Significant differences were found in mentum deformities in F 1 between the control and the treatment with water from the site affected by mining mercury (differences of proportions test; p = 0.03). In contrast, no differences were found in mentum deformities in F 1 among the other treatments and the control (differences of proportions test; p >0.05). In F 2 significant differences were noted among the treatments with water affected by mining mercury-cyanide, cattle raising, and agriculture with the control (differences of proportions test; p <0.05). In tests with samples of water affected by mining mercury and the reference site, no differences were observed with the control (differences of proportions test; p >0.05). In addition, the greatest deformity in F 1 was registered in the treatment with water from the site with mining mercury (6.9%) and in F 2 in the assay with water from the site with cattle raising with 12.5% ( Table 1 ).

The study registered four types of deformities in the mentum of C. columbiensis only in the treatments; the control did not show any deformities. The deformities found were absence of teeth, increased number of teeth, fusion and space between teeth ( S1 – S4 Figs). The most frequent type in all the treatments was the absence of teeth and increased number of teeth, space between teeth and fusion of teeth were the least-frequent deformities ( Fig 2 ).

3.2 Morphometric variation of the mentum and wing

Landmark digitalization error was at 0.4% for size and 0.7% for shape of the mentum and wing of C. columbiensis, which are acceptable values, given that they do not exceed 10%. The analysis of variance revealed significant differences in the shape of the C. columbiensis mentum among the different treatments (p <0.000), but not among generations (p >0.05). The control group was different from all the treatments (p = 0.000), similar results were found for the reference site (p <0.01). The permutation test conducted on the regression between the centroid size and the shape vectors did not indicate any allometric effect on the fluctuating asymmetry in the C. columbiensis mentum (p >0.05), where size has a variation below 10%.

The CVA of the mentum shape explained 82% of the variance with the first two canonical axes (Fig 3A). The positive end of the CV1 grouped the individuals treated with water from the site affected by mining mercury-cyanide; those from the control group were grouped toward the negative CV1 axis. Results of the samples from the assays with water affected by mining mercury, cattle raising, agriculture, and of reference are grouped in the neutral point of CV1 and CV2.

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larger image TIFF original image Download: Fig 3. Canonical variate analysis in the shape of the Chironomus columbiensis mentum. A) Explain 82% of the total variance of the data in its two first exes, ellipses = 95%CI. B–E shows the shape variation of the mentum of Chironomus columbiensis in its four first canonic axes, which explain 95.5% of the total variance of the data. Light blue line: consensus composition of the shape. Dark blue line: disparities of the shape represented in each component. CV1: 68.2%, CV2: 13.8%, CV3: 7, and CV4: 6.5%. M = mercury and MC = mercury-cyanide. https://doi.org/10.1371/journal.pone.0210348.g003

Fig 3B–3E shows the shape variation of the mentum, represented in the four first canonic axes that explain 95.5% of the total variance. The CV1 with blunt teeth that lose sharpness (Fig 3B) represents the principal pattern of the mentum shape variation, with 68.2% of the total data variance. This pattern of the morphological variations occurs in all the treatments, separating them from the control group. The CV2, with 13.8% of the total mentum shape variation, consists in increased size of the lateral teeth and loss of sharpness of the inter-lateral teeth and of the central trifid tooth (Fig 3C). This pattern of morphological variations occurs in samples from systems affected by mining mercury, cattle raising, agriculture, and the site without anthropogenic impact (WAI), separating them from the control group. The CV3, with 7% of the total mentum shape variation, is altered by an increase in the length of the first two lateral teeth (Fig 3D). This pattern differentiates the samples treated with water from the site of cattle raising and that from the control. Lastly, CV4 –with 6.5% of the total mentum shape variation–consisted in an increase of the length and reduction of the width of the inter-lateral teeth (Fig 3E) and differentiates the agricultural treatment from the control group.

The ANOVA and MANOVA revealed significant differences in the fluctuating asymmetry of the C. columbiensis mentum among the different treatments and control (p <0.0001), but not among generations (p >0.6737). Furthermore, directional asymmetry was detected in all the treatments (p <0.0001); teeth on the left side of the mentum were frequently less sharp than those on the right side. The control group was different from all the treatments (p = 0.000). Similar results were found for the reference site (Dunnett’s test; p <0.001), with greater variation in individuals from the treatment with water from the sites with mining mercury-cyanide, agriculture, cattle raising, mining mercury, and reference site, in their order. The permutation test conducted on the regression between the centroid size and the shape asymmetry vectors did not indicate any allometric effect on the fluctuating asymmetry in the C. columbiensis mentum (p >0.05), where size had a variation below 10%.

The canonical variate analysis of the fluctuating asymmetry explained 83% of the variance with the first two canonic axes (Fig 4A). The positive end of CV1 grouped individuals from the control, while individuals from the group of mining mercury-cyanide were grouped towards the CV1 negative axis. Samples from the treatments with mining mercury, cattle raising, agriculture, and of reference are grouped in the neutral point of the CV1 and CV2.

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larger image TIFF original image Download: Fig 4. Canonical variate analysis of the fluctuating asymmetry of the Chironomus columbiensis mentum. A) Explain 83.04% of the variance of the data in its two first exes, ellipses = 95%CI. B–E shows the fluctuating asymmetry of Chironomus columbiensis in its four first canonic axes, which explain 93.4% of the total data variance. Light blue line: consensus decomposition of the shape. Dark blue line: disparities of the shape represented in each component. CV1: 39.8%, CV2: 21.3%, CV3: 17.7%, and CV4: 14.6%. M = mercury and MC = mercury-cyanide. https://doi.org/10.1371/journal.pone.0210348.g004

Fig 4B–4E shows the variation of the mentum’s symmetrical shape, represented in the first four canonic axes that explain 93.4% of the total variance.

The CV1, with 39.8% of the total data variance, illustrates that the principal deformity consists in a reduction of the first left lateral tooth and an increase of the last right lateral tooth. Additionally, a slight increase is observed of the left lateral trifid tooth, along with a slight reduction of the right (Fig 4B), principally in treatments with mining mercury, mining mercury-cyanide, agriculture, and the reference site, presenting significant differences with the control group. The CV2, with 21.3% of the total variance of the mentum shape shows, mainly in the agriculture treatment, displacement of the trifid tooth and the inter-lateral teeth to the left side of the mentum with greater proportion from the right side (Fig 4C). The CV3, with 17.7% of the total variance, reflects an increase of the left trifid tooth and a reduction of the right (Fig 4D) in treatments with mining mercury, mining mercury-cyanide, and agriculture. Lastly, CV4, with 14.6% of the total data variation, illustrates an increase of the lateral teeth from the left side and a reduction of the right (Fig 4E) in treatments with mining mercury and cattle raising.

With respect to the wing’s shape variation, analyses of principal components suggest the existence of sexual dimorphism in the wing of C. columbiensis adult individuals (Fig 5). In addition, sexual dimorphism does not alter the results of the treatments (Fig 5A), experiments (Fig 5B), and generations (Fig 5C) when working with equal numbers of males and females (Fig 5D). Fig 6 shows the points that represent the shape consensus and the deformation vectors, showing the magnitude and direction of the partial deformations represented in the first two principal axes. The PC1, with 66.6% of the total variance, indicates that the principal deformity consists in the wing’s width reduction in males, while the PC2 (16.4%) shows a reduction of the wing’s basal part in females (Fig 6).

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larger image TIFF original image Download: Fig 5. Principal components analysis of the wing of Chironomus columbiensis showing the sexual dimorphism among the individuals. M = mercury and MC = mercury-cyanide. Ellipses = 95%CI. A) Individuals per treatments; B) Individuals per experiments; C) Individuals per generation; D) Individuals per gender. https://doi.org/10.1371/journal.pone.0210348.g005

The ANOVA revealed significant differences in wing shape among the different treatments and the control (p < 0.00), but not among the generations (p = 0.1). The control was different from all the treatments (p < 0.001) and the treatments with water affected by agriculture and mining mercury-cyanide had the highest morphological variations. Besides, the results from the assay with the sample from the reference site were different from the treatments with water affected by the anthropic activities (p < 0.001). The CVA explained 90.3% of the variance with the first two axes (Fig 7A). While between the positive end of CV2 and the negative end of CV1 the individuals treated with water affected by agriculture are grouped, the positive axis of CV2 and CV1 gather the individuals from the assay with water from the reference site and the control. Samples from both treatments with water samples from the two mining sites and the cattle raising site are grouped in the negative axis of CV2 and in the neutral point of CV1.

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larger image TIFF original image Download: Fig 7. Canonical variate analysis of the Chironomus columbiensis wing. A) Explaining 90.3% of the variance of the data in its two first exes, ellipses = 95%CI. B–E shows the deformations represented in the first four canonic analyses, explaining 98.7% of the variance. Light blue line: consensus decomposition of the shape. Dark blue line: disparities of the shape represented in each component. CV1 = 60%, CV2: 22.3%, CV3: 5.2%, and CV4: 3.2%. M = mercury and MC = mercury-cyanide. https://doi.org/10.1371/journal.pone.0210348.g007

The first four CVAs show the most-relevant variations compared with the consensus of the shapes (Fig 7B–7E). While the light blue line shows the consensus of the shape of the Procrustes fit, the blue lines show the partial deformations represented in the four first canonic axes that explain 98.7% of the variance. The CV1 is the principal pattern of the wing’s shape variation (increased width and reduced basal part) with 68% of the total data variance (Fig 7B). This development instability pattern was registered in the groups treated with water from all the sites affected by anthropic activities, separating them from the control group and from the group treated with the reference sample. The CV2, with 22.3% of the total variation of the wing shape, was due to the increased width and reduced length in the basal part (Fig 7C). This development instability was evidenced in the treatments with samples of water from the sites affected by mining and cattle raising activities and from the reference site, separating them from the control group. The CV3, with 5.2% of the total wing-shape variation, is attributed to the increased length in the terminal part of the wing (Fig 7D) and was present in the assays with water affected by cattle raising and from the site without visible impact, separating them from the control. The CV4, with 3.2% of the total wing-shape variation, was due to the reduced width of the wing (Fig 7E) and was observed in the samples subjected to water from the WAI site and separating such from the control.

In summary, the morphology of the mentum and wing of C. columbiensis had greater variation in the treatment with mining mercury-cyanide, followed by agriculture, mining-mercury, cattle raising, and the WAI site, compared to the control.