Principle components analysis (PCA) was applied to analyse multivariate data; however, most multivariate analyses were uninformative due to the large amount of missing data from the fossil specimens.
We found considerable intraspecific variation and well-supported phylogenetic structure in P. insularum (Fig. 1), although missing data from a few museum specimens degraded clade support in the P. insularum-P.
Representativeness and generalization of the results to the entire MSM population in Norway might not be feasible, as Internet sampling is subject to selection bias, misrepresentation as a member of the sampled population, repeated participation, missing data, inability to gather biological specimens etc. [ 9].
The genes 18S, EF-1α, and COI were not collected from 11, 7, and 2 specimens, respectively, and, except for one sample of Pedicinus pictus (Pedicinus pictus 2), none of the specimens analyzed were missing data from more than one molecular marker (Additional File 1).
Reasons for missing data stemmed from insufficient supplies or specimen volumes and were thus assumed to be missing at random [ 20]; with the exception of weight, which could be related to severity of illness and short-term mortality.
Because of missing data of either CD8 or FOXP3 expression, 409 specimen for CD8 and 412 specimens for FOXP3 were available for analysis.
For A39 the first portion still could not be amplified in the herbarium specimens, and hence was scored as missing data for those individuals.
Although specimen age did negatively affect both the number of quality reads and the amount of missing data per sample (Fig. 1), this effect was less pronounced for specimens >10 yr old.
Although most data collection was primarily prospectively performed, pathology data were obtained retrospectively once printed specimen pathology reports became available, or missing data were found on later review.
Missingness, or the percentage of these SNPs exhibiting missing data in a given sample, was significantly higher in older specimens (r = 0.22, P = 9.2 × 10−7) (Fig. 1B).
