Diet composition, guild structure and trophic relationships of wintering birds of prey in an estuarine wetland (The Evros Delta National Park, Greece)

Seven species of birds of prey studied in the Evros Delta National Park belong to a common assemblage of a trophic guild. However, their diet study revealed different groups of feeding niches. A cluster analysis provided three groups with very similar diets: The eastern imperial eagle ( Aquila heliaca ) and greater spotted eagle ( Clanga clanga ) constituted a group feeding mainly on birds with their diets including 96% (13 bird species) and 98% (16 bird species), by number respectively, most bird prey being water birds. A second group, characterized by a considerable variety of prey types dominated by small mammals and birds, included the western marsh harrier ( Circus aeruginosus ) (67% mammals, 27% birds -at least 12 species ), black kite ( Milvus migrans ) (46% mammals, 39% birds -at least 11 species) and common buzzard ( Buteo buteo ) (birds 36% -at least 7 species, mammals 22%). In all three species, birds dominated by biomass, whereas other prey such as arthropods (mainly insects), amphibians and reptiles had a low contribution by mass where they occurred. A third group included the common barn owl ( Tyto alba ), feeding mainly on small mammals (97%, at least 12 species) and little owl ( Athene noctua ). The little owl’s diet consisted of mammals (29%, -at least 6 species), birds (5% -at least 7 species) and, though insects preponderated by number (64%), mammals were dominant by biomass in both owls. Diversity indices estimated on class level were low, ranked as B. buteo > M. migrans > A. noctua > C. aeruginosus > A. heliaca = T. alba > A. clanga . Dietary similarities may be partly due to common habitat preferences and the role of other potential factors to niche partitioning is discussed.


Introduction
In the natural world, a species ecological "role" within habitat, microhabitat, or range of suitable environmental conditions is considered as a "niche" (Ricklefs 2010), where closely related species likely to compete for ecological resources (Wiens et al. 2005). Species similar tolerances of environmental conditions might lead to closer association when viewed at broader environmental scales (Cavender-Bares et al. 2009;Ricklefs 2010). Thus, species can be grouped according to the manner in which they exploit a common resource, providing a manageable, functional unit forming the "guild" for studying patterns of adaptation and the organization of natural communities (Root 2001). When a guild includes species of the same taxonomic assemblages, it is usually considered as an "assemblage guild" (Jaksić 1981). The identification and analysis of ecological guilds have been fundamental to understand the processes that determine the structure and organization of communities.
The feeding habits of seven species of wintering birds of prey forming a trophic guild were investigated within the framework of a conservation project in the Evros Delta, in northeastern Greece. The aims were a) to provide information on the diet of these conservation-needing species as, at least some of which have been little studied in the eastern Mediterranean, and b) to clarify the trophic relationships of the raptors' within their trophic guild in the area.

Study area and methods
The Evros Delta (40 ₒ 84' N, 26 ₒ 07' E), is the easternmost Greek wetland designated under the Ramsar convention and also as a National Park. Additionally it is an Important Bird Area and a Special Protection Area in the EU Natura 2000 network (HOS 2009). The study area includes a great variety of habitats, such as extensive saltmarshes and salt flats, sand dunes and sandy islets, mudflats, lagoons, reedbeds, tamarisk and riparian forest, permanent and temporary freshwater marshes and extensive cultivations (Babalonas 1979). The area is famous for its wintering raptors, as well as for the large populations of water birds (Handrinos et al. 2015). This study was based on pellet analysis collected opportunistically from roosting sites of seven species in the winter months (September to February) from 2001 to 2005. Pellet collection locations of eastern imperial eagles (Aquila heliaca) and greater spotted eagles (Clanga clanga) were a pinewood communal roost, of black kites (Milvus migrans) and marsh harriers (Circus aeruginosus), a riparian forest communal roost and a reed bed respectively, of common buzzards (Buteo buteo) individual tree roosts and of barn owl (Tyto alba) and little owl (Athene noctua) abandoned buildings. Pellets were collected at relatively random dates for most species. At the eagle roosts, at least 40 greater spotted eagle and 4 imperial eagles were counted. The roosts of the black kites and marsh harriers were used by up to 60 and 70 birds, respectively. Pellet analysis may not reflect the actual number of prey items taken, but are a good estimate of the relative frequencies of prey types for comparative purposes (Steenhof & Kochert 1988).
The pellets were analyzed using reference books (Mammals, Lawrence & Brown 1973;Chaline 1974. Birds, Brown et al. 1987. Reptiles, Arnold & Burton 1980. Insects, Chinery 1981. Mean weight of each prey taxon was taken from the literature, (Mammals, Macdonald & Barrett 1993. Birds, Perrins 1987. Reptiles, Helmer & Scholte 1985. Statistical analyses were performed with Statistica 12. The trophic diversity of prey was estimated on a class level by using the antilog of the Shannon-Weiner index (Shannon & Weaver 1963), while an evenness index was calculated (according to Simpson 1949) in order to standardize the trophic diversity for comparisons among species. Also, a Pianka's index was calculated, in order to study the diet overlap of the species (Pianka 1974). A manner of determining the trophic guilds and assign species to them is to perform a cluster analysis to classify species according to their similarities in feeding patterns (González -Salazar et al. 2014).Thus, to compare the prey differentiation among species, cluster analysis based on the biomass proportions of all prey classes was performed. For these analyses, the Primer 5.1.2 software was used with Bray-Curtis similarity as distance measure and group mean as linkage measure.
There was a significant difference between the two eagles in their main prey types (grouped as, large waterfowl Anserinae, small waterfowl Anatinae, other water birds and others) (χ 2 = 8.224, P < 0.05), with the eastern imperial eagle taking relatively more large waterfowl than the greater spotted eagle.
The main prey of the common barn owl were mammals (97% by number and c. 91% by biomass, with at least 12 species), while birds formed 3% and 9% of the diet respectively (including at least 10 species, Table 3). Other prey types (insects and amphibians) were insignificant. The main mammalian prey by biomass was Macedonian mouse (Mus macedonicus) (33%), sibling vole (32%) and the lesser whitetoothed shrew (Crocidura suaveolens) (10%). The most important bird species was the common starling (4%). Little owl took a variety of prey, including by numbers c. 64% insects, 29% mammals, 5% birds with at least 7 species involved, 1% reptiles and < 1% each chilopods, diplopods and arachnids (Table 3). In terms of biomass, mammals were the dominant prey (72%, including at least 6 species), with main species being the sibling vole (38%) and small mice (Mus spp.) (22%). Birds made up 22% of the biomass of which 13% were undetermined passerines. Insects, mainly beetles (Coleoptera) accounted for only 6% of the total biomass, while other prey was insignificant.
The relative proportions of the main prey groups in terms of both numbers and biomass and the respective diversity indices for the species studied are summarized in Fig. 1. Diversity indices ranked as B. buteo > M. migrans > A. noctua > C. aeruginosus > A. heliaca = T. alba > A. clanga. In general, the common buzzard and black kite had the most diverse diets at the class level with the highest evenness distribution among prey groups, whereas the two eagles and the barn owl had the lowest. (Fig 1).
Cluster analysis revealed three main branches, one (lower in Fig. 2) contained the two owl species, reflecting their main diet on small mammals; a second (middle) involved three species with the most diverse diets; and a third (upper) included the two eagle species with similar diets based on water birds. The cluster indicates a greater similarity between the first two groups which diverge from the third one. Pianka's indexes (Table 4) indicated a virtually complete diet overlap in the diet of the two eagles, with significant overlaps also in the medium-sized raptors. The owls hardly overlapped with the eagles, but they did overlap with the other raptors due to the estimation of the index on prey class levels, whereas differences occurred on specific levels.

Discussion
Birds were the most important food of the eastern imperial eagle in the Evros Delta in similarity to wintering birds to Jordan (Appendix). In contrast, mammals were most important during breeding season in the species eastern European range, where all other studies were carried out (Appendix). Numerous bird and mammal species were usually involved in each area. In contrast to these studies, no carrion was found in the species diet in our area probably due to a high availability of overwintering waterfowl (hunted in saltmarshes). Imperial eagles are flexible in utilizing the most abundant prey sources, and it is believed to facilitate conservation efforts (Hovárth et al. 2010).
In the study area the diet of greater spotted eagle was based on the abundant waterfowl hunted over saltmarshes and coastal areas. As in our area, water birds preponderate in the eagle's diet in other wintering areas (Amvrakikos, Spain), where, on the other hand, mammals were most important during the breeding season. In all these other areas there was higher prey diversity (Appendix).
In our area, western marsh harriers preyed mainly upon mammals, whereas in other European countries the main food was birds (Appendix), probably due to reduced availability in winter from the effects of weather of some small mammal populations (Solonen & Ahola 2010); mammals were also main prey in France (Ingenbleek et al 2004). In all areas numerous species participate in the harrier's diet though some such as insects seem unimportant as mass. This prey diversity in our area (Alivizatos et al. 2011) is reflected by diverse habitat preferences such as reedbeds, freshwater marshes and saltpans (Bobola et al. 2018).
Regarding black kite, a comparison of our findings to those of other areas, shows a great similarity in the preponderance of mammal prey in relation to birds either in winter or in other period (Appendix). In Spain, the relative abundance of prey differs based on dry or wet winter periods (Garcìa et al. 1998). In most diet studies in winter in Spain, mammals were the dominant prey the black kite (summarized in Garcìa et al. 1998). The main bulk of diet in all periods in Europe is composed by mammal and/or fish carrion. Other prey (reptiles, insects) where present, constitutes a small amount as mass. The use of carrion and rubbish dumps has been reported in a variety of studies (Viñuela & Veiga 1992, De Giacomo & Guerrieri 2008, Mazumdar et al. 2016. Refuse tips and dumps for dead livestock and their management during the winter are considered important for the conservation of the species (Garcìa et al. 1998). In our study it is likely that the importance of carrion in the kite's diet has been underestimated, particularly if these birds used dumps out of the study area. In contrast, in the Evros Delta, black kites showed a strong selection for rivers, canals, freshwater marshes and reedbeds (Bobola et al. 2018), constituting a diverse habitat use that explains the diversity in prey taken.
In contrast to our results, in studies in Europe carried out both in the nesting period and in winter, common buzzards preyed mainly upon small mammals (Appendix). In addition the results of most of the nine diet studies in central Europe (one in winter) again indicate a mostly mammal-dominated diet (Voříšek et al. 1997). The buzzard in Ireland has adapted to the lack of vole species by adopting a wide dietary niche (Rooney & Montgomery 2013). Buzzards are able to shift their diet (Reif et al. 2001), hence the variety in prey items observed in all studies, though mammals and birds remain the most important. Amphibians and reptiles in our samples may suggest that a part prey was taken early in the study period. Common buzzards preferred a wide variety of habitat types in the Evros Delta, freshwater marshes, followed by rivers, canals, grassland and reedbeds (Bobola et al. 2018). This bird, being a typical perch-hunting predator used perches highly available at the edges of such habitats. A high proportion of non-waterbird avian prey might be explained by the absence of perches at cultivations, so that common buzzards resorted to flight-hunting over cultivations in pursuit of flocks of small land birds where they congregate to exploit seeds left behind after the harvest (Bobola et al. 2018).
In the study area and in Europe, the common barn owl overwhelmingly feeds on small mammals in all seasons, whereas other prey types are negligible (Appendix). Previous studies in other parts of Greece, including wetlands, revealed that both in winter and summer the most important prey was small mammals and the same picture held true in studies from around Mediterranean (see reviews in Bontzorlos et al. 1995, Goutner & Alivizatos 2003Obuch & Benda 2009). Birds follow far behind in importance while other prey is unimportant.
Insects numerically dominate the little owl's diet in our study area as in other areas being higher mostly around the Mediterranean and in summer diet (Appendix), probably due to increased insect availability. Nevertheless, all studies report that mammals are the most important prey, birds of secondary importance, whereas other prey types form only a minor part of the diet.
In some areas diurnal and nocturnal birds of prey co-exist in various trophic guilds and their diets have been found roughly similar on major prey groups though differentiate on prey species levels (Jaksić 1983;Marti & Cochert 1995). In our area, owls and the other diurnal birds of prey (particularly, black kite, western marsh harrier and common buzzard) commonly shared small mammal and birds in their diet. This may be due to that both falconiforms and strigiforms extend their hunting activities to crepuscular hours, thus sharing prey of that activity period; also to that reputedly diurnal and nocturnal prey becomes vulnerable to predators of asynchronous hunting time by extending their activities in the field (Jaksić 1982). Nocturnality in owls may be more attributable to avoidance of interference interactions with hawks than to avoidance of exploitation competition for food (Carothers & Jaksić 1984). Anyway, more other factors are involved in the extent of overlap between diets of predators with differential activity: the size of predators and prey active at day or night, foraging mode of the predator, diversity of prey, and relative length of day and night (Marti et al. 1993). The dietary similarities among black kite, western marsh harrier and common buzzard found in this study may also be partly attributable to that coexisting predators with similar diet composition tend to be more alike in body size (Marti et al. 1993). Common preferences of these species in hunting habitats (Bobola et al. 2018) also explain diet similarities.
Data on the abundance of prey species are available only in the case of the waterfowl (main prey of the two eagles), from the Midwinter Waterfowl Counts (Handrinos et al. 2015). These data are difficult to compare statistically with those from our pellet analysis, but it appears that the greater spotted eagle takes the main waterfowl species in proportion to their abundance as hunted over their main gathering areas, while the eastern imperial eagle shows some preference for the larger species (geese, shelducks and mallards).
A variety of studies have indicated that the trophic guilds of predators beyond falconiforms and strigiforms involve other taxa such as carnivores and snakes (Jędrzejewski et al. 1989;Marti et al. 1993;Jaksić et al. 1996;among others). In our study area, common carnivores such as foxes (Vulpes vulpes) and European wild cats (Felis sylvestris) may be winter counterparts of the raptors' trophic guild, a situation that necessitates further study.