models to serve as a visual representation of conspecific and heterospecific animals. This is the first study to use both avian wooden models and song playback to investigate the importance of visual and acoustic signals for inter- and intraspecific discrimination. Our successful use of avian wooden models provides an alternative to sacrificing study animals to create taxidermic mounts. Although our wooden models were realistic (see Figure 3.1), and the colour matched the plumage reflectance of museum specimens, the lack of movement may have hindered the response of the birds. If the models produced movements such as wing flaps or tail cocking, they may have elicited stronger aggressive responses (Anderson et al., 2013). Experiments involving robotic birds (e.g. Patricelli et al., 2006; Balsby & Dabelsteen, 2002) show that movements can influence responses to model presentation experiments. Moreover, a previous study showed that birds use not only colour but also surface texture as a signal for species discrimination (Nemec et al., 2014). Red-backed shrikes (Lanius collurio) attacked a taxidermic model of a predator Eurasian jay (Garrulus glandarius) more often than a plush model, and attacked a silicone model the least (Nemec et al., 2014). Although our wooden models had feather- like texture carvings and looked more realistic than the plush model used in the
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Shuker 2011). Examples of RI can range from straightforward heterospecific copulation attempts, (i.e. de Bruyn et al. 2008) to more subtle behavioural changes such as forgoing normal mate-quality assessment in favour of reliable species discrimination (Pfennig and Pfennig 2009). The presence of heterospecific signals in the environment can interfere with sexual signalling (e.g. in the frog, Allobates femoralis; Amezquita et al. 2006, and Neo- tropical singing mice, Scotinomys sps.; Pasch et al. 2013), influencing both mate-attraction and mate-searching. While much of the literature on the effects of RI has focused on responses to heterospecific copulation attempts, including avoidance behaviours leading to changes in habitat use (McLain and Shure 1987), or erroneous mate choice (Butler and Stein 1985; Dame and Petren 2006), heterospecific matings do not actually have to occur for RI to be present. The presence of heterospecifics may influence an organism’s repro- ductive behaviour in more than one way (Miller et al. 2013), especially if they are unable to differentiate between hetero- and conspecifics. For example, studies in Drosophila melanogaster have shown that heterospecific song can influence female gene expression and that these changes are broadly comparable to those caused by conspecific song (Im- monen and Ritchie 2012).
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Microsatellites are often considered ideal markers to investigate ecological pro- cesses in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leo- pardus and P. maculatus (Serranidae). Coral trout are important fisheries spe- cies throughout the Indo-Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co- amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent–offspring relationships in natural populations, with over 99.6% accuracy in parent–offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28–96% of loci. The multiplex PCRs developed here provide a reliable and cost-effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild popu- lations and aquaculture brood stocks for these closely related fish species.
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All PCRs from the individual markers used in this study have proven to be applicable directly on clinical specimens (4, 8, 11–14, 16) and allow species identification on the basis of 1 PCR ampli- con instead of the 7 amplicons used in MLST, without the need for parasite culturing, a technique that is time consuming and often not successful. The hsp70 target was identified as the best choice for high-resolution species discrimination, as it identifies the same species and species complexes as does the gold standard MLST applied here. A downside of hsp70 sequencing is the size of the PCR product, which is 1,286 bp. In our experience, some samples require amplification of 2 overlapping PCR fragments, either in a single round or by nested PCR (16), but in most cases, the 1,286-bp fragment can be amplified from a single PCR. To obtain reliable Sanger sequence reads from both strands, 4 primers are needed. It is noteworthy that 3 shorter hsp70 fragments have been described and evaluated in clinical samples (13, 14, 16). These are almost as effective for sequence-based typing as the 1,286-bp re- gion used here (see Fig. S5 to S8 in the supplemental material), although the most suitable fragment is governed by the required discrimination. The miniexon provides nearly the same resolu- tion as does hsp70, but its advantages are that the amplicon is much smaller and more copies are present in the genome, which may facilitate amplification of samples with low parasite loads (5). Sequencing can be completed with 2 primers, but it is often tech- nically challenging or impossible mainly due to variations be- tween the different copies in the same genome (38). The rDNA ITS1 is equally advantageous in copy number and size (8) but provides poor resolution in the L. (Viannia) subgenus. Also for this marker, sequencing is challenging mainly because of ho- mopolymer tracks. Finally, the 7SL RNA marker provides poor typing resolution in both the Old and New World species and is not recommended for accurate species identification. It should be noted that an extended 7SL RNA fragment (11) might improve the resolution obtained with this marker.
do not discriminate against heterospecific freeze-killed T. sarcophagae females but only against freeze-killed M. uniraptor females (Fig. 2a). As a cosmopolitan species documented to encounter T. sarcophagae  as well as other Pteromalid species in close sympatry [53, 71, 72], pronounced discrimination behavior would have been ex- pected particularly in N. vitripennis. Although acceptance of heterospecific mates has been shown in N. vitripennis in behavioral assays with other Nasonia species [44, 63, 73], the extension of this non-selective behavior to a phylogen- etically even more distant genus was surprising. Concern- ing population-specific variation, N. vitripennis CHC profiles have been found to be remarkably stable across populations irrespective of their geographic origin stem- ming from Europe or North America (Buellesbach, Diao, Beukeboom & Schmitt., unpublished data). Moreover, males from an N. vitripennis population collected in North America also showed non-discriminatory behavior against freeze-killed T. sarcophagae females (Additional file 1), suggesting that CHC profiles and associated behaviors con- stitute genetically fixed, species-specific traits with low in- traspecific variation, which is also in accordance with studies on other Nasonia species [41, 43, 72].
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In the past few decades, the mapping and monitoring of rangeland degradation in South Africa has primarily focused on commercial rangeland (Palmer and van Rooyen, 1998; Shackleton et al., 2005), meaning communal rangeland has not as yet enjoyed the same degree of attention (Mansour et al., 2012). The continued degradation of communal rangeland is a major threat to livestock production, biodiversity and human livelihoods (Hoffman and Todd, 2000). Different agronomic and ecological techniques have been developed over the past two decades to evaluate and monitor rangeland based on the relative abundance and distribution of increaser species. These techniques have achieved differing degrees of success for evaluating and monitoring rangelands over small geographic areas. However, these agronomic and ecological techniques require intensive and difficult fieldwork in terms of species identification and this exercise is often too expensive and time-consuming because rangeland often cover large spatial extents and are, moreover, frequently to be found in isolated, inaccessible areas (Trollope, 1990). The best method, which includes generating real-time, consistent, repeatable and spatially explicit data, is required for mapping and evaluating rangeland degradation. In this regard, remote sensing techniques offer a practical and economical means for
the development of methodology to discriminate blood samples of different species  with the application of Raman spectroscopy. A recent publication in the Journal of Analytical Chemistry reports on this method of identi- fication of species based on the Raman spectroscopic an- alysis of blood . We have recently reported on poten- tial application of Raman spectroscopy for determining the burial time based on bone remains . Here we re- port on a preliminary investigation of species differentia- tion based on Raman spectroscopy of bones. Our hyp- othesis is based on the literature which demonstrates that vibrational spectroscopic analysis of bone components and mineralized tissue can discriminate species of origin [24-28]. Species discrimination using Raman spectros- copy of animal tusk has been previously investigated by several researchers. Animal tusk, or dentine, is a mineral- ized tissue very similar to bone. Dentine tends to be more mineralized and have less collagen than bone tissue. Shi- moyama et al. (1997) reports that hard and soft mammoth dentine were discriminated using near infrared (NIR) FT-Raman spectroscopy . Brody et al. (2001) report the discrimination of dentine from six mammalian species, with slight overlap, also using FT-Raman spectroscopy  and chemometeric processing. Edwards et al. report using a similar approach to discriminate between Asian and African elephant dentine in a forensic setting . Discrimination in these studies is likely due to organic to inorganic ratio differences between species.
Bacterial strains. A total of 188 strains belonging to the species listed in the database of the VITEK 2 software were investigated. Twelve strains were ref- erence strains from the American Type Culture Collection (ATCC): Campy- lobacter coli ATCC 43478; Campylobacter jejuni ATCC 33560; Gardnerella vagi- nalis ATCC 14018; Haemophilus paraphrophilus ATCC 49917; Haemophilus influenzae ATCC 10211, ATCC 49247, and ATCC 49766; Haemophilus parain- fluenzae ATCC 33392; Neisseria gonorrhoeae ATCC 49759, ATCC 700825, and ATCC 49226; and Neisseria meningitidis ATCC 13090. Eight strains were ob- tained from the German Collection of Microorganisms and Cell Cultures (DSMZ): Capnocytophaga ochracea DSM 7271; Cardiobacterium hominis DSM 8339; Haemophilus actinomycetemcomitans (formerly Actinobacillus actinomyce- temcomitans) DSM 8324 and DSM 11122; Neisseria cinerea DSM 4630; Neisseria elongata DSM 17712; Neisseria lactamica DSM 4691; and Neisseria sicca DSM 17713. Fifty strains were selected from the strain collection of the German National Reference Center for Meningococci, i.e., five strains of N. gonorrhoeae, five strains of N. lactamica (3), and 40 genetically diverse strains of N. meningi- tidis (8, 9). Twenty-six strains of N. meningitidis were serotyped as serogroup B, six as serogroup C, four as serogroup Y, one as serogroup 29E, one as serogroup A, one as serogroup W135, and one as serogroup X. The remaining 118 strains were clinical isolates from unrelated patients belonging to the clinical strain collection of the Institute of Hygiene and Microbiology of the University of Wu ¨rzburg. The sources of the isolates included blood, cerebrospinal fluid, uro- genital, and respiratory samples. For the study, the strains were transferred from storage at ⫺70°C, placed on chocolate agar supplemented with PolyVitex (bio- Me ´rieux, Nu ¨rtingen, Germany), and subcultured a second time in a CO 2 incu-
Besides identification of bacterial species by species-specific PCR-based assays, identification may also be performed by coupling the amplification of a highly conserved gene with hybridization to internal probes or DNA sequencing (1, 14, 20, 22). Many conserved genes have been selected as targets for this purpose. Among them, the 16S rRNA gene has been used to detect a wide variety of eubacteria because the presence of conserved regions and variable regions in this gene provides the possibility of developing PCR-based assays suitable for detecting and identifying bacteria at the species level or higher taxonomic levels (22). Other genes have been exploited for similar purposes. The sod gene, coding for superoxide dis- mutase, has been used as a target to amplify 28 species of mycobacteria and to differentiate one from another with probes recognizing species-specific regions (39). A similar ap- proach has been used for the detection of staphylococci by targeting the chaperonin 60 (cpn60) gene (13, 14). These meth- ods are specific, but their sensitivity remains unclear. The latter is critical when such tests are used to detect bacteria directly from clinical specimens. Berg et al. (1) have devel- oped a PCR-based detection system for M. fermentans by tar- geting the tuf gene; the system has a high sensitivity. A similar assay has also been used for the identification of M. pneu- moniae (20).
droplets associated with single cones (supplementary material Table S1). Variation in the spectral sensitivity of LWS single cones was best attributed to the existence of vitamin A2 chromophores in Z. vivipara that extended spectral sensitivity into the near infrared (Archer, 1999; Hárosi, 1994; Whitmore and Bowmaker, 1989), whereas most diurnal lizards and terrestrial vertebrates use exclusively vitamin A1 chromophores in their visual pigments (Jacobs, 2010; Yokoyama, 2000). Vitamin A2 chromophore was previously recorded in Anolis carolinensis (Loew et al., 2002; Provencio et al., 1992) and a mixture of A1 and A2 chromophores was also shown by chromatography in Podarcis sicula (Provencio et al., 1992) and two chameleon species, Chamaeleo dilepis and Furcifer pardalis (Bowmaker et al., 2005). The presence of vitamin A2 in the eye of Z. vivipara remains to be confirmed by electrophysiology (Loew et al., 2002). San-Jose et al. (San-Jose et al., 2013) recently found that vitamin A2 was the dominant vitamin A compound in common lizards, where it is stored in the liver. They did not attribute this result to differential feeding but to a preferential synthesis and increased accumulation of vitamin A2 in Zootoca vivipara, which is usually absent in most species (San-Jose et al., 2013). Our results and findings in other lizard species thus suggest that the ability to synthesise vitamin-A2-based visual pigments sporadically appeared during the adaptive radiation of lizards.
Several earlier experiments on spatial vision in the honeybee revealed that shape recognition and discrimination are better in the lower frontal eye region than in other frontal eye regions (e.g. Wehner, 1972b; Lehrer, 1998, 1999). In the present study, four pairs of shapes differed from each other exclusively in either the lower or the upper visual field: each of the two triangles combined with either the square or the diamond. Because each pair was trained reciprocally, there are four tests involving the upper visual field and four tests involving the lower one. With both the solid black shapes and the patterned shapes and in both A. mellifera and M. rotundata, the two categories of test rendered practically identical mean CF values. Thus, the lower visual field has no priority over the upper one in the present task, which supports the idea that discrimination in this case is based on cues present at the edges. In natural orientation tasks, the most useful and omnipresent edge, namely the horizon, never projects on the ventral eye region.
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An. kleini, and so on) . The PCR assay was established by sequences of second internal transcribed spacer (ITS2) region of the ribosomal DNA (rDNA) to identify An. sinensis from its cryptic species members of Hyrcanus Group . The genetic structure of An. sinensis popula- tions in China were also detected by molecular markers, and the weak genetic structure may be a consequence of low genetic differentiation and high gene flow among populations in central China [7–10]. However, there are still some issues to be elucidated in the molecular classifi- cation of An. sinensis, such as natural hybrid between An. kleini and An. sinensis was discovered in the Republic of Korea [11, 12] and China (unpublished data). So, the evolutionary relationship of An. sinensis, such as speci- ation and other issues need to be elucidated further.
Several researchers have demonstrated infec- tion by parasites such as Echinococcus spp (19), Toxocara spp (20), F. hepatica (1, 21), Ostertagia ostertagi (22), Oesophagostomum bifurcum and Ne- cator americanus (23), and Opisthorchis viverrini (24) using PCR assay of feces. There is no ev- idence regarding molecular discrimination of Fasciola species agents in fecal specimens from living ruminants in Iran, though there are many studies that deal with the genetic charac- terization of adult forms of Fasciola species from slaughtered animals in different regions of Iran (4, 14-16).
Geographic variation in song may reduce or eliminate the ability of some populations to recognize each other as conspecifics, possibly leading to assortative mating, reproductive isolation, and speciation. Song playback experiments, used to evaluate the significance of geographic variation in song, have been particularly useful in discovering divergence among previously un known populations of sibling species. In this study, I report the results of song playback to male Mourning Warblers (Geothlypis philadelphia) from populations throughout the breeding range and discuss the implications for population divergence. Four regions in the breeding range contain unique song types or regiolects: western, eastern, Nova Scotia, and Newfoundland. Results of reciprocal song playback experiments showed that males from the western and Newfoundland regiolects respond more aggres- sively to songs in their own regiolect than those in the other regiolects. Interior populations, i.e., eastern and Nova Scotia regions, showed little or no difference in aggressive response toward their own versus other regiolects. this pattern may be due to a combination of geographic proximity of populations belonging to different regiolects, song learning, experience, and contact during migration. Song discrimination by populations from the western Prairie Provinces and Newfoundland is consistent with the existence of at least partial reproductive isolation at the geographic extremes of the breeding range.
the same species), making no further distinctions among nonkin. We found, though, that B. subtilis behaves differently toward close versus distant species, an unappreciated consequence of its multicellular lifestyle that likely impacts many aspects of its ecology. The cutoff point for kin discrimination behavior corresponded to those species that secreted a surfactant molecule that a nonswarming mutant of B. subtilis could poten- tially beneﬁt from (i.e., steal) but was prevented from doing so when in close contact. (Note that we have demonstrated only stealing by B. subtilis, not stealing of B. subtilis, but we assume that other surfactant-compatible species could reciprocate the exploi- tation.) There is thus a good correlation between species that can exploit public goods and antagonism between them. This suggests to us that the two could be linked: either the compatibility of public goods impacts antimicrobial range, or the antimicrobial range (selected for other reasons) affects the use of certain public goods. Alternatively, the two traits could arise independently from their shared correlation with phylogeny (and thus general physiology), which may require studies in other bacteria to deﬁni- tively determine. Likewise, the incompatibility of public goods could merely be a result of evolutionary drift as a consequence of species evolving in isolation from one another and thus under no selective pressure to maintain the same surfactants. More detailed studies of the evolution of kin discrimination genes, public good genes, and the phylogenetic background in which they appear will hopefully shed light on the possible coevolution of these genes separate from their inherent history. It would be particularly interesting to compare the rate of change of the lipopeptide synthetases that produce surfactants to the spectrum of antimicrobials made by each species.
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Figure 2 shows the dendrogram obtained after clustering by UPGMA when the data obtained with all four primer pairs were analyzed as a composite. A total of four primary clusters were observed among the 62 Francisella strains tested. The five F. tularensis subsp. tularensis strains were assigned to primary cluster A. Two subclusters were seen, one for isolate 62 (sub- cluster A2) and the other for the remaining F. tularensis subsp. tularensis isolates (subcluster A1). A genetic similarity of 91% was observed between these two subclusters. In addition, a minor variation between isolate 1 and the other three isolates in subcluster A1 was seen. Primary cluster B included all the F. tularensis subsp. holarctica isolates. Three major subclusters were detected within cluster B (with similarities among them of about 94%). Subcluster B1 contained four isolates recovered from monkeys and humans in the United States (greater than 96% similarity), subcluster B2 contained the Czech and Rus- sian isolates (greater than 98% similarity), and subcluster B3 included the Spanish and French isolates, as well as an isolate (isolate 52) of unknown origin (greater than 99% similarity). The isolates in primary cluster B shared less than 90% simi- larity with the isolates in primary cluster A. The two F. tula- rensis subsp. novicida isolates, which were slightly different from each other, were assigned to primary cluster C and had about 72% similarity to the other F. tularensis subspecies. Fi- nally, a genetically distant cluster (cluster D) was generated for F. philomiragia (less than 24% similarity to the F. tularensis strains). This species had just 15 AFLP bands in common with the other strains (data not shown).
the positive ion mode for all of the samples analyzed in this study. After Pareto scaling and mean-centering, these data were displayed as scores and loadings in a coordi- nate system of principal components resulting from data dimensionality reduction. The three-component PCA score plots (Fig. 2A) showed that the 33 Curcumae Radix samples could be clearly classified into four differ- ent clusters depending on their species, according to the differences in their global chemical profiles. Although the clusters corresponding to C. wenyujin and C. phaeocau- lis were close to each other, they could be clearly distin- guished by PC2. Sevenfold cross validation was used to assess the validity of the model. Notably, all of the obser- vations in the current study fell within the Hotelling T2 (0.95) ellipse. Furthermore, the R 2 X (cum) and Q 2 (cum)
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To date, DNA-DNA hybridization and G+C content determination  remain the gold standard meth- ods for the definition of bacterial species, despite the development of 16S rRNA PCR and sequenc- ing which have deeply changed bacterial taxono- my . Over recent years, high throughput ge- nome sequencing provided a wealth of genetic information . In an effort to include genomic data in bacterial taxonomy we recently used a polyphasic approach  that includes genomic data, MALDI-TOF spectrum and main phenotypic characteristics to describe new bacterial species [5,6] .
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A study was conducted to evaluate the new VITEK 2 system (bioMérieux) for identification and antibiotic susceptibility testing of gram-positive cocci. Clinical isolates of Staphylococcus aureus (n ⴝ 100), coagulase- negative staphylococci (CNS) (n ⴝ 100), Enterococcus spp. (n ⴝ 89), Streptococcus agalactiae (n ⴝ 29), and Streptococcus pneumoniae (n ⴝ 66) were examined with the ID-GPC identification card and with the AST-P515 (for staphylococci), AST-P516 (for enterococci and S. agalactiae) and AST-P506 (for pneumococci) suscepti- bility cards. The identification comparison methods were the API Staph for staphylococci and the API 20 Strep for streptococci and enterococci; for antimicrobial susceptibility testing, the agar dilution method according to the procedure of the National Committee for Clinical Laboratory Standards (NCCLS) was used. The VITEK 2 system correctly identified to the species level (only one choice or after simple supplementary tests) 99% of S. aureus, 96.5% of S. agalactiae, 96.9% of S. pneumoniae, 92.7% of Enterococcus faecalis, 91.3% of Staphylococcus haemolyticus, and 88% of Staphylococcus epidermidis but was least able to identify Enterococcus faecium (71.4% correct). More than 90% of gram-positive cocci were identified within 3 h. According to the NCCLS break- points, antimicrobial susceptibility testing with the VITEK 2 system gave 96% correct category agreement, 0.82% very major errors, 0.17% major errors, and 2.7% minor errors. Antimicrobial susceptibility testing showed category agreement from 94 to 100% for S. aureus, from 90 to 100% for CNS, from 91 to 100% for enterococci, from 96 to 100% for S. agalactiae, and from 91 to 100% for S. pneumoniae. Microorganism-antibiotic combinations that gave very major errors were CNS-erythromycin, CNS-oxacillin, enterococci-teicoplanin, and enterococci-high-concentration gentamicin. Major errors were observed for CNS-oxacillin and S. agalactiae- tetracycline combinations. In conclusion the results of this study indicate that the VITEK 2 system represents an accurate and acceptable means for performing identification and antibiotic susceptibility tests with med- ically relevant gram-positive cocci.
Since harbours are protected waterways, often with lim- ited water circulation and surrounded by urban and in- dustrial activities, pollutants frequently accumulate on the bottom over time (Reish and Gerlinger 1997). Ceuta harbour is considerably polluted, but provided with a channel which increases the water renewal inside the harbour (Guerra-Garca 2001). Due to this channel, the polychaete species richness inside Ceuta harbour is un- usually high compared with harbours in southern Spain, such as Saladillo harbour, studied by Estacio (1996) and Estacio et al. (1997). This harbour, located in Algeciras Bay, shows similar levels of sediment pollution to Ceuta harbour. However, it has only one entrance and the mac- rofaunal communities are very poor inside this harbour. Even polychaete species, traditionally considered to be more resistant to pollution than crustaceans and molluscs, occur at considerably lower numbers in Saladillo harbour than in Ceuta harbour.