Duration of embryo development and the prevalence of haematozoan blood parasites in birds

This study is concerned with statistical methods used for the analysis of comparative data (in which observations are not expected to be independent because they are sampled across phylogenetically related species). The phylogenetically independent contrasts (PIC), phylogenetic generalized least-squares (PGLS), and phylogenetic autocorrelation (PA) methods are compared. Although the independent contrasts are not orthogonal, they are independent if the data conform to the Brownian motion model of evolution on which they are based. It is shown that uncentered correlations and regressions through the origin using the PIC method are identical to those obtained using PGLS with an intercept included in the model. The PIC method is a special case of PGLS. Corrected standard errors are given for estimates of the ancestral states based on the PGLS approach. The treatment of trees with hard polytomies is discussed and is shown to be an algorithmic rather than a statistical problem. Some of the relationships among the methods are shown graphically using the multivariate space in which variables are represented as vectors with respect to OTUs used as coordinate axes. The maximum-likelihood estimate of the autoregressive parameter, p, has not been computed correctly in previous studies (an appendix with MATLAB code provides a corrected algorithm). The importance of the eigenvalues and eigenvectors of the connection matrix, W, for the distribution of p is discussed. The PA method is shown to have several problems that limit its usefulness in comparative studies. Although the PA method is a generalized least-squares procedure, it cannot be made equivalent to the PGLS method using a phylogenetic model.

We compared information obtained by both microscopy and nested mitochondrial cytochrome b PCR in determining prevalence of haemosporidian infections in naturally infected birds. Blood samples from 472 birds of 11 species belonging to 7 families and 4 orders were collected in Europe, Africa and North America. Skilled investigators investigated them using the PCR-based screening and microscopic examination of stained blood films. The overall prevalence of haemosporidian infections, which was determined combining results of both these methods, was 60%. Both methods slightly underestimated the overall prevalence of infection, which was 54.2% after the PCR diagnostics and 53.6% after microscopic examination. Importantly, both these tools showed the same trends of prevalence of Haemoproteus spp. (21% by PCR and 22% by microscopy), Plasmodium spp. (17% and 22%) and Leucocytozoon spp. (30% and 25%) in the same sample, testifying that microscopy is a reliable tool in determining patterns of distribution of blood haemosporidian parasites in naturally infected birds. We encourage using optical microscopy in studies of blood parasites in parallel to the now widely employed molecular methods. Microscopy is relatively inexpensive and provides valuable information about directions how molecular methods can be further improved and most effectively applied, especially in the field studies of parasites. Importantly, blood films, which are used for microscopic examination, should be of good quality; they should be examined properly by skilled investigators. In spite of relatively long duration of microscopy of each sample, such examination provides opportunities for simultaneous determination and verification of taxonomically different parasites. Presently, different PCR protocols must be used for the detection of parasites belonging to different genera; this is expensive and time-consuming.