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    Comprehensive characterisation of Culicoides clastrieri and C. festivipennis (Diptera: Ceratopogonidae) according to morphological and morphometric characters using a multivariate approach and DNA barcode

    Molecular analyses
    Results of molecular analyses
    The sequences obtained are available in GenBank (Supplementary Information 1). Sequence alignments were 399 bp for COI and 587 bp for 28S including gaps.
    Phylogenetic analysis
    Our molecular analysis (Fig. 1) with both markers generated seven supported clusters, six of which were in agreement with the morphological determination (i.e. C. alazanicus, C. brunnicans, C. circumscriptus, C. furcillatus, C. nubeculous and C. pictipennis). However, one cluster (i.e. two species) corresponded to undistinguished C. clastrieri and C. festivipennis.
    Figure 1

    Block diagram of the study.

    Full size image

    In addition, the COI mtDNA tree shows that C. furcillatus is the sister of the “C. clastrieri/festivipennis” clade. Indeed, C. pictipennis is the sister species of C. brunnicans while C. circumscriptus is positioned between the two clades.
    Moreover, the 28S rDNA tree shows that C. pictipennis is the sister of the “C. clastrieri/festivipennis” clade. The other species are positioned in several places without a clade.
    Intra- and inter-specific comparison
    The COI Genbank sequences show little intraspecific divergence in both C. clastrieri (0.1 ± 0.1%) and C. festivipennis (1.2 ± 0.4%). The interspecific difference between C. clastrieri and in C. festivipennis is 0.7 ± 0.2%.
    Small intraspecific divergences with COI sequences were observed in our sample: C. alazanicus (1.2 ± 0.4%), C. brunnicans (0.7 ± 0.2%), C. circumscriptus (2.2 ± 0.5%), C. clastrieri (0.3 ± 0.1%), C. festivipennis (0.4 ± 0.1%), C. furcillatus (1.5 ± 0.4%), C. nubeculosus (0.2 ± 0.1%) and C. pictipennis (1.1 ± 0.3%).
    Finally, C. festivipennis and C. clastrieri—grouped in the same main clade—showed small interspecific distances (0.4 ± 0.2%); these were not identified as separate species based on DNA barcodes. We therefore decided to create a new group (C. clastrieri/festivipennis clade) based on interspecific distance. The overall mean genetic distance (K2P) computed for the different species of Culicoides was found to be 16.6 ± 1.4%. Interspecific K2P values for different (Table 1) species and taxa ranged from 27.3% (between C. furcillatus and C. nubeculosus; between C. circumscriptus-and C. furcillatus) to 17.2 ± 2.1% (between C. circumscriptus and the C. clastrieri/festivipennis clade) for our samples. For the COI Genbank sequences, we observed approximatively the same proportion and the same species (Table 1). We remarked very little interspecific divergence between our sample of the C. clastrieri/festivipennis clade and the C. clastrieri/festivipennis Genbank clade (0.6 ± 0.4%).
    Table 1 Estimation of pairwise distance (± SD) of the Culicoides species for the COI domain of the mtDNA and D1D2 region of the rDNA.
    Full size table

    Analysis from 28S rDNA sequences did not show any intraspecific divergence whatever the taxa (0.000) with the exception of C. nubeculosus (0.1 ± 0.1%) and C. festivipennis/C.clastrieri (0.1 ± 0%). The overall mean genetic distance (K2P) computed for the different species of Culicoides was found to be 2.1 ± 0.03%. Interspecific K2P values for different species (Table 1) and taxa ranged from 1.2% (between C. circumscriptus and C. furcillatus; C. furcillatus and C. brunnicans, the main C. clastrieri/festivipennis clade and C. furcillatus) to 5.3 ± 0.9% (between C. circumscriptus and C. nubeculosus).
    Morphometric and morphological analyses
    In all, 148 specimens identified as C. alazanicus (n = 10), C. brunnicans (n = 27), C. circumscriptus (n = 27), C. clastrieri (n = 21), C. festivipennis (n = 20), C. furcillatus (n = 14), C. nubeculosus (n = 19) and C. pictipennis (n = 20) were analysed with 11 wing landmarks/specimens (Fig. 2).
    Figure 2

    Trees obtained from nucleotide analysis of: (a) COI mtDNA; (b) 28S rDNA (with MP method) sequences of C. alazanicus, C. brunnicans, C. circumscriptus C. clastrieri, C. festivipennis, C. furcillatus, C. nubeculosus and C. pictipennis and bootstrap values are shown in nodes (1000 replicates).

    Full size image

    Principal component analyses
    Principal component analysis (PCA) was used to observe possible grouping trends.
    Firstly, we performed a first normed PCA using the “Wing landmarks” model. The first three axes accounted for 76%, 15% and 8% of the total variance, which suggests a weak structuration of the data. This was confirmed by a scatterplot of PCA axes 1 and 2 that was unable to separate the species (Fig. 3).
    Figure 3

    Principal component analysis (PCA): percentage of variance explained for each PCA dimension and results.

    Full size image

    Secondly, we performed a first normed PCA on the “Wing morphological characters” model. The various specimens of each species are represented by a single point suggesting a close correlation of wing morphological characters. This model, without variance, is not validated and does not permit species separation.
    We studied the “Full wing (landmarks and morphological, characters)” model through a normed PCA on raw data. C. clastrieri could be clearly separated from C. festivipennis. The first five axes accounted for 40%, 25%, 12%, 10% and 5% of the total variance. The scatterplot separated unambiguously and without overlap C. clastrieri-C. festivipennis on the one hand and the six species on the other hand (Fig. 3).
    Finally, we performed a first normed PCA on the “Full model” (Morphological characters—wing, head, abdomen, legs—and wing landmarks). The first nine axes accounted for 26%, 23%, 22%, 10%, 8%., 4%, 3%, 2% and 1% of the total variance, which reveals good structuration of the data. This was confirmed by a scatterplot of PCA axes 1 and 2 that presents the same topology as the wing morphological model (Fig. 3).
    This supports discrimination according to the species’ wing pattern. Similarly, and some body pattern characters could be used to identify Culicoides from the clastrieri/festivipennis clade better and quicker. With that objective in mind, we performed analyses on three datasets: (1) “Wing landmarks” (11 landmarks); (2) “Full wing” (38 items) and (3) the “Full model” that includes 71 items.
    Discriminant analyses
    PLS-DA and sPLS-DA models were used in order to discriminate the extremes (i.e. the most sensitive and most robust groups) using the three datasets (species, models and components) as described. The accuracy and the balanced error rate (BER) for the two models were compared and are summarised in Supplementary Information 2 and Fig. 4.
    Figure 4

    Balanced error rate (BER) choosing the number of dimensions. Performance and ncomp selection.

    Full size image

    The tuning step of the number of components to select showed that 16 components were necessary to lower the BER (Fig. 4A,B) for the “Wing landmarks” data. The AUC values with 16 components are as follows: C. alazanicus (0.97, p  More

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    Monkeys fight more in polluted air

    Monkey conflict data
    We obtained social conflict data ofNorthern China Rhesus Monkeys from Hongshan Forest Zoo of Nanjing, China. Nanjing (31° 14′–32° 37′ N, 118° 22′–119° 14′ E) is located in the central region of the lower Yangtze River and southwest of Jiangsu Province. It is an important national gateway city for the development of the central and western regions in the Yangtze River Delta, with an area of 6587 km2 covering a population of more than 8 Million. Average annual temperature is about 15.4 °C. Annual precipitation is 1106 mm, 60% of which occurs from Jun to Sep.
    There are about 90 monkeys in the Hongshan Zoo in 2017, about 35 adults, 20 sub-adults and 35 juveniles or new-borns. The round monkey park was located in the central part of the zoo, with an area of about 2000 m2. Although a thick and 3-m high glass wall has been built to prevent artificial feedings, visitors sometimes throw food into the monkey park, causing a social conflict due to the food competition. Usually the zookeeper feeds these monkeys twice a day at about 9:30 am and 3:30 pm respectively.
    We established a monitoring camera web (Haikang DS-7104N-SN/P) covering the monkey park in September 2016 and video-recorded the whole population since then. We defined social conflicts of monkeys as aggressive or fighting behaviors between individuals, including chasing (one chases another until it escapes), wrestling (one grapples and wrestles with another, until one escapes or gives up), biting (one opens its mouth and bites or tries to bites another), scratching (One scratches or scrapes another using its hands), threating (One warns or threats another through calling or behavioural display), etc. The age of participants and the occurrence time were recorded for each aggression46. We considered a conflict ends if there is no continuation within 10 s after the aggression. Since these monkeys are inactive during the night, we only recorded their diurnal aggressive behaviors from 6:30 till 18:30 and then summed the fights as daily social conflicts. One-year round data were collected from Mar 2017 to Feb 2018.
    Air Quality Index
    We obtained Air Quality Index (AQI) data of Nanjing from the Data Centre of the Ministry of Environmental Protection of the People’s Republic of China (MEP, http://datacenter.mep.gov.cn/)17. Based on established criteria (GB3095-2012). AQI is calculated for six major air pollutants separately: particle matter  More

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    Anthropogenic interferences lead to gut microbiome dysbiosis in Asian elephants and may alter adaptation processes to surrounding environments

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