Critical steps to ensure the successful reintroduction of the Eurasian red squirrel

Critical steps to ensure the successful reintroduction of the Eurasian red squirrel.— Wildlife reintroduction strategies aim to establish viable long–term populations, promote conservation awareness and provide economic benefits for local communities. In Portugal, the Eurasian red squirrel (Sciurus vulgaris) became extinct in the 16th century and was reintroduced in urban parks in the 1990s, mainly for aesthetic and leisure purposes. We evaluated the success of this reintroduction in two urban parks and here described the critical steps. We assessed habitat use, population density and abundance, and management steps carried out during reintroduction projects. Reintroductions have been successful to some extent given squirrels are present 20 years after release. However, populations in both parks are declining due to the lack of active management and poor quality habitat. Successful reintroduction of Eurasian red squirrel in areas without competition of alien tree squirrels involves three critical main stages. The pre–project stage includes studies on habitat quality, genetic proximity between donors and closest wild population, and health of donor stocks. In the release stage, the number of individuals released will depend on resource variability, and the hard release technique is an effective and economically viable method. Post–release activities should evaluate adaptation, mitigate mortality, monitor the need for supplementary feeding, provide veterinary support, and promote public awareness and education.


Introduction
Animal translocation is an ancient process used by humans to relocate species from one place to another (Griffith et al., 1989;Hodder & Bullock, 1997;Armstrong & Seddon, 2007;Seddon et al., 2007;Ewen et al., 2012).Griffith et al. (1989) defined animal translocation as the intentional release to establish, re-establish or increase the population of a given species.Reintroduction is currently one of the most popular translocation strategies used in the management of species (Armstrong & Seddon, 2007;Seddon et al., 2007;Ewen et al., 2012).Wildlife reintroductions are conducted to establish viable populations, enhance long-term survival of a given species, settle long-term economic benefits for local communities, and to promote conservation awareness (IUCN, 1998).Reintroductions should be carefully planned by a multidisciplinary team, and follow a three-step protocol, focusing on the pre-project activities, release stages and post-released activities (IUCN, 1998).Such projects require complex planning, implementing and monitoring species and habitats according to their biology, socio-economic impact on local communities, and legal requirements (Caughley & Gunn, 1996;IUCN, 1998;Armstrong & Seddon, 2007;Seddon et al., 2007;Ewen et al., 2012;Harrington et al., 2013).Parameters of success change in each project but should follow the principles of long-term survival of species while providing benefits for the local community and fostering conservation awareness (IUCN, 1998(IUCN, , 2012)).The potential positive impact of reintroductions depends on temporal, spatial, and taxonomic factors (Ewen et al., 2012), and if reintroductions are not properly carried out they can damage both donor and receptor populations as well as ecosystems (Hodder & Bullock, 1997;Armstrong & Seddon, 2007;Seddon et al., 2007;Ewen et al., 2012).Therefore, publication and dissemination of successful and unsuccessful cases contribute to improve current reintroduction protocols (Armstrong & Seddon, 2007;Seddon et al., 2007;Ewen et al., 2012;IUCN, 2012).
Mammals, together with birds, are the most frequently chosen groups for releases with conservation purposes (Griffith et al., 1989;Seddon et al., 2005Seddon et al., , 2007)).Although most of reintroductions focus on ungulates and carnivores (Seddon et al., 2005), rodents such as the edible dormouse Glis glis in Poland (Jurczyszyn, 2006) and the European ground squirrel Spermophilus citellus in Central Europe (Matějů et al., 2010) have also been released in the last 20 years.The most commonly reported reintroductions among rodents are those concerning the Eurasian red squirrel Sciurus vulgaris reintroductions, with a considerable number of programmes being implemented in Europe over the last 30 years (Swinnen, 1988;Fornasari et al., 1997;Wauters et al., 1997aWauters et al., , 1997b;;Poole & Lawton, 2009).
Although the Eurasian red squirrel is a widespread Palearctic species (Lurz et al., 2005;Shar et al., 2008;Bosch & Lurz, 2012), some of its populations, particularly in the United Kingdom and Italy, are threatened or extinct due to habitat loss, hunting, disease and competition with alien tree squirrels Sciurus carolinensis, Callosciurus erythraeus and C. finlaysonii (Gurnell, 1987;Wood et al., 2007;Bosch & Lurz, 2012;Bertolino & Lurz, 2013).In Portugal, the Eurasian red squirrel became extinct in the 16 th century due to significant habitat loss, and it only reappeared in extreme northern areas around the 1980s (Mathias & Gurnell, 1998;Ferreira et al., 2001).One decade later, isolated reintroductions occurred in some urban parks, but no monitoring has been conducted since then to understand population dynamics and their status or to evaluate management and success.In order to determine whether Eurasian red squirrel reintroductions carried out in Jardim Botânico da Universidade de Coimbra and in Parque Biológico de Gaia were successful or not, we estimated population viability through density, abundance, and habitat use in released sites.We also evaluated stepwise reintroduction in both urban parks, based on the IUCN guidelines (IUCN, 1998(IUCN, , 2012) ) to highlight critical steps and suggest actions ensure the long-term persistence and viability of these Eurasian red squirrel populations.

Study area
The Parque Biológico de Gaia (PBG), which was created in 1983, initially covered 2 ha but has been extended to include 35 ha (Oliveira, 2013).It is situated in Vila Nova de Gaia, northern Portugal (41º 05' N and 8º 33' W; fig.1A) and it is managed by a municipal company (Oliveira, 2013).This urban park is composed of open areas, a wildlife rehabilitation center and monospecific forests of black alder Alnus glutinosa, oak Quercus robur or cork oak Q. suber (fig.1A).It also has enclosures distributed throughout open areas in the park containing wildlife that could not be rehabilitated or that provide examples of native and exotic fauna.The aim is to promote environmental education, as the case of the Eurasian red squirrel.

Eurasian red squirrel survey
From 7th October to 11th November 2013, 15 walking transects of 100 m were established in each study site.All transects were surveyed in the morning and afternoon for seven days to avoid biases from squirrel behavior (Gurnell et al., 2001).Transects were performed one after other to avoid double counting of individuals moving from one transect to another.Transects were selected to include all habitat types and to cover most of the area at each of the two urban parks, but with at least 20 m distance from each other also to avoid double counting (fig.1; Gurnell et al., 2001).We counted squirrels using the distance sampling method with direct observation using binoculars 8-16 x 40 (Gurnell et al., 2001), given that both parks had great visibility with small and clear forests.Squirrel surveys were conducted between 8:00 and 16:00 in autumn when higher numbers of squirrels can be found (Tonkin, 1983;Wauters et al., 1992;Bosch & Lurz, 2012).The distance from the observer to the squirrel was measured using a telemeter, and compass bearings were taken to determine the angle between the animal and the transect line (Buckland et al., 1993;Gurnell et al., 2001).We measured the distance of squirrels once and did not consider individuals again after moving to a different position.
Population density and abundance were estimated using Distance Sampling 6.0 software (Thomas et al., 2010).Estimates were stratified and based on Conventional Distance Sampling.Half-normal, hazard and negative exponential rate models for the detection function were fixed against the records using a cosine function (Thomas et al., 2010).Models assumed certainty of detection and measurements (Thomas et al., 2010).The selection of the best model and adjustment term were based on the lowest Akaike information criterion (AIC).

Habitat use
Eurasian red squirrels prefer mature native forests that can provide them with an abundant supply of food (Bosch & Lurz, 2012).We assessed vegetation type, location of dreys (i.e.squirrel nests) and food availability to understand habitat use in both urban parks.The survey was conducted in the PBG in October 2013 and in the JBUC in November 2013.Vegetation type (fig. 1) was mapped with a geographic information system in ArcView GIS 9.2 software (ESRI, 2008).The geographical limits of forests and gardens having the same composition and dominance were confirmed in the field.The number of trees to determine dominant species was verified in 10 x 10 m quadrats randomly within the study sites.Due to different area sizes, 80 quadrats were located in PBG and 60 in JBUC.Dreys were mapped to determine preferences in relation to vegetation type (fig.1).Drey counts were obtained by direct observation in a 3 km transect at each site.Transects to count dreys were larger than transects to count individuals because dreys were fixed and double counting was unlikely.We determined the position of dreys, tree species chosen and drey height (Cagnin et al., 2000;Kopij, 2009).Old or abandoned dreys were excluded from counts (Wauters & Dhondt, 1988;Cagnin et al., 2000;Kopij, 2009).The significance of the distribution of dreys in relation to height was measured using a one-way ANOVA in Bioestat 5.0 software (Ayres et al., 2007).Tukey's post hoc test (F) was applied to determine the significance of any differences (Zar, 1999).
Food availability focused on three aspects: number of feeders, relative abundance and richness of edible mushrooms, and energetic content of natural seeds (cones, acorns, hackberries, and nuts).Feeders with supplementary food were counted directly.Relative abundance of Basidiomycota was estimated by counting fungal bodies or remains with characteristic squirrel bites in stipe and cap on the ground in the same 10 x 10 m quadrats where the vegetation type was measured.Only mushrooms eaten by squirrels during the surveys or reported in the literature were considered as a component of Eurasian red squirrel diet (Fogel & Trappe, 1978;Bertolino et al., 2004).Fungi identification and nomenclature follows Crous et al. (2004).
In each 10 x 10 m quadrat, the number of trees with fruits of each species was counted.Only tree species already reported in the literature (Lurz et al., 2005;Bosch & Lurz, 2012) or those seen being consumed during fieldwork were considered as a component of the Eurasian red squirrel diet.A quadrat of 5 x 5 m was placed below every tree bearing fruit inside the 10 x 10 m quadrat to count fallen cones, acorns, hackberries or nuts.The remains of fruits consumed by Eurasian red squirrels were also recorded and identified by characteristic squirrel bites.Only natural sources of seeds were evaluated given that the composition of seeds offered in feeders varied widely.Seed counts provided an estimate of seed availability (calculated as 10 3 seeds / ha, Bosch & Lurz, 2012).We used data on seed production and calorific content obtained from the literature (Grodziński & Sawicka-Kapusta, 1970;Demir et al., 2002;Wauters et al., 2002;Bosch & Lurz, 2012;Stock et al., 2013) to measure the mean energy value (10 3 kJ / ha -1 ) and standard deviation (± SD) related to seed counts per habitat.

Reintroduction management
Reintroductions were considered successful as viable populations were established, long-term benefit for local communities were achieved, and improvements in conservation awareness were made, in accordance with IUCN guidelines (IUCN, 1998(IUCN, , 2012)).Qualitatively data on management were assessed by unstructured interviews with park managers and employees, and by consulting official documents.We investigated release histories according to motivation, year of release, reintroduction technique (e.g., soft release in which animals are first acclimatized with new habitat in enclosures before release, or hard release in which individuals are directly released into the new environment; see Ewen et al., 2012), supplementary feeding, veterinary support, choice of donor population, number of individuals released, Table 1.Best-fitting models according to Akaike information criterion (AIC) and degree of freedom (df) values to estimate the population density of Eurasian red squirrels at the Parque Biológico de Gaia (PBG) and at the Jardim Botânico da Universidade de Coimbra (JBUC), Portugal, in autumn 2013.

Eurasian red squirrel population
During the surveys, we observed 61 individuals in the PBG and four in the JBUC.The best relative fit model and adjustment term for the population in the PBG was a hazard-rate cosine based on the lowest AIC score (table 1).In contrast, the best fit for the JBUC population was a negative exponential cosine model based on the lowest AIC score (table1).Estimated abundance and density were higher in PBG (N = 47 squirrels, D = 1.33 squirrel/ha) than in JBUC (N = 2 squirrels, D = 0.17 squirrel/ha).The detection probability in the PBG was 44% whereas in the JBUC it was 51.5%.The encounter rate was 56% and 48.5% for the PBG and JBUC, respectively.

Habitat use
Seven squirrel dreys were found placed in the oak and cork oak forest at PBG (fig.1B).Squirrels placed a significant portion of dreys in the height of 13 m in forests dominated by Q. robur and Castanea sativa (F = 8.35, df = 11, P < 0.01).Seven dreys were found around 14 m high in the oak forest in the JBUC (F = 15.74,df = 14, P < 0.01).One drey was found on Pseudotsuga menziesii in the alien conifer garden (fig.1B).
During autumn 2013, only three tree species were fruiting in the PBG: Q. robur, C. sativa and P. pinaster (fig.1A, table 2).The black alder forest had higher seed productivity (143.8 ± 163.6 x 10 3 seeds/ha) and energetic content (9,524 x 10 3 kJ/ha -1 ) due to the high concentration of fruiting C. sativa (table 2).We counted 471 fungal bodies from 28 species in the PBG.Only 16% of mushrooms were edible to the Eurasian red squirrel (table 3).Russula spp.showed significant relative abundance of edible fungi in the PBG, with R. cyanoxantha and R. decipiens together accounting for 59.4% (table 3).Ongoing supplementary feeding in the PBG consisted of five feeders daily supplied with birdseed to attract birds, but these were also used by squirrels.Squirrels were observed eating mainly sunflower seeds.
Only the oak and mixed forests had fruiting trees in the JBUC during surveys (table 2), namely Pinus pinea, Quercus robur, and Celtis australis.Fruits of this last tree were seen being eaten by squirrels during fieldwork.The oak forest had higher seed productivity (974.0 ± 534.9 x 10 3 seeds/ha) and energetic content (20,779 x 10 3 kJ / ha -1 ) due to the high productivity of seeds per cone of P. pinea (table 2).In the JBUC, we counted 33 fungal bodies of seven species and 70% of them were edible to the Eurasian red squirrel (table 3).As in PBG, the genus Russula was also

Reintroduction management
Table 4 summarizes reintroductions attendance to IUCN stepwise.Both reintroductions in PBG and JBUC aimed at enhancing parks aesthetics and enable people to become familiar with this species (table 4).Park managers used the Eurasian red squirrel historical population observations of Antunes (1985) as proving of the species historical range in Portugal.Both urban parks acquired squirrels from commercial creators with veterinary control and support which lowered possibilities of diseases or parasites.
The PBG released 12 squirrels In 1997, and a further 40 couples between 1998 and 2001 using a hard release approach.The animals were from Azé (France).The squirrels in the PBG have continuous veterinary support, because a wildlife rehabilitation center is located therein (table 4), and continuous feeding is provided through bird feeders.Two main failures were detected in the reintroduction project in PBG: the absence of genetic comparison between donors and the closest wild population, and a lack of long-term, technical monitoring.
Twelve squirrels from Madrid (Spain) were hard released at JBUC in 1994.Four squirrel feeders in the forest were active only during the first year (table 4).As in the PBG, the reintroduction project at  4).

Discussion
To date, reintroductions of Eurasian red squirrels in Portugal have been successful to some extent given that squirrels are still present in the urban parks almost 20 years later.However, the populations of squirrels are decreasing in both urban parks.Studies found densities from 0.03 to 1.80 squirrels/ha in mixed woodlands (Wauters & Dhondt, 1988;Cagnin et al., 2000;Magris & Gurnell, 2002;Vilar, 1997), figures that are similar to our estimate (0.17 squirrels/ha in JBUC and 1.33 squirrels/ha in PBG).Considering urban parks of limited area and resources, the density in the PBG is similar to densities found in Belgium (Wauters et al., 1997a) and Spain (Vilar, 1997, while the density in the JBUC is lower.The difference in density of the reintroduced populations is mainly related to post-release management since the PBG had squirrel population reinforcements but the JBUC did not.Habitat quality also regulates species abundance and density and is of great importance for the success or failure of reintroductions (Ewen et al., 2012).Squirrel dreys in both urban parks were predominantly placed in native oak forests and near food sources, reinforcing the need for high quality habitat and food diversity for the maintenance of these populations.The studied parks had few fruiting trees compared with other studies (Bosch & Lurz, 2012).Forests in the JBUC had a higher energetic content than those in the PBG but the diversity of native food items was poorer.In contrast, forests in the PBG had less energetic content, but they presented richer and more abundant additional food items, such as edible mushrooms.Additionally, the PBG had continuous supplementary food, mainly through bird feeders also used by squirrels, whereas the JBUC only had feeders in the year following the reintroduction.
In terms of species identity for conservation purposes, genetic proximity was only adequately considered in the JBUC where the subspecies Sciurus vulgaris infuscatus was reintroduced, while in the PBG the subspecies S. v. fuscoater was released.Although both subspecies occur in the Iberian Peninsula, only S. v. infuscatus occurs naturally in Portugal (Mathias & Gurnell, 1998;Lurz et al., 2005;Bosch & Lurz, 2012).Further studies on Eurasian red squirrel distribution, taxonomy and genetic diversity in the Iberian Peninsula should consider the influence of S. v. fuscoater presence in Portugal, as has been done to other subspecies in the United Kingdom (see Hale & Lurz, 2003;Hale et al., 2004).
Post-project monitoring was not explicitly considered in either park.It is there not fully understood whether dispersal to vicinity (table 3) was natural or due to stress of limited resources.Deficiency in post-release actions, such as monitoring health and abundance, is responsible for the long-term decrease in Eurasian red squirrel populations in both urban parks but active adaptive management could improve the current situation (Ewen et al., 2012;Runge, 2013).Future actions should consider improving habitat quality by means of specific feeders for Eurasian red squirrels, and replacement of alien trees for native oak forest.Monitoring population health, adaptation and demographic variation will endorse the long-term success of the reintroductions.In addition, managers should ensure active human community involvement so that effective education would not only foster knowledge of species but also concern for its needs (IUCN, 2012).
Critical steps for successful reintroduction of Eurasian red squirrels in areas without competition of alien tree squirrels should follow three stages, consisting of pre-project activities, release stages and post-release activities (IUCN, 1998(IUCN, , 2012)).Pre-project activities should include studies on (1) habitat quality, (2) genetic proximity between donors and the closest wild population, and (3) the health of donor stocks.
In the release stage, (1) the number of individuals released should consider 35 to 85 individuals to achieve a long-term viable population in an area of high resource variability, and 55 to 175 individuals in areas of low resource variability (Wood et al., 2007) and (2) hard release technique proved to be a good and cheaper method to Eurasian red squirrel reintroductions (Swinnen, 1988;Fornasari et al., 1997).Finally, post-release activities should (1) evaluate population adaptation, (2) mitigate mortality, (3) monitor the need for supplementary feeding, (4) provide veterinary support, and (5) promote continuous public awareness and education (IUCN, 1998(IUCN, , 2012)).
Reintroductions for aesthetic and leisure purposes are not usually concerned about strictly following conservation protocols unless required by law.However, these reintroductions for aesthetic and leisure purposes have significant effects on wildlife management and conservation (Hodder & Bullock, 1997).Therefore, we strongly suggest that reintroductions with aims other than conservation should also have standardized international guidelines, regulations and monitoring.

Fig. 1 .
Fig. 1.Study area in northern and central Portugal with inset showing vegetation type and distribution of dreys (white circles) in the Parque Biológico de Gaia at Vila Nova de Gaia (A), and in the Jardim Botânico da Universidade de Coimbra at Coimbra (B).

Fig. 1 .
Fig. 1.Zona de estudio situada en el norte y el centro de Portugal con recuadros que muestran el tipo de vegetación y la distribución de los nidos de ardilla (círculos blancos) en el Parque Biológico de Gaian en Vila Nova de Gaia (A) y en el Jardín Botánico de la Universidad de Coimbra, en Coimbra (B).