New Bird Sexing Strategy Developed in the Order Psittaciformes Involves Multiple Markers to Avoid Sex Misidentification: Debunked Myth of the Universal DNA Marker.

Sexing of birds is indispensable for scientific, breeding and conservation programs but is difficult in many species and is particularly problematic in the case of nestlings showing no sexual dimorphism. Most useful and efficient methods of sex determination are based on unique features of the Z and W sex chromosomes detected via PCR to distinguish males (ZZ) and females (ZW). During the last twenty-five years researchers searched for the universal marker capable of sexing a maximally wide spectrum of species in a single PCR assay. We screened the phylogenetically representative set of 135 Psittaciformes species including 59 species sexed for the first time. Two known (P2P8, CHD1iA) PCR markers and four additional W/Z polymorphisms (CHD1iE, CHD1i16, CHD1i9 and NIPBLi16) located within the Chromo Helicase DNA binding CHD1 or the Nipped-B homolog NIPBL genes were applied. We present the electrophoretic patterns obtained for the PCR products of the analyzed markers including most typical and atypical patterns allowing sex determination, as well as those obtained when the given marker failed in sexing. Technical aspects of molecular sex determination are discussed: the optimization of amplification conditions, direct PCR and potential misinterpretations. A truly universal marker has not been found, and therefore, we propose a sexing strategy based on multiple CHD1i16, NIPBLi16, CHD1i9 and CHD1iE markers. This new strategy confirms the sex of a given bird with at least two markers detecting independent Z/W polymorphisms, reduces the number of necessary PCR reactions and minimizes the risk of sex misidentification.

The first complete mitochondrial genome of Pyrrhura sp.--question about conspecificity in the light of hybridization between Pyrrhura molinae and Pyrrhura rupicola species.

The genus Pyrrhura contains at least 24 species widespread mainly through South America. Pyrrhura molinae (Green-cheeked Conure) and Pyrrhura rupicola (Black-capped Conure) are treated as separate species based on morphological differences. Geographical ranges of these two species overlap in southern Peru. However, it is not clear if they occur there sympatrically or it is a hybridization zone. Some taxonomists conjecture their conspecific character, as possible hybrid individuals have been found in Puno region. We sequenced mitochondrial genome of specimen with phenotype typical for P. molinae. To confirm its species belonging, we aligned CYTB and control region (CR) sequences with adequate sequences of 21 Pyrrhura species. Surprisingly, analyzed P. molinae individual grouped together with two different individuals of P. rupicola species. Such incompatibility of genetic data with morphology may be only the result of these two species hybridization.

Complete mitochondrial genome of endangered Maroon-fronted Parrot (Rhynchopsitta terrisi) - conspecific relation of the species with Thick-billed Parrot (Rhynchopsitta pachyrhyncha).

Maroon-fronted Parrot (Rhynchopsitta terrisi) is an endangered parrot endemic to pine-oak forests in north-eastern Mexico. According to all present classifications, R. terrisi as well as Rhynchopsitta pachyrhyncha are treated as a separate species based on some morphological and behavioral discrepancies. Despite this formal separation of the two species, some taxonomists conjecture their conspecific character. However, mutual taxonomic position of both species/subspecies was never verified by molecular research. We sequenced full mitochondrial genome of R. terrisi and constructed phylogenetic tree using sequences of mitochondrial ND2 gene from R. terrisi. R. pahyrchyncha and some other representative species of the tribe Arini. Our results indicated that in contrast to formal classification, both Rhynchopsitta taxa should be treated as subspecies.

Complete mitochondrial genome of Blue-crowned Parakeet (Aratinga acuticaudata)--phylogenetic position of the species among parrots group called Conures.

Blue-crowned Parakeet (Aratinga acuticaudata) is a South American parrot species with a taxonomic position not confirmed by molecular studies. We sequenced full mitochondrial genome and constructed phylogenetic tree using sequences of mitochondrial ND2 gene from A. acuticaudata and some other representatives of Conures group. Our results confirmed previously described distribution of Aratinga species into three clades, but surprisingly did not classify Blue-crowned Parakeet to any of them. We found that A. acuticaudata shares the closest relationship with Diopsittaca nobilis and forms a separate clade together with Guaruba guarouba and Leptosittaca branickii species with a significant node. Our results confirm lack of monophyly of the genus Aratinga and underline the need of its taxonomic revision.