Molecular and statistical approaches to the delimitation of Eimeriidae species: A case of extreme polymorphism in eimerian oocysts from plumbeous pigeons Patagioenas plumbea Vieillot, 1818 (Columbiformes) in South America

The current work aimed to analyze, morphologically, statistically and molecularly, oocysts shed from plumbeous pigeons Patagioenas plumbea Vieillot, 1818 from a locality at 2197m of altitude near the Agulhas Negras peak, the highest point of the State of Rio de Janeiro, southeastern Brazil. The oocysts were extremely polymorphic, being subspheroidal, ovoidal or ellipsoidal, in addition to having the random presence/absence of characteristic features associated with the oocyst wall, such as micropyle, micropyle cap, lateral micropyle and outer veil/rough wall. Linear regression confirmed the extreme polymorphism of oocysts, showing that if all combinations of taxonomic characters in oocysts (morphotypes) were overestimated, 19 different species could be identified/described. In contrast, the means comparison analysis between oocysts with presence/absence of characteristic features and the histograms showed equivalences and regularity in the distribution in the classes of measures, which indicate the presence of a single species in the measured oocysts. Molecular analyzes was performed from the isolation of individual oocysts of different morphotypes, which had their genetic material extracted, amplified and sequenced in 4 non-overlapping loci in cox1, cox3 genes and fragments of small and large subunit rDNA of mitochondrial DNA. The sequences were 100% identical between the morphotypes, with the exception of a very small divergence observed at the locus that partially covers the cox3 gene. The phylogenetic analysis was inconclusive for the locus within cox1 gene traditionally used for eimeriid coccidians, however the other loci should have a promising future for phylogenetic studies when more sequences for the same genic regions are deposited in GenBank. Finally, the multifactorial analysis of the current work supported that the polymorphic oocysts shed from P. plumbea are a single species, which was named as Eimeria patagioenasae, making this the twenty-second eimerian description from Columbiformes.