In Silico Analysis of Seven PCR Markers Developed from the CHD1, NIPBL and SPIN Genes Followed by Laboratory Testing Shows How to Reliably Determine the Sex of Musophagiformes Species.

Sex determination in birds, due to the very common lack of sexual dimorphism, is challenging. Therefore, molecular sexing is often the only reliable way to differentiate between the sexes. However, for many bird species, very few genetic markers are available to accurately, quickly, and cost-effectively type sex. Therefore, in our study, using 14 species belonging to the order Musophagiformes, we tested the usefulness of seven PCR markers (three of which have never been used to determine the sex of turacos), developed based on the CHD1, NIPBL, and SPIN genes, to validate existing and develop new strategies/methods of sex determination. After in silico analysis, for which we used the three turaco nuclear genomes available in GenBank, the suitability of the seven selected markers for sexing turacos was tested in the laboratory. It turned out that the best of the markers tested was the 17th intron in the NIPBL gene (not previously tested in turacos), allowing reliable sex determination in 13 of the 14 species tested. For the one species not sexed by this marker, the 9th intron in the CHD1 gene proved to be effective. The remaining markers were of little (4 markers developed based on the CHD1 gene) or no use (marker developed based on the SPIN gene).

The Length Polymorphism of the 9th Intron in the Avian CHD1 Gene Allows Sex Determination in Some Species of Palaeognathae.

In palaeognathous birds, several PCR-based methods and a range of genes and unknown genomic regions have been studied for the determination of sex. Many of these methods have proven to be unreliable, complex, expensive, and time-consuming. Even the most widely used PCR markers for sex typing in birds, the selected introns of the highly conserved CHD1 gene (primers P2/P8, 1237L/1272H, and 2550F/2718R), have rarely been effective in palaeognathous birds. In this study we used eight species of Palaeognathae to test three PCR markers: CHD1i9 (CHD1 gene intron 9) and NIPBLi16 (NIPBL gene intron 16) that performed properly as Psittaciformes sex differentiation markers, but have not yet been tested in Palaeognathae, as well as the CHD1iA intron (CHD1 gene intron 16), which so far has not been used effectively to sex palaeognathous birds. The results of our research indicate that the CHD1i9 marker effectively differentiates sex in four of the eight species we studied. In Rhea americana, Eudromia elegans, and Tinamus solitarius, the electrophoretic patterns of the amplicons obtained clearly indicate the sex of tested individuals, whereas in Crypturellus tataupa, sexing is possible based on poorly visible female specific bands. Additionally, we present and discuss the results of our in silico investigation on the applicability of CHD1i9 to sex other Palaeognathae that were not tested in this study.

Mitogenomes of Accipitriformes and Cathartiformes Were Subjected to Ancestral and Recent Duplications Followed by Gradual Degeneration.

The rearrangement of 37 genes with one control region, firstly identified in Gallus gallus mitogenome, is believed to be ancestral for all Aves. However, mitogenomic sequences obtained in recent years revealed that many avian mitogenomes contain duplicated regions that were omitted in previous genomic versions. Their evolution and mechanism of duplication are still poorly understood. The order of Accipitriformes is especially interesting in this context because its representatives contain a duplicated control region in various stages of degeneration. Therefore, we applied an appropriate PCR strategy to look for duplications within the mitogenomes of the early diverged species Sagittarius serpentarius and Cathartiformes, which is a sister order to Accipitriformes. The analyses revealed the same duplicated gene order in all examined taxa and the common ancestor of these groups. The duplicated regions were subjected to gradual degeneration and homogenization during concerted evolution. The latter process occurred recently in the species of Cathartiformes as well as in the early diverged lineages of Accipitriformes, that is, Sagittarius serpentarius and Pandion haliaetus. However, in other lineages, that is, Pernis ptilorhynchus, as well as representatives of Aegypiinae, Aquilinae, and five related subfamilies of Accipitriformes (Accipitrinae, Circinae, Buteoninae, Haliaeetinae, and Milvinae), the duplications were evolving independently for at least 14-47 Myr. Different portions of control regions in Cathartiformes showed conflicting phylogenetic signals indicating that some sections of these regions were homogenized at a frequency higher than the rate of speciation, whereas others have still evolved separately.

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.

New view on the organization and evolution of Palaeognathae mitogenomes poses the question on the ancestral gene rearrangement in Aves.

BACKGROUND: Bird mitogenomes differ from other vertebrates in gene rearrangement. The most common avian gene order, identified first in Gallus gallus, is considered ancestral for all Aves. However, other rearrangements including a duplicated control region and neighboring genes have been reported in many representatives of avian orders. The repeated regions can be easily overlooked due to inappropriate DNA amplification or genome sequencing. This raises a question about the actual prevalence of mitogenomic duplications and the validity of the current view on the avian mitogenome evolution. In this context, Palaeognathae is especially interesting because is sister to all other living birds, i.e. Neognathae. So far, a unique duplicated region has been found in one palaeognath mitogenome, that of Eudromia elegans. RESULTS: Therefore, we applied an appropriate PCR strategy to look for omitted duplications in other palaeognaths. The analyses revealed the duplicated control regions with adjacent genes in Crypturellus, Rhea and Struthio as well as ND6 pseudogene in three moas. The copies are very similar and were subjected to concerted evolution. Mapping the presence and absence of duplication onto the Palaeognathae phylogeny indicates that the duplication was an ancestral state for this avian group. This feature was inherited by early diverged lineages and lost two times in others. Comparison of incongruent phylogenetic trees based on mitochondrial and nuclear sequences showed that two variants of mitogenomes could exist in the evolution of palaeognaths. Data collected for other avian mitogenomes revealed that the last common ancestor of all birds and early diverging lineages of Neoaves could also possess the mitogenomic duplication. CONCLUSIONS: The duplicated control regions with adjacent genes are more common in avian mitochondrial genomes than it was previously thought. These two regions could increase effectiveness of replication and transcription as well as the number of replicating mitogenomes per organelle. In consequence, energy production by mitochondria may be also more efficient. However, further physiological and molecular analyses are necessary to assess the potential selective advantages of the mitogenome duplications.

Resolving Phylogenetic Relationships within Passeriformes Based on Mitochondrial Genes and Inferring the Evolution of Their Mitogenomes in Terms of Duplications.

Mitochondrial genes are placed on one molecule, which implies that they should carry consistent phylogenetic information. Following this advantage, we present a well-supported phylogeny based on mitochondrial genomes from almost 300 representatives of Passeriformes, the most numerous and differentiated Aves order. The analyses resolved the phylogenetic position of paraphyletic Basal and Transitional Oscines. Passerida occurred divided into two groups, one containing Paroidea and Sylvioidea, whereas the other, Passeroidea and Muscicapoidea. Analyses of mitogenomes showed four types of rearrangements including a duplicated control region (CR) with adjacent genes. Mapping the presence and absence of duplications onto the phylogenetic tree revealed that the duplication was the ancestral state for passerines and was maintained in early diverged lineages. Next, the duplication could be lost and occurred independently at least four times according to the most parsimonious scenario. In some lineages, two CR copies have been inherited from an ancient duplication and highly diverged, whereas in others, the second copy became similar to the first one due to concerted evolution. The second CR copies accumulated over twice as many substitutions as the first ones. However, the second CRs were not completely eliminated and were retained for a long time, which suggests that both regions can fulfill an important role in mitogenomes. Phylogenetic analyses based on CR sequences subjected to the complex evolution can produce tree topologies inconsistent with real evolutionary relationships between species. Passerines with two CRs showed a higher metabolic rate in relation to their body mass.

New Insight into Parrots' Mitogenomes Indicates That Their Ancestor Contained a Duplicated Region.

Mitochondrial genomes of vertebrates are generally thought to evolve under strong selection for size reduction and gene order conservation. Therefore, a growing number of mitogenomes with duplicated regions changes our view on the genome evolution. Among Aves, order Psittaciformes (parrots) is especially noteworthy because of its large morphological, ecological, and taxonomical diversity, which offers an opportunity to study genome evolution in various aspects. Former analyses showed that tandem duplications comprising the control region with adjacent genes are restricted to several lineages in which the duplication occurred independently. However, using an appropriate polymerase chain reaction strategy, we demonstrate that early diverged parrot groups contain mitogenomes with the duplicated region. These findings together with mapping duplication data from other mitogenomes onto parrot phylogeny indicate that the duplication was an ancestral state for Psittaciformes. The state was inherited by main parrot groups and was lost several times in some lineages. The duplicated regions were subjected to concerted evolution with a frequency higher than the rate of speciation. The duplicated control regions may provide a selective advantage due to a more efficient initiation of replication or transcription and a larger number of replicating genomes per organelle, which may lead to a more effective energy production by mitochondria. The mitogenomic duplications were associated with phenotypic features and parrots with the duplicated region can live longer, show larger body mass as well as predispositions to a more active flight. The results have wider implications on the presence of duplications and their evolution in mitogenomes of other avian groups.

The influence of molecular markers and methods on inferring the phylogenetic relationships between the representatives of the Arini (parrots, Psittaciformes), determined on the basis of their complete mitochondrial genomes.

BACKGROUND: Conures are a morphologically diverse group of Neotropical parrots classified as members of the tribe Arini, which has recently been subjected to a taxonomic revision. The previously broadly defined Aratinga genus of this tribe has been split into the 'true' Aratinga and three additional genera, Eupsittula, Psittacara and Thectocercus. Popular markers used in the reconstruction of the parrots' phylogenies derive from mitochondrial DNA. However, current phylogenetic analyses seem to indicate conflicting relationships between Aratinga and other conures, and also among other Arini members. Therefore, it is not clear if the mtDNA phylogenies can reliably define the species tree. The inconsistencies may result from the variable evolution rate of the markers used or their weak phylogenetic signal. To resolve these controversies and to assess to what extent the phylogenetic relationships in the tribe Arini can be inferred from mitochondrial genomes, we compared representative Arini mitogenomes as well as examined the usefulness of the individual mitochondrial markers and the efficiency of various phylogenetic methods. RESULTS: Single molecular markers produced inconsistent tree topologies, while different methods offered various topologies even for the same marker. A significant disagreement in these tree topologies occurred for cytb, nd2 and nd6 genes, which are commonly used in parrot phylogenies. The strongest phylogenetic signal was found in the control region and RNA genes. However, these markers cannot be used alone in inferring Arini phylogenies because they do not provide fully resolved trees. The most reliable phylogeny of the parrots under study is obtained only on the concatenated set of all mitochondrial markers. The analyses established significantly resolved relationships within the former Aratinga representatives and the main genera of the tribe Arini. Such mtDNA phylogeny can be in agreement with the species tree, owing to its match with synapomorphic features in plumage colouration. CONCLUSIONS: Phylogenetic relationships inferred from single mitochondrial markers can be incorrect and contradictory. Therefore, such phylogenies should be considered with caution. Reliable results can be produced by concatenated sets of all or at least the majority of mitochondrial genes and the control region. The results advance a new view on the relationships among the main genera of Arini and resolve the inconsistencies between the taxa that were previously classified as the broadly defined genus Aratinga. Although gene and species trees do not always have to be consistent, the mtDNA phylogenies for Arini can reflect the species tree.

Complete mitochondrial genome of bronze-winged parrot (Pionus chalcopterus chalcopterus, Psittaciformes).

Medium-sized neotropical parrots from Pionus genus are represented by at least eight species. However, their taxonomy should be revised because some external morphological characters together with genetic data recognize 19 taxa. At present, only two mitochondrial markers are available for most of these taxa and obtained phylogenies are not well resolved. Therefore, we sequenced Pionus chalcopterus chalcopterus mitogenome to gain more molecular data required for future studies of the taxonomical status and phylogenetic relationships between Pionus taxa. Performed phylogenetic analyses showed seven monophyletic clades including at least two sequences assigned to one species. However, not all subspecies sequences were monophyletic.

The complete mitochondrial genome of red-fronted parrot (Poicephalus gulielmi) revealed a new gene rearrangement within the order Psittaciformes.

Vertebrate mitogenomes are thought to be selected for compactness. Therefore, the increasing number of avian mitogenomes comprising duplicated regions is surprising. Such regions were proposed for at least 26 parrot genera based on the length of PCR products. However, complete mitogenomes with the duplications were shown only for six genera. These duplications evolved probably from the ancestral tRNATHR/tRNAPRO/ND6/tRNAGLU/CR and were subjected to subsequent degeneration. Here, we report the mitogenome of Poicephalus gulielmi (the subfamily Psittacinae) with a unique duplication tRNATHR/pseudoND6/CR1/tRNAPRO/ND6/tRNAGLU/CR2. This region is different from all other identified regions and resembles mostly the arrangements in Amazona and Pionus from the subfamily Arinae.

Complete mitochondrial genome of chestnut-fronted macaw (Ara severus, Psittaciformes).

Six genera from Arini tribe form a morphologically diverse group named macaws, which differ from other Arini in the presence of bare facial area. Macaws are further distinguished by the bare face pattern, plumage colouration and body size. Six of the eight macaw species from Ara genus can be easily segregated into three pairs according to their colouration. An exception is Ara severus, which differs from others in their size and morphology. The lack of appropriate molecular markers precludes determination of its phylogenetic position. Therefore, the mitogenome of Ara severus presented in this report will be indispensable to refine these phylogenetic relationships.

Complete mitochondrial genome of the greater Antillean parrot Amazona ventralis (Hispaniolan amazon).

Androglossini is one of the four tribes recognized within the group of neotropical parrots. The tribe includes 10 genera of which Amazona genus is presently represented by about 30 species. The number of species may increase in the future because paraphyly of some Amazona species was recently demonstrated and some subspecies were proposed to be elevated to the species rank. Evolutionary history of Amazona genus also remains unresolved because published phylogenies suggest contradictory scenarios concerning directions of islands-mainland colonization. Therefore, we sequenced mitogenome of Amazona ventralis from Greater Antilles to gain molecular data essential in future examination of this genus diversification.

The first complete genome of 'true' Aratinga genus in comparison to mitogenomes of other parrots from Arini tribe.

Arini with 19 genera is the most diversified tribe of the neotropical parrots from Arinae subfamily. So far, among this tribe, the genus Aratinga appeared to be the most problematic for the taxonomists. The typical representative of this genus is Aratinga solstitialis, whose complete mitochondrial genome were sequenced and compared with five other representatives of Arini mitogenomes. Despite the conservatism in their general organization, some changes in A + T% composition of individual genes, start/stop codon usage and intergenic regions accumulated during evolution.

Complete mitochondrial genome of Red-throated Conure (Psittacara rubritorquis): its comparison with mitogenome of Socorro Conure (Psittacara brevipes).

According to some taxonomists the Red-throated Conure (Psittacara rubritorquis) is considered a subspecies of Green Conure (Psittacara holochlora). Some other classifications treat rubritorquis as a separate species based on relatively minor morphological differences between both species/subspecies. So far, taxonomic position of P. rubritorquis was determined by molecular researches using only ND2 gene sequence or incomplete combined mitochondrial ND2, COI and CYTB gene sequences. Obtained outcomes found that P. rubritorquis should be treated as a subspecies of P. holochlora. However, the lack of P. h. brewsterii and P. h. strenua samples as well as incompleteness of combined mitochondrial sequence do not exclude opposite scenario. Therefore, we sequenced P. rubritorquis mitogenome to gain a source of molecular data appropriate for future examination of evolutionary diversification of the P. holochlora group.

Complete mitochondrial genome of endangered Socorro Conure (Aratinga brevipes) - taxonomic position of the species and its relationship with Green Conure.

Socorro Conure (Aratinga brevipes.Aratinga holochlora brevipes) is a parrot endemic to the Island of Socorro. According to some taxonomists the species is considered a subspecies of Green Conure (Aratinga holochlora). Some other classifications treat brevipes as a separate species based on relatively minor morphological differences between both species/subspecies. However, taxonomic position of Aratinga brevipes was never determined by molecular research. We sequenced full mitochondrial genome of the species and constructed phylogenetic tree using sequences of mitochondrial ND2 gene from A. brevipes and some other representatives of Conures group. Our results showed, that despite Aratinga brevipes is closely related to Aratinga holochlora, this Conure should be treated as a separate species.