David Martín-Gálvez

New methods to identify conserved microsatellite loci and develop primer sets of high cross-species utility - as demonstrated for birds

We have developed a new approach to create microsatellite primer sets that have high utility across a wide range of species. The success of this method was demonstrated using birds. We selected 35 avian EST microsatellite loci that had a high degree of sequence homology between the zebra finch Taeniopygia guttata and the chicken Gallus gallus and designed primer sets in which the primer bind sites were identical in both species. For 33 conserved primer sets, on average, 100% of loci amplified in each of 17 passerine species and 99% of loci in five non‐passerine species. The genotyping of four individuals per species revealed that 24–76% (mean 48%) of loci were polymorphic in the passerines and 18–26% (mean 21%) in the non‐passerines. When at least 17 individuals were genotyped per species for four Fringillidae finch species, 71–85% of loci were polymorphic, observed heterozygosity was above 0.50 for most loci and no locus deviated significantly from Hardy–Weinberg proportions.

High-utility conserved avian microsatellite markers enable parentage and population studies across a wide range of species

BackgroundMicrosatellites are widely used for many genetic studies. In contrast to single nucleotide polymorphism (SNP) and genotyping-by-sequencing methods, they are readily typed in samples of low DNA quality/concentration (e.g. museum/non-invasive samples), and enable the quick, cheap identification of species, hybrids, clones and ploidy. Microsatellites also have the highest cross-species utility of all types of markers used for genotyping, but, despite this, when isolated from a single species, only a relatively small proportion will be of utility. Marker development of any type requires skill and time. The availability of sufficient “off-the-shelf” markers that are suitable for genotyping a wide range of species would not only save resources but also uniquely enable new comparisons of diversity among taxa at the same set of loci. No other marker types are capable of enabling this. We therefore developed a set of avian microsatellite markers with enhanced cross-species utility.ResultsWe selected highly-conserved sequences with a high number of repeat units in both of two genetically distant species. Twenty-four primer sets were designed from homologous sequences that possessed at least eight repeat units in both the zebra finch (Taeniopygia guttata) and chicken (Gallus gallus). Each primer sequence was a complete match to zebra finch and, after accounting for degenerate bases, at least 86% similar to chicken. We assessed primer-set utility by genotyping individuals belonging to eight passerine and four non-passerine species. The majority of the new Conserved Avian Microsatellite (CAM) markers amplified in all 12 species tested (on average, 94% in passerines and 95% in non-passerines). This new marker set is of especially high utility in passerines, with a mean 68% of loci polymorphic per species, compared with 42% in non-passerine species.ConclusionsWhen combined with previously described conserved loci, this new set of conserved markers will not only reduce the necessity and expense of microsatellite isolation for a wide range of genetic studies, including avian parentage and population analyses, but will also now enable comparisons of genetic diversity among different species (and populations) at the same set of loci, with no or reduced bias. Finally, the approach used here can be applied to other taxa in which appropriate genome sequences are available.

CUCKOO PARASITISM AND PRODUCTIVITY IN DIFFERENT MAGPIE SUBPOPULATIONS PREDICT FREQUENCIES OF THE 457bp ALLELE: A MOSAIC OF COEVOLUTION AT A SMALL GEOGRAPHIC SCALE

Abstract The level of defense against great spotted cuckoo (Clamator glandarius) parasitism in different European populations of magpie (Pica pica) depends on selection pressures due to parasitism and gene flow between populations, which suggests the existence of coevolutionary hot spots within a European metapopulation. A mosaic of coevolution is theoretically possible at small geographical scales and with strong gene flow, because, among other reasons, plots may differ in productivity (i.e., reproductive success of hosts in the absence of parasitism) and defensive genotypes theoretically should be more common in plots of high productivity. Here, we tested this prediction by exploring the relationship between parasitism rate, level of defense against parasitism (estimated as both rejection rate and the frequency of the 457bp microsatellite allele associated with foreign egg rejection in magpies), and some variables related to the productivity (average laying date, clutch size, and number of hatchlings per nest) of magpies breeding in different subpopulations. We found that both estimates of defensive ability (egg rejection rate and frequency of the 457bp allele) covaried significantly with between-plot differences in probability of parasitism, laying date, and number of hatchlings per nest. Moreover, the parasitism rate was larger in more productive plots. These results confirm the existence of a mosaic of coevolution at a very local geographical scale, and the association between laying date and number of hatchlings with variables related to defensive ability and the selection pressure arising from parasitism supports the prediction of coevolutionary gradients in relation to host productivity.

A quantitative trait locus for recognition of foreign eggs in the host of a brood parasite

Avian brood parasites reduce the reproductive output of their hosts and thereby select for defence mechanisms such as ejection of parasitic eggs. Such defence mechanisms simultaneously select for counter‐defences in brood parasites, causing a coevolutionary arms race. Although coevolutionary models assume that defences and counter‐defences are genetically influenced, this has never been demonstrated for brood parasites. Here, we give strong evidence for genetic differences between ejector and nonejectors, which could allow the study of such host defence at the genetic level, as well as studies of maintenance of genetic variation in defences. Briefly, we found that magpies, that are the main host of the great spotted cuckoo in Europe, have alleles of one microsatellite locus (Ase64) that segregate between accepters and rejecters of experimental parasitic eggs. Furthermore, differences in ejection rate among host populations exploited by the brood parasite covaried significantly with the genetic distance for this locus.

Variation in innate immunity in relation to ectoparasite load, age and season: a field experiment in great tits (Parus major)

SUMMARY It remains largely unknown which factors affect the innate immune responses of free-living birds. Nevertheless, the degree of innate immunity may play a crucial role in an individuals survival as it procures the first defence against pathogens. We manipulated the ectoparasite load of great tit (Parus major) nests by infesting them with hen fleas (Ceratophyllus gallinae) before egg laying. We subsequently quantified natural antibody (NAb) concentration and complement activation in nestlings and adult females during breeding and post-breeding periods. NAb concentrations increased in nestlings and adult females breeding in flea-infested nest boxes during the nestling provisioning period, but not in breeding females during incubation. In contrast, parasite abundance did not affect levels of complement activity in females. NAb levels of nestlings were already fully developed at the end of the nestling stage, but complement activation was only observed post-fledging. Concentrations of NAbs and complement activation of adult females were significantly lower during the breeding season compared with post-breeding levels, but did not differ between incubation and chick rearing. Further experimental studies in species that vary in life-history strategies will allow us to unravel the mechanisms underlying the observed variation in innate immune defences.

Food acquisition by common cuckoo chicks in rufous bush robin nests and the advantage of eviction behaviour

The common cuckoo, Cuculus canorus, is a brood parasite that monopolizes parental care of its host species: soon after hatching, the chicks remove the host offspring. Although cuckoo chicks trick their foster parents into providing enough food, it is unknown whether cuckoo begging behaviour represents an advantage over that of the host chicks in a hypothetical competitive scenario. We studied the feeding behaviour of rufous bush robins, Cercotrichas galactotes, when rearing their own and parasitic chicks in natural and in experimental nests where a cuckoo and a host brood were presented simultaneously to parents. In natural parasitized and nonparasitized nests, the feeding rate for cuckoo chicks did not differ from that of a single host chick of the same age, but cuckoos were fed with a different diet and with larger prey. Thus, cuckoo chicks received a similar amount of food to that received by a whole host brood. Cuckoo chicks in experimental nests did not receive a diet, prey size or feeding rate different to that received by a single host chick and thus received considerably less food than cuckoo chicks in unmanipulated nests. These results suggest that cuckoo chicks could not outcompete host chicks, at least when parasitizing this host species, and thus that their eviction behaviour is beneficial. We discuss various explanations for the inability of cuckoos to outcompete host chicks.

Benefits associated with escalated begging behaviour of black-billed magpie nestlings overcompensate the associated energetic costs

SUMMARY Several experimental results support the existence of costs associated with exaggerated begging behaviour, which are assumed by some theoretical models of honest signalling in parent–offspring communication. However, to understand how honest begging behaviour is evolutionarily maintained in nature, the long-term cost–benefit output associated with exaggerated signals should also be estimated. As far as we know, the net cost–benefit balance of begging display has not previously been explored. Here, we used an appetite stimulant, cyproheptadine hydrochloride, to increase the feeling of hunger in some magpie nestlings. Supporting the use of cyproheptadine to manipulate hunger level and thereby begging behaviour, we found that experimental nestlings increased the frequency of begging and received more food than their control nestmates. Contrary to the expectation that physiological costs per se counteract the associated benefits of escalated begging signals, we found that near-fledging experimental magpies showed a better physical condition than control nestlings. These findings stress the interesting question of why magpie nestlings do not show to adults an escalated level of hunger if it implies an advantage. We discuss the responsibility of inclusive fitness costs and indirect genetic effects for the maintenance of honesty in parent–offspring communication.

DNA sampling from eggshell swabbing is widely applicable in wild bird populations as demonstrated in 23 species

There is increasing interest in noninvasive DNA sampling techniques. In birds, there are several methods proposed for sampling DNA, and of these, the use of eggshell swabbing is potentially applicable to a wide range of species. We estimated the effectiveness of this method in the wild by sampling the eggs of 23 bird species. Sampling of eggs was performed twice per nest, soon after the clutch was laid and again at the end of egg incubation. We genotyped DNA samples using a set of five conserved microsatellite markers, which included a Z‐linked locus and a sex‐typing marker. We successfully collected avian DNA from the eggs of all species tested and from 88.48% of the samples. In most of the cases, the DNA concentration was low (ca. 10 ng/μL). The number of microsatellite loci amplified per sample (0–5) was used as a measure of the genotyping success of the sample. On average, we genotyped 3.01 ± 0.12 loci per sample (mean ± SE), and time of sampling did not seem to have an effect; however, genotyping success differed among species and was greater in those species that used feather material for lining their nest cups. We also checked for the occurrence of possible genotyping errors derived from using samples with very low DNA quantities (i.e. allelic dropout or false alleles) and for DNA contamination from individuals other than the mother, which appeared at a moderate rate (in 44% of the PCR replicates and in 17.36% of samples, respectively). Additionally, we investigated whether the DNA on eggshells corresponded to maternal DNA by comparing the genotypes obtained from the eggshells to those obtained from blood samples of all the nestlings for six nests of magpies. In five of the six magpie nests, we found evidence that the swab genotypes were a mixture of genotypes from both parents and this finding was independent of the time of incubation. Thus, our results broadly confirm that the swabbing of eggshells can be used as a noninvasive method for obtaining DNA and is applicable across a wide range of bird species. Nonetheless, genotyping errors should be properly estimated for each species by using a suite of highly polymorphic loci. These errors may be resolved by sampling only recently laid eggs (to avoid non‐maternal DNA contamination) or by performing several PCR replicates per sample (to avoid allelic dropout and false alleles) and/or by increasing the amount of DNA used in the PCR through increasing the volume of the PCR or increasing the concentration of template DNA.

Brood parasitism correlates with the strength of spatial autocorrelation of life history and defensive traits in Magpies

Environmental characteristics of neighboring locations are generally more similar than those of distant locations. Selection pressures due to parasitism and other environmental conditions shape life history traits of hosts; thus, the probability of parasitism should be associated with the strength of spatial autocorrelation in life history and defensive traits of their hosts. Here we test this hypothesis in three different subpopulations of Magpie (Pica pica) parasitized by the Great Spotted Cuckoo (Clamator glandarius) during three breeding seasons. In some of the years and study plots, we found evidence of positive spatial autocorrelations for clutch size and parasitism rate, but not for laying date. As predicted, brood parasitism was associated with the strength of these spatial autocorrelations. Magpies that bred close to each other in areas of high risk of parasitism responded similarly to experimental parasitic eggs. Moreover, an elevated risk of parasitism eliminated the spatial autocorrelation for clutch size, which became randomly distributed. We discuss possible mechanisms explaining these associations, which may have important consequences for estimating evolutionary responses of hosts to parasitic infections and, therefore, for epidemiological, ecological, and evolutionary studies of host-parasite relationships.

Do first-time breeding females imprint on their own eggs?

The egg-recognition processes underlying egg rejection are assumed to be based on an imprinting-like process (a female learning the aspect of her own eggs during her first breeding attempt). The imprinting-like process and the misimprinting costs have been the objective of many theoretical models and frequently have a leading role in papers published on brood parasitism; however, an experiment has never been undertaken to test the existence of this imprinting-like process by manipulating egg appearance in first-time breeding females. Here, we present the first such experimental study using the house sparrow (Passer domesticus), which is a conspecific brood parasite and which has a good ability to reject conspecific eggs, as a model species. We found that contrary to what the hypothesis predicts first-time breeding females did not reject their own eggs in their second breeding attempt. This lack of response against unmanipulated eggs could indicate that females have an innate preference for their own eggs. However, in a second experimental group in which first-time breeding females were allowed to learn the aspect of their (unmanipulated) own eggs, none ejected manipulated eggs during their second clutch either—a finding that does not support the idea of recognition templates being inherited, but instead suggests that recognition templates could be acquired again at each new breeding attempt. Our results demonstrate that it is likely that egg discrimination is not influenced by egg appearance in the first breeding attempt.