Avian mating systems vary substantially among and within species, with DNA-based studies revealing that genetic mating system frequently deviates from the social monogamy observed in many bird species [1
]. Genetic mating systems have been traditionally characterized by means of DNA-based parentage assignments, comparing the genotypes of caretaker adults to those of putative offspring within a brood [2
]. In many species, however, obtaining an appropriate sample of candidate parents is not straightforward or possible in natural populations. For example, birds such as parrots, grouse, birds of prey and waterbirds are elusive animals, difficult to capture, often inhabit inaccessible or remote places, and can move long distances, imposing difficulties to direct blood sampling of putative parents [3
]. Kinship analyses made possible by use of hypervariable markers such as microsatellites have greatly facilitated the study of avian mating systems [1
]. Even in the absence of biological samples from putative parents, kinship reconstruction techniques may provide insights into relative relatedness and mating system when putative groups of siblings can be determined a priori
], as is the case for all nestlings within a brood.
Waterbirds of the order Ciconiiformes and Pelecaniiformes are colonial breeders that prove interesting models in which to investigate alternative reproductive tactics. In general, colonially breeding species are predicted to have high extra-pair paternity (EPP) rates due to the close proximity to potential sexual partners [8
]. Extra-pair copulation (EPC), i. e. copulation between one individual and another of the opposite sex that is not its social partner, a pre-requisite for EPP, can be frequent in breeding colonies [9
]. High densities of reproductive individuals clustered together are, in turn, one of the most important ecological factors affecting EPP at the species level [11
]. Colonial nesters also can be prone to conspecific brood parasitism (CBP), due to the availability, within colonies, of potential hosts' nests in high numbers [12
]. In spite of showing an interesting breeding behavior, the genetic mating systems of colonial waterbirds, particularly those of Neotropical species, are not well studied [16
One reason for the dearth of studies of genetic mating systems in waterbirds is that many species exhibit adult behaviour and life-history characteristics that preclude the ascertainment of biological samples for DNA analysis. For example, breeding adults of some storks, spoonbills and egrets show restless behaviour and fly away from their nests as soon as researchers enter the breeding colonies to conduct fieldwork, preventing their capture and hindering the collection of blood samples for DNA analyses. Although non-invasive methods have been applied in some avian species for the collection of parental genetic material, those methods may not be suitable for the study of natural breeding colonies of waterbirds. Egg-swabbing, for instance, requires relatively extensive early-egg manipulation [17
]. This approach will increase the amount of time spent by researchers in the breeding colonies in this critical precocious stage of development, and consequently will increase nest disturbance and augment the risk of nest loss. Likewise, some waterbirds' breeding colonies, such as those located in the Pantanal wetland of Brazil, have a high incidence of aerial predators (e.g., the Southern Caracara, Turkey Vulture and Black-collared Hawk) [18
]. Presence of researchers in and around nests often induces adults to fly away, offering predators enhanced opportunities to assault eggs. The use of blood sucking insects is another non-invasive technique that has been successfully applied to sample material of incubating birds [19
]. This methodology requires checking dummy eggs every ca. 30 min to see if the insects have had their blood meal and to collect blood [19
]. Storks, herons and spoonbills nests are built in trees as tall as 20 m (e.g., in breeding colonies from the Brazilian Pantanal and Amapa state), which can be accessed only by professional tree-climbers. Thus, direct application of non-invasive sampling techniques is precluded in such situations.
In this study, we inferred kinship patterns among offspring in broods of three geographically co-distributed species, Wood Stork (Mycteria americana Linnaeus, 1758), Roseate Spoonbill (Platalea ajaja Linnaeus, 1758) and Great Egret (Ardea alba egretta, Gmelin 1789), to investigate genetic mating system in the absence of parental data. Our prediction was that if social mating system (monogamy) equates to genetic mating system in these species, all nestlings found inside a nest will be genetic full-siblings. We combined different approaches based on multilocus microsatellite genotypes to reconstruct kinship among nestlings: estimates of relatedness coefficients, formulation of relationship-hypotheses, significance testing of alternative hypotheses, and maximum-likelihood sibship reconstruction techniques. Using this framework, we found evidence that alternative reproductive strategies may be present in natural populations of Wood Storks and Roseate Spoonbills, whereas Great Egrets did not deviate from a hypothesis of genetic monogamy. Although co-distributed, differences among these species may be due to variation in their life histories. In addition, we explored the relative merits and drawbacks of our approach for reconstructing genetic mating system in the absence of parental information, including its applicability to future studies.