of hybridization  and red‐legged partridge hybrids (Alectoris rufa) appear to have facilitated hybridization with wild populations of the latter  appears to have predisposed commercial cultivars to hybridization and introgression with native congeners , could avert extirpation in many such cases  that hybridize with native European polecats (M. putorius) in Britain , threatening their genetic integrity . Examples include escaped domesticated ferrets  involved taxa that were not native to the region where hybridization occurred. Many of these include species that have been intentionally introduced and released, such as the aforementioned introductions of rainbow trout from its native range in the Pacific basin into lakes and rivers throughout the world  and the native flannelmouth sucker (C. latipinnis) in the Colorado River Basin have facilitated introgression between the latter and a previously isolated congener, C. discobolus  of introduction. These factors can lead to high and potentially asymmetric hybridization rates  and invasive green iguanas (I. iguana), which have been introduced from French Guyana as stowaways on boats  and native California cordgrass (S. foliosa) threatens the latter with local extinction throughout the San Francisco Estuary , there were too few data to confirm (or refute) the predictions of Currat et al. . Possibly this is due to inconsistent reporting of disturbance rather than a weak effect.Anderson  emphasizKunzea sinclairii, a rare shrub endemic to the rhyolitic rock outcrops on Great Barrier Island in northeastern New Zealand, is compatible with the more abundant close relative, K. ericoides, but hybrids are limited to disturbed sites created by fire or logging  hybrid swarms that formed following habitat disturbance in the 1940s due to grazing, and/or trail and road construction. When he revisited the populations 22 years later, two of the sites had returned two predisturbance conditions and were dominated by plants that resembled one of the parental species. In contrast, one site remained disturbed and hybridization was still evident. Such observations also support a management strategy that includes habitat preservation and restoration. Mature, diverse, and undisturbed communities appear to be resistant to hybrid establishment and success. However, maintaining such communities will be challenging in the face of a growing human population, growing resource use, and climate change (below).Possibly the most compelling evidence linking habitat disturbance and hybridization derives from studies documenting the cessation of hybridization following habitat restoration. For example, Heiser  reports Glaucomys volans) have expanded their range north in response to climate change. This has brought them into contact with their congener, the northern flying squirrel (G. sabrinus) where hybridization has occurred  of the Flathead River system (USA and Canada)  were extensively stocked from the late 1800s to 1969, but by the 1980s only low (<2%) levels of hybridization with cutthroat trout were detected. As climate warming increased, so did the level of hybridization, and Muhlfed et al. showed that precipitation and summer stream temperature explained current introgression levels.The threat of hybridization to species conservation may be amplified by climate change due to the breakdown of spatial, temporal, behavioral, or postzygotic reproductive barriers (reviewed by Chunco Spea bombifrons) prefer heterospecific Mexican spadefoot toads (Spea multiplicata) under low water conditions because development time is faster in hybrids and maturation time is limited by when the ponds dry out  small, inbred populations . In the F2 generation, however, individuals have only half the heterozygosity of F1s, so a further reduction in fitness is expected unless there is highly favorable dominance x dominance epistasis.These empirical results have been augmented by theory, which enable more general predictions and recommendations about outbreeding depression and its management. In an early study, Lynch  developeTigriopus californicus) populations along geographic and genetic divergence clines  gene flow is limited over time or in degree; or (ii) selection is strong enough to maintain or re‐assemble the genomic and phenotypic differences between the parental lines.We defined the ‘genetic rescue’ outcome in our literature survey as hybridization resulting in a net fitness gain to one or both taxa without threat of extinction. In classifying cases, we considered direct evidence for genetic rescue as well as more speculative reasoning by study authors. We note that this outcome could also be considered genetic swamping under our schema, as it is the contribution of alleles from one taxon to another that is responsible for increased fitness, for exampleGenetic rescue was a rare outcome in our literature survey: definitive in only one case, where hybridization was a human‐mediated, intraspecific, single event in a population suffering from inbreeding depression (Benson et al. In a selective meta‐analysis, Frankham  found in, Pickup et al. , Miller et al. What is clear from both theoretical and empirical studies is that genetic rescue is highly dependent on the context of the hybridization (e.g.In 1996, two important reviews were published that established hybridization as an extinction threat (Levin et al. We specifically provide quantitative support for the long‐recognized association between human activities, hybridization, and extinction risk. Surprisingly, husbandry and species introductions contributed more to this association than did habitat disturbance, although this might be a consequence of ascertainment bias. We also confirmed predictions that extinction risk is higher in the absence of reproductive barriers and when hybridization/introgression is in the direction of the threatened species.Our analysis/review further indicates that genetic swamping is more frequent than demographic swamping, extinction risk from hybridization is likely higher in hybridizing vertebrates than plants, the fitness consequences of hybridization can be surprisingly benign, and outbreeding depression will be short in duration if there is a single bout of immigration and immigrants carry locally adapted alleles.Important questions remain. For example, it seems likely that climate change will amplify the threat of hybridization to species conservation, but support for this conjecture is currently weak. More generally, how do changes in the environment affect rates of hybridization and associated risks versus benefits? Theoretical and experimental studies imply that genetic rescue through hybridization may be equally or more probable than demographic swamping, but our literature survey failed to support this claim. At a more philosophical level, should we be opposed to extinction via genetic swamping if the hybrid entity that emerges is more variable and fit than one or both parental species? Such an entity might be better able to withstand future ecological and evolutionary challenges.From a more practical standpoint, can our results inform management strategies? We contend that they can, in part by strengthening barriers to the introduction of hybridization‐prone exotics, sharpening decision making concerning the release of captive‐bred individuals and the appropriate use of hybridization for genetic rescue, and prioritizing restoration of mature and diverse habitats that are resistant to hybrid production and establishment.Our review/analysis also points to gaps in our knowledge about the role/prevention of hybridization in species extinction and information and experiments that are needed to fill these gaps. Although there has been a large increase in the quantity of research on hybridization in species conservation since the mid‐1990s Fig. , the quaData underlying results in this paper can be found in Table S1.Figure S1 Number and kinds of markers employed in the studies of hybridization and extinction included in our literature survey. The number of publications is also shown.Click here for additional data file.Table S1 Description of case studies employed in literature survey.Click here for additional data file.


The results obtained using four relevant and model viruses (enveloped/non enveloped RNA/DNA viruses) still serve to improve the freeze drying conditions needed for the development and the distribution of a large virus collection.As a partner of the European Virus Archive (EVA) FP7 project, our laboratory maintains a large collection of freeze-dried viruses. The distribution of these viruses to academic researchers, public health organizations and industry is one major aim of the EVA consortium. It is known that lyophilization requires appropriate stabilizers to prevent inactivation of the virus. However, few studies have investigated the influence of different stabilizers and lyophilization protocols on the thermostability of different viruses. In order to identify optimal lyophilization conditions that will deliver maximum retention of viral infectivity titre, different stabilizer formulations containing trehalose, sorbitol, sucrose or foetal bovine serum were evaluated for their efficacy in stabilizing a representative panel of freeze dried viruses at different storage temperatures  for one week, the two latter mimicking suboptimal shipping conditions. The Tissue Culture Infectious Dose 50% (TCID EVA (European virus Archive) is a non-profit organization, funded by the European Commission through the FP7 CAPACITIES Project GA n° 228292. EVA mobilizes a European network of scientific laboratories with expertise in virological procedures to collect, characterize and distribute viruses and virus-derived products such as antibodies, antigens, diagnostic and research reagents and assay . As the Lyophilization of cells in water or a simple salt solution typically results in poor survival. Consequently, using the same assumptions, a variety of protective media has been developed for lyophilization of viruses, including augmented growth media or sugar, and peptide solutions . Most coThe freeze drying protocol can also determine the properties of a specific formulation by influencing the crystallization of additives and subsequently acting on the collapse temperature. It is known that significant crystallization of the bulking agent will decrease drying time but can also reduce stabilizing effects of the amorphous stabilizer especially with proteins. In fact, different freezing or drying rates can produce vastly different freeze-dried products, both in appearance and viability of the micro-organism and an optimized lyophilization protocol will provide excellent drying efficiency while maintaining the virus integrity . AlthougThis study focuses on optimizing both the protocol and the formulation for the lyophilization of enveloped and non-enveloped viruses possessing RNA or DNA genomes. One aim was to select the standardized method allowing optimal recovery of viral infectivity after one week at +4°C or +20°C, conditions mimicking transportation without cold chain preservation. The performance of the protocols is evaluated by measurement of the infectivity of the resulting freeze-dried material. Using a representative panel of viral specimens, several chemical stabilizers were assayed to evaluate the thermostability of the freeze-dried preparation. Commonly known cryo/lyoprotectants, including sucrose, trehalose, sorbitol or foetal bovine serum in two freeze-drying programmes, were compared for their stabilizing property on four representative viruses. After different temperature storage conditions representing those that may be encountered during viral transport and distribution, the recovery of viral infectivity titre was analyzed to identify the optimal conditions for the lyophilization protocol.European-virus-archive.com).The four virus strains used in this study were isolated from human clinical samples collected for diagnosis in the Virology laboratory of the University Hospital of Marseille . These sHuman herpesvirus 1 (strain MRS2010 7351047) and Human enterovirus B type Echovirus 13 (strain 142604/2000) were propagated on primary human fibroblast MRC-5 cells (ATCC number CCL-171) grown in Basal Medium Eagle (BME) supplemented with 10% foetal calf serum (FCS), 2 mM L-glutamine, 100U/ml Penicillin, 100μg/ml Streptomycin sulfate, under 5% CO2. Chikungunya virus (strain LR2006_OPY1) was propagated on Vero cells (ATCC number CCL-81) grown in minimal essential medium (MEM) supplemented with 5% FCS, 2 mM L-glutamine,100U/ml Penicillin, 100 μg/ml Streptomycin sulfate, under 5% CO2. Human Adenovirus type C (strain MRS2012 23910121) was propagated on a Hep-2 cell line (ATCC number CCL-23) grown in minimal essential medium (MEM) supplemented with 5% FCS, 2 mM L-glutamine,100U/ml Penicillin, 100 μg/ml Streptomycin sulfate, 1% non essential amino-acids, under 5% CO2.Four different stabilizer solutions were tested in this study. For each, two concentrations were prepared depending on the data found in the literature. Sorbitol, trehalose and sucrose were obtained from Sigma (France). foetal bovine serum (FBS) was obtained from Invitrogen (France). For each assay, 100μl of infected cell culture supernatant was mixed with 100μl of stabilizer solution to obtain final concentrations at 5% and 10% with sorbitol and trehalose, at 3.5% and 7% with sucrose, and at 25% and 50% with FBS.2 flasks were respectively infected with Chikungunya virus (CHIKV), Herpesvirus type 1 (HSV-1), Human Echovirus 13 (EV-13) or Human Adenovirus type C (HAdV-C) with a multiplicity of infection (MOI) between 0.1 and 0.01. When a 50% cytopathic effect (CPE) was observed, cell supernatant media were clarified using low-speed centrifugation (1500g for 10 min) and supplemented with 25mM HEPES (Sigma). 200μL aliquots of the appropriate virus, in appropriate stabilizing media, were added to sterile 2-mL lyophilization vials  and the aliquots were stored at -70°C prior to freeze drying.Confluent Vero, MRC-5 and Hep-2 cells in 175-cm3, 200μl of viral preparation were dispensed in sterile 2mL vials partially sealed with vented rubber stoppers before freezing in a dry ice/ethanol cooling bath.Lyophilization was carried out in a BSL3 laboratory using a CHRIST alpha 1–4 LSC freeze-dryer. To avoid cross contamination and to ensure identical conditions, each freeze-dried batch production was performed on a single virus strain with different stabilizer formulations. Briefly, as described in section Two lyophilization programs were used for comparison; (i) LP1 "optimized;", this is used routinely in our laboratory and was previously developed based on a yellow fever 17 D vaccine virus model (data not shown), which consisted of a 6-hour primary drying step  followed by a secondary drying step , (ii) LP2 "rapid"; this second program consisted of a single 6-hour secondary drying step . All the vials were finally sealed under vacuum following the lyophilization stage.Separate freeze dried virus vials were exposed to different storage temperatures  in a light-protected environment for 7 days. Vials were then rehydrated with 200μL of sterile distilled water before virus titration as described below.To summarize and to simplify the comparison of the different freeze dried batches, the following abbreviation was adopted: LP1/2 (-20/+4/+20°C) for all samples freeze dried using the LP1 or LP2 protocol and stored for one week at -20, +4 or +20°C. Storage at -20°C represents the laboratory standard for EVA collection preservation of freeze dried viruses. A storage at +4°C or +20°C for one week mimics conditions which can be encountered during sample transportation from the EVA to the customer laboratory.50) using standard cell culture procedures. Briefly, when cells reached 80% confluence in 96-well microtitre plates, six replicates were infected with 150μL of tenfold serial dilutions of the virus sample, and then incubated for 7 days at 37°C in a humidified atmosphere of 5% CO2. CPE was monitored using an inverted microscope, and infectivity titres were expressed as TCID50/ml based on the Karber formula. The virus infectivity was then compared and analyzed to determine the optimal sample formulation under the different conditions of preparation and storage. Moreover, to evaluate the program and temperature influence on viral infectivity independently of sample formulation, t-tests were used to estimate the difference between groups of data which were expressed as mean virus titres ± standard deviations (Virus infectivity was quantified by estimating the 50% tissue culture infectivity dose (TCIDviations .50 data obtained for CHIKV.7.8±1.3 TCID50/ml) and LP1 (-20°C) freeze dried samples , when compared with LP1 (+20°C). Also, LP2 (+20°C) produced similar yields of virus. (p = 0.07).Independently of sample formulation, recovered virus infectivity levels were significantly higher (p<0.004) for LP1 (+4°C) . However, for LP1 (-20°C) freeze dried samples, the used of FBS 50% significantly increased the mean viral titre by about 2 log10 TCID50 (108±0.9 TCID50/ml versus 1010.1 TCID50/ml). For LP1 (+4°C) freeze dried samples, formulated with sorbitol 10% resulted in recovery of the highest virus infectivity .Moreover, independently of sample formulation, there were no significant differences for recovered virus infectivity in the LP2 freeze dried samples in relation to the storage temperature and only slight differences in viral infectivity were observed between the various stabilizers by comparison with the mean viral titres for each storage temperature  freeze dried samples, with 10% sorbitol or 50% FBS as stabilizers whereas the lowest viral titres were obtained for both LP1 and LP2 (+20°C) freeze dried samples with no significant difference between the stabilizers. The LP2 (-20°C) is not significantly different from the LP2 (+20°C) result.50 data obtained for HSV-1.9.2±1.3 TCID50/ml). Virus infectivity recovered was also statistically higher (p = 0.004) for the LP2 freeze-drying protocol when samples were stored at -20°C . These results were independent of the sample formulation and they refer to comparison with the mean viral titre obtained following storage at +20°C (107.2±1.5 TCID50/ml) or +4°C (106.7±0.4 TCID50/ml). Moreover for the same samples, the most efficient stabilizers were 10% sorbitol and 5–10% trehalose with a gain in viral infectivity of about 1–2 log10 TCID50/ml by comparison with the mean viral titre (109.2±1.3 TCID50/ml).The results show that independently of sample formulation and storage temperature, there were no significant differences between the mean viral infectivity observed using the LP1 protocol (p>0.2). However, infectivity recovered was significantly higher (p<0.0001) when the LP2 freeze drying protocol was used and the samples were stored at -20°C . Inversely, the lowest virus infectivity was obtained for LP2 (+20°C) freeze dried samples without stabilizer. However, under sub-optimal storage conditions (i.e. after one week at +20°C), 10% sorbitol combined with the LP2 protocol and 3.5% sucrose combined with the LP1 protocol were the more efficient stabilizers, increasing recovered viral titres by about 2 log10 TCID50 when compared with other freeze drying formulations.To summarize, the highest mean viral titres were obtained using the LP2 (-20°C) conditions , the recovery of virus infectivity was significantly higher (p = 0.03) for LP2 (-20°C) freeze dried samples . Similarly and by comparison with the mean viral titres obtained at +20°C (102±1.7 TCID50/ml), the recovery of virus infectivity was significantly higher (p = 0.02) for LP1 (-20°C) freeze dried virus .The two lyophilization protocols (LP1 and LP2) produced closely similar results with no significant difference of mean virus titres recovered (p>0.05) at each storage temperature . Independently the sample formulation and by comparison with the mean viral titres obtained at +20°C (1010 TCID50 by comparison with other freeze dried formulations). Accordingly, the lowest mean virus infectivities were obtained for both LP1 and LP2 (+20°C) freeze-dried samples. Under sub-optimal storage conditions (i.e. after one week at +20°C), 5–10% sorbitol with the LP1 or LP2 protocol was also the more efficient stabilizer by increasing the viral titre recovery of about 2 log10 TCID50 by comparison with other freeze drying formulations.To summarize, the highest mean viral titres were obtained using either the LP1 or LP2 protocol following storage at low temperatures +4°C/-20°C. Moreover, the highest infectivity recovered was obtained for LP1 or LP2 (+4°C) freeze dried samples with formulations containing 5–10% sorbitol (increase of about 2 log50 data obtained for HAdV-C.4.1±0.3 TCID50/ml).Independently of freeze drying formulations and lyophilization protocols, no significant differences were observed by comparing the mean viral infectivity for samples stored at +4°C or +20°C . On the other hand, the virus recovery infectivity was statistically higher (p<0.05) for LP1 (-20°C) freeze dried samples . However, under sub-optimal conditions (one week at +20°C), sucrose 3.5 or 7% in combination with the LP1 lyophilization protocol was the more efficient stabilizer by comparison with other freeze dried formulations. This formulation produced a viral titre recovery as high as those obtained under optimal storage conditions.A fundamental problem in preservation of freezed-dried live viruses is the possible loss of infectivity (i) either during the process of lyophilization (ii) or because of improper observation of the cold chain integrity often encountered during the transportation of viruses between laboratories . There aIn this study, we have considered that -20°C was the optimal temperature for long-term storage. Experiments conducted after one-week storage at either +4°C or +20°C were assayed to simulate failure due to the breakdown in cold-chain maintenance possibly encountered during the shipping of vials containing freeze-dried viruses, and to test whether the type of freeze-drying protocol, and the nature of stabilizers might improve the stability of end products and their ability to resist to uncontrolled conditions.Two different lyophilization protocols were tested in this study: (i) LP1 (12 hours with primary and secondary drying steps) which was the "reference" protocol usually described , 9; (ii)It is known that the level of virus viability after freeze drying varies according to the virus family, and the efficacy of the protective agent (stabilizer). For 20 years sugars such as trehalose and sucrose were reported to enhance the tolerance to desiccation through stabilization of membranes and proteins . SucroseSorbitol is recommended as cryoprotectant during freezing and lyophilization of several viruses such as cytomegalovirus, varicella-zoster virus, herpes simplex virus, respiratory syncytial virus, and 17D Yellow fever vaccine) . Others Usually, enveloped viruses demonstrate greater lability during storage than those lacking envelopes. In fact, decreases in viral titre correlate with demonstrable changes since the viral envelope is assumed to be associated with viral infectivity . LyophilThis study focused on optimizing both the protocol and formulation for the lyophilization of selected model viruses under routine collection production and distribution conditions. Reference (LP1) and “rapid” (LP2) lyophilization protocols were conducted with a variety of sugar-based excipients and under different temperatures storage for one week. Although there is the need for more detailed analysis specifically dedicated for each virus species, the aim of this study was to evaluate a rapid protocol applicable to a broad range of viruses. Regarding this objective we recommend to use the combination of LP2 protocol together with 10%-sorbitol supplementation when aiming at high throughput workflow for preparing material in the intention to distribution. For long-term storage, the LP1 reference protocol can be associated with 10%-sorbitol supplementation.Finally, due to few data available, the results obtained using four relevant and model viruses (enveloped/non enveloped RNA/DNA viruses) would improve the freeze drying conditions needed for the development and the distribution of a large virus collection. Future developments would concern optimization of these methods for different virus families and evaluation of these protocols for perennial virus conservation at the -20°C reference temperature storage.