Polymorphism and transcription of Mhc class I genes in a passerine bird, the great reed warbler

 The class I genes of the major histocompatibility complex (Mhc) are here investigated for the first time in a passerine bird. The great reed warbler is a rare species in Sweden with a few semi-isolated populations. Yet, we found extensive Mhc class I variation in the study population. The variable exon 3, corresponding to the α₂ domain, was amplified from genomic DNA with degenerated primers. Seven different genomic class I sequences were detected in a single individual. One of the sequences had a deletion leading to a shift in the reading frame, indicating that it was not a functional gene. A randomly selected clone was used as a probe for restriction fragment length polymorphism (RFLP) studies in combination with the restriction enzyme Pvu II. The RFLP pattern was complex with 21–25 RFLP fragments per individual and extensive variation. Forty-nine RFLP genotypes were detected in 55 tested individuals. To study the number of transcribed genes, we isolated 14 Mhc class I clones from a cDNA library from a single individual. We found eight different sequences of four different lengths (1.3–2.2 kilobases), suggesting there are at least four transcribed loci. The number of nonsynonymous substitutions (d _N ) in the peptide binding region of exon 3 were higher than the number of synonymous substitutions (d _S ), indicating balancing selection in this region. The number of transcribed genes and the numerous RFLP fragments found so far suggest that the great reed warbler does not have a "minimal essential Mhc" as has been suggested for the chicken. Received: 13 May 1998 / Revised: 18 August 1998     

MHC class I typing in a songbird with numerous loci and high polymorphism using motif-specific PCR and DGGE

The major histocompatibility complex (MHC) has a central role in the specific immune defence of vertebrates. Exon 3 of MHC class I genes encodes the domain that binds and presents peptides from pathogens that trigger immune reactions. Here we develop a fast population screening method for detecting genetic variation in the MHC class I genes of birds. We found evidence of at least 15 exon 3 sequences in the investigated great reed warbler individual. The organisation of the great reed warbler MHC class I genes suggested that a locus-specific screening protocol is impractical due to the high similarity between alleles across loci, including the introns flanking exon 3. Therefore, we used motif-specific PCR to amplify two subsets of alleles (exon 3 sequences) that were separated with by DGGE. The motif-specific primers amplify a substantial proportion of the transcribed class I alleles (2-12 alleles per individual) from as many as six class I loci. Although not exhaustive, this gives a reliable estimate of the class I variation. The method is highly repeatable and more sensitive in detecting genetic variation than the RFLP method. The motif-specific primers also allow us to avoid screening pseudogenes. In our study population of great reed warblers, we found a high level of genetic variation in MHC class I, and no less than 234 DGGE genotypes were detected among 248 screened individuals.     

A 39-kb sequence around a blackbird Mhc class II gene: ghost of selection past and songbird genome architecture

To gain an understanding of the evolution and genomic context of avian major histocompatibility complex (Mhc) genes, we sequenced a 38.8-kb Mhc-bearing cosmid insert from a red-winged blackbird (Agelaius phoeniceus). The DNA sequence, the longest yet retrieved from a bird other than a chicken, provides a detailed view of the process of gene duplication, divergence, and degeneration ("birth and death") in the avian Mhc, as well as a glimpse into major noncoding features of a songbird genome. The peptide-binding region (PBR) of the single Mhc class II B gene in this region, Agph-DAB2, is almost devoid of polymorphism, and a still-segregating single-base-pair deletion and other features suggest that it is nonfunctional. Agph-DAB2 is estimated to have diverged about 40 MYA from a previously characterized and highly polymorphic blackbird Mhc gene, Aph-DAB1, and is therefore younger than most mammalian Mhc paralogs and arose relatively late in avian evolution. Despite its nonfunctionality, Agph-DAB2 shows very high levels of nonsynonymous divergence from Agph-DAB1 and from reconstructed ancestral sequences in antigen-binding PBR codons-a strong indication of a period of adaptive divergence preceding loss of function. We also found that the region sequenced contains very few other unambiguous genes, a partial Mhc- class II gene fragment, and a paucity of simple-sequence and other repeats. Thus, this sequence exhibits some of the genomic streamlining expected for avian as compared with mammalian genomes, but is not as densely packed with functional genes as is the chicken Mhc.     

Screening of Mhc variation in Atlantic salmon (Salmo salar): a comparison of restriction fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE) and sequencing

We compared three different molecular methods currently used for screening of Mhc variation in population studies of Atlantic salmon. Restriction fragment length polymorphism (RFLP) of the entire class II gene detected 22 haplotypes. Seventeen exon 2 sequences were obtained from individuals carrying the 22 haplotypes, two of which had not been detected by RFLP. The six alleles (27%) detected by RFLP and not by exon 2 sequencing probably resulted from sequence variation outside exon 2. Within exon 2, RFLP differentiated 88% of the sequences. Alternatively, denaturing gradient gel electrophoresis (DGGE) performed under two run conditions detected 94% of the sequence variation. Both RFLP using different probes, and the two PCR-based methods using three different primer pairs, suggest that there is only a single Mhc class II B gene in the Baltic populations of Atlantic salmon.     

Songbird genomics: analysis of 45 kb upstream of a polymorphic Mhc class II gene in red-winged blackbirds (Agelaius phoeniceus)

Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative gamma-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.     

MHC variation in birds and reptiles

The major histocompatibility complex (MHC) has been studied in a multitude of mammals by now, but much less is known about its organisation and variation in other vertebrate species. The mammalian MHC is organised as a single gene cluster, but recent studies on birds suggest that this paradigm of MHC organisation has to be supplemented. The domestic chicken thus possesses two separate gene clusters which both contain MHC class I and class II B genes, and we have shown that the ring‐necked pheasant Phasianus colchicus also has two unlinked clusters of class II B genes. We are studying the effect of the MHC on mate choice, survival and reproductive success in natural populations of birds and reptiles. For this reason, we are developing DNA techniques to determine the animals' MHC genotype. The amplification of the hypervariable exon 3 of the class I gene from songbirds and reptiles has provided us with species specific probes that can be used in Southern blot analysis. The first results indicate very extensive variation in all studied species, that is starlings Sturnus vulgaris, great reed warblers Acrocephalus arundinaceus and water pythons Liasis fuscus. The restriction fragment length polymorphism (RFLP) analysis also suggests that the number of MHC genes is significantly larger in these species than in pheasants and domestic chickens. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variation at the major histocompatibility complex in Savannah sparrows

The class I and class II genes of the major histocompatibility complex (Mhc) encode dimeric glycoproteins responsible for eliciting the adaptive immune response of vertebrates. Recent work with birds suggests that the number, size, and arrangement of these genes can differ markedly across species, although the extent of this variation, and its causes and consequences, are poorly understood. We have used a 157-base-pair (bp) portion of the second exon of a class II B gene to probe the Mhc in a free-living population of Savannah sparrows (Passerculus sandwichensis). Segregation analysis of Mhc bands suggests that class II B genes can be found in two independently assorting clusters, as previously described for domestic chickens (Gallus gallus) and ring-necked pheasants (Phasianus colchicus) but unlike gene organization in mammals. The Mhc in Savannah sparrows appears large (with many class II B genes) and variable; we found 42 unique genotypes among 48 adults breeding on Kent Island, New Brunswick, Canada in 1995. Savannah sparrows are long-distance migrants, and these results support recent predictions that migratory birds should show higher levels of Mhc polymorphism and/or a greater number of genes than sedentary species. Savannah sparrows are also socially polygynous with high levels of extra-pair paternity, suggesting that a history of sexual selection might also influence the size and/or structure of the avian Mhc.     

Comparative genomic analysis of two avian (quail and chicken) MHC regions

We mapped two different quail Mhc haplotypes and sequenced one of them (haplotype A) for comparative genomic analysis with a previously sequenced haplotype of the chicken Mhc. The quail haplotype A spans 180 kb of genomic sequence, encoding a total of 41 genes compared with only 19 genes within the 92-kb chicken Mhc. Except for two gene families (B30 and tRNA), both species have the same basic set of gene family members that were previously described in the chicken "minimal essential" Mhc. The two Mhc regions have a similar overall organization but differ markedly in that the quail has an expanded number of duplicated genes with 7 class I, 10 class IIB, 4 NK, 6 lectin, and 8 B-G genes. Comparisons between the quail and chicken Mhc class I and class II gene sequences by phylogenetic analysis showed that they were more closely related within species than between species, suggesting that the quail Mhc genes were duplicated after the separation of these two species from their common ancestor. The proteins encoded by the NK and class I genes are known to interact as ligands and receptors, but unlike in the quail and the chicken, the genes encoding these proteins in mammals are found on different chromosomes. The finding of NK-like genes in the quail Mhc strongly suggests an evolutionary connection between the NK C-type lectin-like superfamily and the Mhc, providing support for future studies on the NK, lectin, class I, and class II interaction in birds.     

Sequence diversity of Mhc genes in lake whitefish

The sequence variation of three exons of the major histocompatibility complex ( Mhc ) was examined in a lake whitefish Coregonus sp., population from the Swiss lake of Hallwil. DNA sequences from the Mhc class I A1 , A2 and class II B1 exons, corresponding to the α1, α2 and β1 domains of the Mhc glycoproteins, were obtained by the polymerase chain reaction followed by cloning and sequencing. The numbers of variable sequences detected for each exon were 15 ( A1 ), 11 ( A2 ) and 20 ( B1 ). Levels of nucleotide similarity ranged from 82 to 99% for the A1 exon, 58–96% for the A2 and 88–99% for the B1 exon. At the A1 and B1 exons, the nonsynonymous substitution rates ( dn ) exceeded synonymous substitution rates ( ds ) greatly within the peptide binding regions, indicating the effect of balancing selection. Sequence diversity at the A2 exon did not seem to be maintained by balancing selection ( ds > dn ). Phylogenetic comparison of whitefish Mhc sequences with sequences from other salmonid species and more distantly related teleosts indicated shared ancestral (trans-species) polymorphism.     

Ancestral Polymorphism of Mhc Class II Genes in Mice: Implications for Balancing Selection and the Mammalian Molecular Clock

To investigate the evolutionary dynamics at Mhc class II DR genes of mice (genus Mus), we sequenced the peptide binding regions (PBRs) of 41 DRB (=Eβ) genes and eight DRA (=Eα) genes from 15 strains representing eight species. As expected trees of these PBR sequences imply extensive maintenance of ancestral DRB alleles across species. We use a coalescent simulation model to show that the number of interspecific coalescent events (c) observed on these trees was higher than the number expected for neutral genealogies and similar sample sizes and is more consistent with balancing selection than with neutrality. Patterns of ancestral polymorphism in mouse DRB alleles were also used to examine the tempo of synonymous substitution in the PBR of mouse class II genes. Both absolute and relative rate tests on DRA and DRB genes imply increased substitution rates at two- and fourfold degnerate sites of mice and rats relative to primates, and decreased rates for the DRB genes of primates relative to ungulate and carnivore relatives. Thus rates of synonymous substitution at Mhc DR genes in mammals appear to be subject to generation time effects in ways similar to those found at other mammalian genes.     

Natural selection of the major histocompatibility complex (Mhc) in Hawaiian honeycreepers (Drepanidinae)

The native Hawaiian honeycreepers represent a classic example of adaptive radiation and speciation, but currently face one the highest extinction rates in the world. Although multiple factors have likely influenced the fate of Hawaiian birds, the relatively recent introduction of avian malaria is thought to be a major factor limiting honeycreeper distribution and abundance. We have initiated genetic analyses of class II beta chain Mhc genes in four species of honeycreepers using methods that eliminate the possibility of sequencing mosaic variants formed by cloning heteroduplexed polymerase chain reaction products. Phylogenetic analyses group the honeycreeper Mhc sequences into two distinct clusters. Variation within one cluster is high, with dN > dS and levels of diversity similar to other studies of Mhc (B system) genes in birds. The second cluster is nearly invariant and includes sequences from honeycreepers (Fringillidae), a sparrow (Emberizidae) and a blackbird (Emberizidae). This highly conserved cluster appears reminiscent of the independently segregating Rfp-Y system of genes defined in chickens. The notion that balancing selection operates at the Mhc in the honeycreepers is supported by transpecies polymorphism and strikingly high dN/dS ratios at codons putatively involved in peptide interaction. Mitochondrial DNA control region sequences were invariant in the i'iwi, but were highly variable in the 'amakihi. By contrast, levels of variability of class II beta chain Mhc sequence codons that are hypothesized to be directly involved in peptide interactions appear comparable between i'iwi and 'amakihi. In the i'iwi, natural selection may have maintained variation within the Mhc, even in the face of what appears to a genetic bottleneck.     

Molecular characterization of three Mhc class II B haplotypes in the ring-necked pheasant

We investigated the class II B genes in free-ranging population of the ring-necked pheasant Phasianus colchicus by a combination of restriction fragment length polymorphism (RFLP), polymerase chain reaction (PCR), and DNA sequencing. Special attention was paid to the variation in the second exon, which encodes the peptide-binding ₁-domain. The population was introduced, but it still exhibited major histocompatibility complex polymorphism with at least three segregating class II B haplotypes and consequently six genotypes. We found two class II B genes associated with each haplotype. The class II B genes of birds had until then only been molecularly characterized in the domestic chicken. the pheasant genes were highly variable, although one of the amplified sequences was found in two different haplotypes. Taken together, the most polymorphic positions (residues 37 and 38) were not identical in any of the predicted protein sequences, but all except one of the motifs had already been foud in the domestic chicken. Structurally important features in mammalian class II B genes were generally conserved also in the pheasant sequences, but the loss of a potential salt bridge constituent (Arg₇₂) in several sequences may suggest a slightly different structure of the adjacent parts of the peptide-binding groove. The pheasant genes are most closely related to the so called B-LBII family in the chicken, indicating that this represents a major line of development among avian class II B genes.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X75403-X75407. Correspondence to: H. Wittzell, Department of Theoretical Ecology, Ecology Building, Lund University, S-223 62 Lund, Sweden.     

Patterns of polymorphism in the MHC class II of a non-passerine bird,the great snipe (<Emphasis Type="Italic">Gallinago media</Emphasis>)

The genomic organisation of the major histocompatibility complex (MHC) seems to vary considerably between different bird species. In order to understand this variation it is important to gather information from different species. We have, for the first time, investigated MHC class II polymorphism in a wader species, the great snipe (Gallinago media). Eleven alleles were found in five sequenced individuals; these come from at least three different loci, but RFLP data suggest that a larger number of genes may be present. For MHC genes, amino acid substitutions followed the, for MHC genes, general pattern of high non-synonymous substitution rates in peptide-binding regions, suggesting that the sequenced alleles may be expressed. The number of genes, lengths of introns and exon sequences of the great snipe MHC seem to be intermediate between those of chicken and passerine birds.     

Hybridization but no evidence for backcrossing and introgression in a sympatric population of great reed warblers and clamorous reed warblers

Hybridization is observed frequently in birds, but often it is not known whether the hybrids are fertile and if backcrossing occurs. The breeding ranges of the great reed warbler (Acrocephalus arundinaceus) and the clamorous reed warbler (A. stentoreus) overlap in southern Kazakhstan and a previous study has documented hybridization in a sympatric population. In the present study, we first present a large set of novel microsatellite loci isolated and characterised in great reed warblers. Secondly, we evaluate whether hybridization in the sympatric breeding population has been followed by backcrossing and introgression.We isolated 181 unique microsatellite loci in great reed warblers. Of 41 loci evaluated, 40 amplified and 30 were polymorphic. Bayesian clustering analyses based on genotype data from 23 autosomal loci recognised two well-defined genetic clusters corresponding to the two species. Individuals clustered to a very high extent to either of these clusters (admixture proportions ≥ 0.984) with the exception of four previously suggested arundinaceus-stentoreus hybrid birds that showed mixed ancestry (admixture proportions 0.495-0.619). Analyses of simulated hybrids and backcrossed individuals showed that the sampled birds do not correspond to first-fourth-generation backcrosses, and that fifth or higher generation backcrosses to a high extent resemble 'pure' birds at this set of markers.We conclude that these novel microsatellite loci provide a useful molecular resource for Acrocephalus warblers. The time to reach reproductive isolation is believed to be very long in birds, approximately 5 Myrs, and with an estimated divergence time of 2 Myrs between these warblers, some backcrossing and introgression could have been expected. However, there was no evidence for backcrossing and introgression suggesting that hybrids are either infertile or their progeny inviable. Very low levels of introgression cannot be excluded, which still may be an important factor as a source of new genetic variation.     

Dynamic risk assessment: does a nearby breeding nest predator affect nest defence of its potential victim?

There is growing evidence that birds are able to discriminate different types of nest intruders and adjust their nest defence behaviour according to intruder dangerousness and distance from the nest (the dynamic risk assessment hypothesis). Here, we tested whether birds’ decisions about nest defence may additionally be affected by an increasing familiarity with a particular nest predator. We tested nest defence responses of great reed warblers Acrocephalus arundinaceus to a nest predator, the little bittern Ixobrychus minutus. Great reed warbler nests located close (≤7 m) to synchronously breeding little bitterns were “neighbour”, other nests were “solitary”. Great reed warbler specific aggression towards a little bittern dummy was much lower (~5-times) at neighbour than solitary nests. In contrast, generalised responses to a control innocuous intruder (the turtle dove, Streptopelia turtur) were statistically identical at neighbour and solitary nests. These patterns are in line with dynamic risk assessment hypothesis. We hypothesise that decreased great reed warbler aggression at neighbour nests also represents a specific behavioural adaptation to nesting in association with the little bittern. Little bitterns breeding closer to great reed warblers showed decreased risks of failure due to predation. However, further research is needed to experimentally test the causal links behind these patterns.     

Inability to regain normal body mass despite extensive refuelling in great reed warblers following the trans‐Sahara crossing during spring migration

Migratory birds wintering in Africa face the challenge of passing the Sahara desert with few opportunities to forage. During spring migration birds thus arrive in the Mediterranean area with very low energy reserves after crossing the desert. Since early arrival to the breeding grounds often is of importance to maximize reproductive success, finding stopover sites with good refuelling possibilities after the Saharan passage is of utmost importance. Here we report on extensive fuelling in the great reed warbler Acrocephalus arundinaceus on the south coast of Crete in spring, the first land that they encounter after crossing the Sahara desert and the Mediterranean Sea in this area. Birds were studied at a river mouth and due to an exceptional high recapture rate (45 and 51% in two successive years), we were able to get information about stopover behaviour in 56 individual great reed warblers during two spring seasons. The large proportion of trapped great reed warbler compared to other species and the large number of recaptures suggest that great reed warblers actively choose this area for stopover. They stayed on average 3–4 d, increased on average about 3.5 g in body mass and the average rate of body mass increase was 4.8% of lean body mass d^–1. Wing length affected the rate of increase and indicated that females have a slower increase than males. The results found show that great reed warblers at this site regularly deposit larger fuel loads than needed for one continued flight stage. The low body mass found in great reed warblers (also in birds with high fat scores) is a strong indication that birds staging at Anapodaris still had not been able to rebuild their structural tissue after the strenuous Sahara crossing, suggesting that rebuilding structural tissue may take longer time than previously thought.     

Untersuchungen an Drossel- und Teichrohrs?nger(Acrocephalus arundinaceus, A. scirpaceus): Bestandsentwicklung, Brutbiologie, ?kologie

Zusammenfassung Von 1973–1978 wurden systematische Beringungen und regelmäßige Nestkontrollen einer Drs-Population im Fränkischen Weihergebiet (Nordbayern) durchgeführt (Auswertung von 487 Nestkarten). Die vorhandene Trs-Population wurde nicht systematisch erfaßt (645 Nestkarten).Der Bestand der einzelnen Teilpopulationen des Drs schwankte im Untersuchungszeitraum; die gesamte Population blieb annähernd konstant.Die Nestabstände benachbarter Drs-Bruten innerhalb eines günstigen Schilfstreifens lagen zwischen 7 m und ca. 300 m. Das kolonieartige Brüten der Trs wird mit Beispielen belegt.Medianer Legebeginn des Drs war der 29. Mai, der des Trs der 13. Juni. Der nach Erreichen des Maximums im Legemuster folgende Abfall war beim Drs deutlich steiler als beim Trs. Die mittlere Gelegegröße des Drs betrug 4,73, die des Trs 3,85 Eier. Bei beiden Arten fand eine Gelegegrößenreduktion mit fortschreitender Brutzeit statt.Das Schlüpfen der Jungen erfolgte beim Drs überwiegend am 12. bis 14. Tag nach Ablage des letzten Eies, beim Trs am 11. bis 13. Die Brutdauer betrug meist 14 (Drs) bzw. 13 (Trs) Tage.Beim Drs waren 59,7 % der Nester erfolgreich. Der Ausfliegeerfolg, bezogen auf erfolgreiche Nester, betrug 73,2 %, der Gesamtbruterfolg demnach 43,7 %. Beim Trs ergaben sich entsprechend die Werte 66,6 %, 82,9 % und 55,2 %. Auch die durchschnittliche Anzahl flügger Jungvögel pro Brutnest lag beim Drs mit 2,00 etwas niedriger als beim Trs mit 2,15. Als Reproduktions-rate des Drs wurde ein Wert von 2,24 flüggen Jungen pro errechnet. Für den Trs wird die Reproduktionsrate wesentlich höher geschätzt.Die Verluste wurden nach Ursachen aufgeschlüsselt, wobei besonders verglichen mit gleichaltrigen Trs die vielfach größere Empfindlichkeit nestjunger Drs gegen Regen und Kälte auffiel.In zwei Fällen konnten für Drs- Zweitbruten mittels Beringung nachgewiesen werden. Beobachtungen an Trs gaben zu Vermutungen von Zweitbruten bei dieser Art Anlaß.Bigamie wurde beim Drs mehrfach mittels Farbberingung nachgewiesen. Aus den Nestabständen konnte im Untersuchungsgebiet nicht auf monogames oder polygames Verhalten der Drs geschlossen werden. Es werden Angaben über Alter und Verhalten polygamer in verschiedenen Jahren gemacht. Paarzusammenhalt am Vorjahresbrutplatz wurde beim Drs mittels Beringung einmal nachgewiesen.Die meisten überlebenden der als Brutvögel beringten Drs kehrten ins Untersuchungsgebiet zurück, von den überlebenden nestjung beringten dagegen etwa ein Drittel. 3 nestjung beringte Trs wurden nach 1 bzw. 2 Jahren im Untersuchungsgebiet zur Brutzeit kontrolliert.Ein Drittel der Drs-Population stammte aus dem Untersuchungsgebiet. 2 nestjung beringte aus 78 km und 500 km Entfernung wurden als Brutvögel kontrolliert. Alter der Brutvögel 1–9 Jahre.An Beispielen wird die räumliche und zeitliche Einnischung beider Arten beschrieben.

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Studies onAcrocephalus arundinaceus andscirpaceus: Population trends, breeding biology, and ecology

Summary Results of 6 years (1973–1978) of systematic ringing and regular nest controls of a great reed warbler population in Northern Bavaria are given (data of 487 nests) as well as results on a population of the reed warbler (data of 645 nests).Numbers of great reed warblers fluctuated in different parts of the study area. The whole population, however, remained fairly constant.Data on the arrival of males in the breeding area are given.Distances between neighbouring nests of great reed warblers varied from 7 to about 300 m. Differences in nest densities in the reed warbler could be found.The mean first egg laying dates in the great reed warbler and the reed warbler were May 29 and June 13 respectively. The great reed warbler showed a distinct steeper decrease in its egg laying pattern than the smaller species. Average clutch size in the great reed warble was 4.73 eggs and 3.85 eggs in the reed warbler. In both species clutch size decreased during the season.Great reed warbler nestlings hatched on the 12th to 14th day after the last egg had been laid, reed warbler nestlings on the 11th to 13th day. The incubation period was mainly 14 days in the great reed warbler and 13 days in the reed warbler.In the great reed warbler 59.7 % of the nests were successful. The fledging success of successful nests was 73.2 %, accordingly the total nest success was 43.7 %. The corresponding data in reed warbler were 66.6 %, 82.9 % and 55.2 %. On average great reed warblers produced 2.00 fledglings per clutch, reed warblers 2.15. In the great reed warbler a reproduction rate of 2.24 fledglings per female was calculated. The reproduction rate in reed warbler was estimated substantially higher.Great reed warbler nestlings were much more sensitive to rain and cold weather than reed warbler nestlings of the same age.Two great reed warbler males were proved to make a second brood. The same is supposed for the other species but could not be proved so far.Polygyny was proved several times in the great reed warbler. Age and behaviour of polygynous males in different years are reported. Two great reed warblers were found breeding with their former mates at last year's breeding place.Most of the great reed warblers ringed as breeding birds returned into the study area. Nearly one third of the great reed warbler nestlings returned for breeding. Three reed warblers ringed as nestlings could be controlled in the study area during breeding period after 1 year and 2 years respectively.Distances of returned great reed warblers in relation to their birth places and their former breeding places are specified.One third of the great reed warbler population originates from the study area. Two females breeding in the study area were ringed as nestlings 78 km and 500 km apart.One-year-old to nine-year-old great reed warblers were found breeding. Data on the age composition of breeding birds are given.Plant species supporting the nests of great reed warbler and reed warbler were investigated. Differences in the spatial and temporal habitat selection of both species are described.

     

Conflicting patterns of mitochondrial and nuclear DNA diversity in Phylloscopus warblers

Molecular variation is often used to infer the demographic history of species, but sometimes the complexity of species history can make such inference difficult. The willow warbler, Phylloscopus trochilus, shows substantially less geographical variation than the chiffchaff, Phylloscopus collybita, both in morphology and in mitochondrial DNA (mtDNA) divergence. We therefore predicted that the willow warbler should harbour less nuclear DNA diversity than the chiffchaff. We analysed sequence data obtained from multiple samples of willow warblers and chiffchaffs for the mtDNA cytochrome b gene and four nuclear genes. We confirmed that the mtDNA diversity among willow warblers is low (pi = 0.0021). Sequence data from three nuclear genes (CHD-Z, AFLP-WW1 and MC1R) not linked to the mitochondria demonstrated unexpectedly high nucleotide diversity (pi values of 0.0172, 0.0141 and 0.0038) in the willow warbler, on average higher than the nucleotide diversity for the chiffchaff (pi values of 0.0025, 0.0017 and 0.0139). In willow warblers, Tajima's D analyses showed that the mtDNA diversity, but not the nuclear DNA diversity, has been reduced relative to the neutral expectation of molecular evolution, suggesting the action of a selective sweep affecting the maternally inherited genes. The large nuclear diversity seen within willow warblers is not compatible with processes of neutral evolution occurring in a population with a constant population size, unless the long-term effective population size has been very large (N(e) > 10(6)). We suggest that the contrasting patterns of genetic diversity in the willow warbler may reflect a more complex evolutionary history, possibly including historical demographic fluctuations or historical male-biased introgression of nuclear genes from a differentiated population of Phylloscopus warblers.     

Dynamics of Mhc evolution in birds and crocodilians: amplification of class II genes with degenerate primers

Genes of the major histocompatibility complex (Mhc) are the most polymorphic functional loci in mammalian populations, but little is known of Mhc variability in natural populations of nonmammalian vertebrates. To help extend such studies to birds and relatives, we present a pair of degenerate primers that amplify polymorphic segments of one chain (the β chain) of the class II genes from the major histocompatibility complex (Mhc) of archosaurs (birds + crocodilians). The primers target two conserved regions lying within portions of the antigen-binding site (ABS) encoded by the second exon and amplify multiple genes from both genomic DNA and cDNA. The pattern of nucleotide substitution in ABS codons of 51 sequences amplified and cloned from five species of passerine birds and an alligator (Alligator mississippiensis) indicates that archosaurian class II β genes are subject to selective forces similar to those operating in mammalian populations. Hybridization of a genomic clone generated by the primers revealed highly polymorphic bands in a sample of Florida scrub jays (Aphelocoma coerulescens coerulescens). Because the primers amplify only part of the ABS from multiple class II genes, they will be useful primarily for generating species specific clones, thereby providing a critical inroad to more detailed structural and evolutionary studies.