INTRODUCTION

The Northern Bald Ibis, Geronticus eremita (Linnaeus 1758), is a migratory bird belonging to the Threskiornithidae family. This species had a fragmented distribution with populations in Central Europe, North Africa, and the Middle East until the seventeenth century (Bowden et al. 2008, Böhm and Pegoraro 2011, Böhm et al. 2021). However, it was eradicated from Central Europe nearly 400 years ago due to habitat loss, climate change, and direct persecution (Böhm and Pegoraro 2011), which makes it one of the threatened bird species (Böhm et al. 2021). The remaining wild or semi-wild populations outside Europe now occur in the western (Morocco) and the eastern Mediterranean (Syria and Turkey). Since 2018, the Northern Bald Ibis has been classified as Endangered on the IUCN Red List of Threatened Species (BirdLife International 2018). The last known wild population in Morocco comprises about 708 relict free-flying adult birds (Oubrou and El Bekkay 2018, Böhm et al. 2021). There are many captive populations, with total number reaching more than 1700 individuals including non-breeding birds in 2018. All captive populations since 1950s originate from the Moroccan population (Böhm et al. 2018). Although monitoring in the field has been extremely difficult since 2015 due to security issues, the Syrian population now appears to be functionally depleted (Böhm et al. 2021).

The Birecik colony in SE Turkey is the main extant population in the East (Fig. 1). The rapid decline of the population in this colony since 1950 was due to excessive use of organochlorines such as DDT (Akçakaya et al. 1992, Hatipoğlu 2016). By 1973, only 23 breeding adults had remained with ongoing high mortality and reproductive failure (Arihan 1999). In 1977, the General Directorate of Nature Conservation and National Parks of Turkey and the Society for Protection of Nature (DHKD) started a project to save the Northern Bald Ibis population in Birecik. Therefore, 41 birds were caught and held in semi-captivity between 1977–1989 whereas a similar number of birds remained in the wild. However, in the 1990s no wild individuals were left that could breed in nature without human support (Akçakaya 1990, Yeniyurt et al. 2016). Captive birds are released in early spring and they forage in the surrounding habitats as free-flying birds until July, when they are moved back into captivity to prevent migration and increase survival rates (Akçakaya 1990, Yeniyurt et al. 2016). By 2021, there were 83 breeding pairs and 101 fledglings in Birecik (unpublished report of DKMP). The distinct migratory behavior of the Birecik population which historically moved - and when given the chance, also now moves - south to winter in southern Arabia and Ethiopia (e.g., Serra et al. 2015) while the western population does not migrate but rather is resident or lives a nomadic existence (Cramp and Simmons 1998, Matheu et al. 2020).

Understanding the genetic background of wild or captive populations of threatened species is essential to increase the success of any conservation approach (Attard et al. 2016). High genetic diversity helps populations cope with environmental fluctuations such as diseases, habitat alteration, or climate change (Tracy et al. 2011). Ensuring adequate genetic diversity is particularly important when using displaced or captive-bred individuals to establish a new population (Johnson et al. 2021). Additionally, selection of the most suitable individuals from the ex-situ pool is an important factor that often determines the success of reintroduction projects (Saura et al. 2008, Wirtz et al. 2018).

Genetic analyzes are especially relevant during any reintroductions as it is fundamental to analyze the genetic variability of founders to prevent inbreeding and to ensure a good probability of persistence (Witzenberger and Hochkirch 2011). With this scope and by using both nuclear and mitochondrial markers, we aimed to analyze the genetic diversity of Northern Bald Ibis (Geronticus eremita) within the Birecik population and compare it to European captive and Moroccan wild populations.

METHODS

Sampling and DNA extraction

No field survey or specimen collecting was performed for this study. Blood samples of 100 Geronticus eremita semi-wild individuals (51 adults and 49 chicks) were collected by the veterinarians in Birecik (37.04 N, 37.99 E).

Genomic DNA was isolated using Qiagen Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany) by following the manufacturer's protocol.

Microsatellite analysis

Samples were genotyped at five microsatellite loci developed for the Northern Bald Ibis (Wirtz et al. 2016). Locus-specific forward primers fluorescently labeled (HEX or FAM) at 5′-end were used in multiplex reaction. PCR was conducted using 5x HOT FIREPol® Blend Master Mix (HOT FIREPol DNA polymerase, 5x blend master mix, 2.5 mM MgCl2, 2 mM dNTP, BSA, blue and yellow dyes; Solis Biodyne) in a total reaction volume of 25 µl, containing 1 µl of genomic DNA, 0.3 µM of each primer pair and, 1× Master Mix (Solis Biodyne). Touch-down PCR was implemented under the following conditions: 15 mins 95 ^oC heat activation followed by 30 cycles of 20 sec at 95 ^oC, 1 min at TA ^oC (optimum annealing temperature for multiplex set, Table 1), 1 min at 72 ^oC, and a final extension of 10 mins at 72 ^oC. Two-percent agarose gel electrophoresis was used to qualify PCR amplifications. Genotyping was repeated three times per sample if the low peak signal was detected. An ABI-PRISM 3100 sequencer (Applied Biosystems) was used to determine allele sizes.

Mitochondrial ND5 analysis

The NADH hydrogenase subunit 5 (ND5) gene 732 bp in length was amplified in 49 adult individuals using the primers Av12976tSerF 5′-CAA GAA CTG CTA ACT CTT GTA TCT G-3′and Av13734ND5R 5′-AAT CCA AAT TGG GCT GAT TTT CC-3′ designed by Wirtz et al. (2018). 5x Hot FIREPol® Blend Master Mix (Solis Biodyne) was used for PCR amplification of this marker. The total reaction volume of 25 µl contained 1x Blend Master Mix, 0.3 µM of each primer, and 1 μl of genomic DNA. Amplification for ND5 was conducted as follows: initial denaturation at 95°C for 15 mins, 38 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 1 min and elongation at 72°C for 4 mins. The final elongation was done for 10 mins at 72°C. Two-percent agarose gel electrophoresis was used to qualify PCR amplification. All sequencing reactions were performed by using ABI terminator 3.1. Kit (Applied Biosystems Inc., Foster City, CA, USA). PCR products were sequenced in both directions to increase accuracy of reading. An ABI 3730x1 Genetic Analyzer (Applied Biosystems) was used for electrophoresis and determination of fluorescently labeled nucleotides.

Data analysis

Microsatellite analysis

Raw sequence data was examined with the PEAK SCANNER 2.0 (Applied Biosystems) software to identify peaks and fragment sizing with the standard. FreeNA (Chapuis and Estoup 2007) was used to test for null alleles. Genotyping errors, allelic dropout, and scoring of stutter peaks were assessed statistically using MICROCHECKER 2.2.3 (Van Oosterhout et al. 2004). In order to determine linkage disequilibrium, gene diversity and allelic richness were calculated by FSTAT 2.9.3 (Goudet 2001). Deviations from Hardy-Weinberg Equilibrium was tested using GenAlEx 6.5 (Peakall and Smouse 2012). The software ML-RELATE (Kalinowski et al. 2006) was used to estimate relatedness and relationships between pairs of individuals. This method calculates the maximum likelihood estimates of relatedness (r) using a downhill simplex routine. The software assigns pairs of samples into four common pedigree relationships: unrelated (U), half-siblings (HS), full-siblings (FS), and parent- offspring (PO). However, there is no class for lower-level relatedness (e.g., cousins and nieces) in these analyzes. It is, therefore, reasonable to assume that these may be misclassified as HS, at least more frequently than unrelated individuals. In addition, polymorphic information content (PIC), indicating the possible utility of markers in identifying individuals, was estimated with CERVUS 3.0.7 (Marshall et al. 1998, Kalinowski et al. 2007); where PIC > 0.5 is highly informative, 0.5 > PIC > 0.25 is moderate informative, and PIC < 0.25 is somewhat informative (Botstein et al. 1980). To reveal any recent bottlenecks, the software BOTTLENECK 1.2.02 (Cornuet and Luikart 1996, Piry et al. 1999) was used. Under the two-phase mutation model (TPM), the statistical significance of heterozygosity excess was measured by means of Wilcoxon’s sign rank test; the mode-shift graphical method was applied to estimate allele frequency shift after a bottleneck event (Luikart et al. 1998). It is expected that alleles carrying intermediate frequencies (about 0.1–0.2) are more widespread than alleles carrying low frequencies (< 0.1; Ganapathi et al. 2012).

Mitochondrial ND5 analysis

The alignment of all ND5 sequences was performed using the Clustal W algorithm (Thompson et al. 1997) in MEGA X (Kumar et al. 2018). Alignments were checked by eye for accuracy and corrected manually. Haplotype diversity (Hd) was calculated using DnaSP program v.6 (Rozas et al. 2017). Mitochondrial DNA sequences representing distinct haplotypes were deposited in GenBank.

RESULTS

Relatedness of study individuals

DNA isolation from blood samples of 100 individuals was successfully performed. All microsatellite loci were amplified successfully in all individuals. No loci were detected to contain null alleles (r < 0.2) when tested with FreeNa (10,000 replicates; Table 2). The probable presence of linkage disequilibrium in the population of 100 individuals was calculated using FSTAT version 2.9.4. Looking at possible comparisons between pairs of loci in the population, no significant linkage disequilibrium was detected after Bonferroni correction and each locus was evaluated independently. When MICROCHECKER was used to statistically assess the presence of genotypic errors, allelic dropouts, and artifacts (such as stutters), none of the five polymorphic loci proved to have any genetic errors in 100 individuals. Therefore, all five loci were used in further analysis.

Of 4950 possible pairwise combinations among 100 individuals, ML-RELATE predicted 1545 family relationships. These were classified as 294 full-siblings, 552 half-siblings, and 699 parent-offspring relationships. Unrelated (U) pairs of samples were 3405. None of the individual sample pairs assigned to the three relatedness classes were significant on their own.

Nuclear genetic diversity, inbreeding, and bottleneck

Five nuclear loci were successfully genotyped for all samples (n = 100). A total of 23 alleles were detected in all loci throughout the population. These five loci exhibited high genetic diversity based on allelic richness (A_R), gene diversity, and observed heterozygosity (H_o; Table 2). The allelic richness was highest in loci A06 and A09 (Table 2). All loci were determined to be in Hardy-Weinberg Equilibrium (HWE). No signs of inbreeding were detected (Table 2). The 5 loci used were found to be highly informative loci (PIC > 0.5) in the G. eremita population (Table 2). According to the Wilcoxon test, the observed proportion of heterozygotes showed no deviation from expectation under mutation-drift equilibrium using a TPM (one-tailed for H excess: P = 0.5) in the pooled sample, which indicates that the Northern Bald Ibis population was not going through a recent bottleneck. The proportion of alleles in different allele frequency classes (0–0.1 low; 0.9–1 high allele frequency class) showed the normal L-shaped distribution rather than the mode-shifting distribution which would be expected for bottleneck populations (Fig. 2).

Mitochondrial genetic diversity

The NADH hydrogenase subunit 5 (ND5) gene was subjected to sequencing in 49 adults from the Birecik population, resulting in the identification of one substitution when compared to the western population. The same nucleotide (A) at position 75 was identified in all individuals of the Birecik population and also previously documented in Wirtz et al. (2018) as eastern haplotype 1 (Accession no: MF682061.1; Wirtz et al. 2018) in five individuals of Turkish and Syrian origin. Consequently, the Birecik population exhibits a single, fixed haplotype (Fig. 3).

DISCUSSION

Loss of genetic diversity due to high rates of inbreeding are both important issues in captive breeding as they often lead to reduced population growth and increased risk of extinction (Willoughby et al. 2015, Choi et al. 2020). Our results show that despite a relatively small founder base, the captive population of Northern Bald Ibis is not strongly affected by a loss of genetic diversity. Although Wirtz et al. (2018) studied more loci than we did, they identified nearly the same level of allelic richness (A_R, range 3.53–4.09) for their study populations: captive individuals (zoo), wild birds (WB), the “Waldrappteam” population (WRT), and the “Proyecto Eremita” population (PE). Similarly, using the same set of microsatellite loci, a comparable level of average heterozygosity (H_o) of 0.575 was observed in Brazilian Roseate Spoonbill populations (Miño and Del Lama 2007). The level of heterozygosity (H_o, range 0.500-0.760) we report is also comparable to other Bald Ibis populations (Wirtz et al. 2016, 2018). Similar to our findings, Wirtz et al. (2018) showed that although only a small sample of eight individuals originated from the eastern wild population, they had higher allelic richness than any ex-situ group studied or the western wild population.

Despite the founder population being no more than 20 pairs, no statistically significant inbreeding coefficient (F_IS varied from - 0.119 to 0.127) was determined for the Birecik population. This may be explained by an already very diverse gene pool to start with and/or individuals preferring to mate with individuals they are more distantly related to (Smith et al. 1997, Hoffman et al. 2007). Negative inbreeding coefficients (F_ıs) were found even for populations with as few as two founders as in the case of the Crested Ibis population in South Korea (Choi et al. 2020), implying it is possible for some populations to overcome adverse effects of low founder size. Although, Wirtz et al. (2018) also detected no sign of inbreeding, the relatively higher F_IS value for the European zoo population may be explained by deviations from the HWE for this population due to the Wahlund effect. Selecting mates from unrelated individuals is the dominant pattern in birds and mammals (Smith et al. 1997, Hoffman et al. 2007). However, a full understanding of the consequences of inbreeding in wild populations requires not only the detection of relatedness but also long-term measures of reproductive success and survival (Brzeski et al. 2014).

mtDNA ND5 sequencing revealed an only one haplotype (eastern haplotype 1) in the eastern population. This was found to be a single nucleotide (eastern haplotype 1: Accession no: MF682061.1) different from the haplotype (western haplotype 1: MF682062.1) found in the western population. Although substitution at position 75 of the gene was not linked to the origin of the sample by Wirtz et al. (2018), this mutation was found to be present in all Birecik individuals and absent in western birds (Wirtz et al. 2018). Despite the presence of a single mutation observed between the eastern and western populations, this specific haplotype is exclusively observed in the eastern population of the Northern Bald Ibis (Birecik, Turkey).

There have been massive efforts to reintroduce the Northern Bald Ibis into Austria, Switzerland, and southern Germany, using captive-bred individuals of the western origin, which have succeeded in establishing a population of about 200 free-ranging birds (Fritz 2021). For the geographically isolated eastern colony in Birecik, on the other hand, efforts until now have mainly focused on growing its numbers (Arihan 1999, Hatipoğlu 2016). However, in order to reduce the risk of disease or other catastrophes wiping out the only colony, it is proposed that new colonies need to be set up at suitable sites elsewhere (e.g., Schenker and Erhardt 2023). Our results suggest such new breeding colonies are unlikely to suffer due to inbreeding since the semi-captive population appears to have enough diversity to select founders with different enough genetic backgrounds.

Overall, the documented geographic, genetic, and behavioral differences confirm that the Birecik semi-wild population constitutes the last representative of the eastern population and is considered as an important separate piece of its western relatives. The presence of a high nuclear genetic diversity indicates that the Birecik semi-wild population is a critical candidate for reintroduction to release of the species to locations other than Birecik.

LITERATURE CITED

Akçakaya, H. R. 1990. Bald ibis Geronticus eremita population in Turkey: an evaluation of the captive breeding project for reintroduction. Biological Conservation 51(3):225-237. https://doi.org/10.1016/0006-3207(90)90153-G

Akçakaya, H. R., and Y. Barış. 1992. Birecik’teki Kelaynak (Geronticus eremita) populasyonunun yok olma nedenleri ve koruma çalışmalarının değerlendirilmesi. Doğa-Turkish Journal of Zoology 16:1-12.

Arihan, O. 1999. Northern Bald Ibis in Turkey: experiences from captive breeding and reintroduction programs. Pages 27-35 in C. Böhm, editor. Northern Bald Ibis Geronticus eremita, 2nd EEP Studbook, Alpenzoo, Innsbruck, Austria.

Attard, C. R. M., L. M. Moller, M. Sasaki, M. P. Hammer, C. M. Bice, C. J. Brauer, D. C. Carvalho, J. O. Harris, and L. B. Beheregaray. 2016. A novel holistic framework for genetic-based captive breeding and reintroduction programs. Conservation Biology 30(5):1060-1069. https://doi.org/10.1111/cobi.12699

BirdLife International. 2018. Species factsheet: Northern Bald Ibis Geronticus eremita. https://datazone.birdlife.org/species/factsheet/northern-bald-ibis-geronticus-eremita

Böhm, C., C. G. Bowden, P. J. Seddon, T. Hatipoğlu, W. Oubrou, M. El Bekkay, M. A. Quevedo, J. Fritz, C. Yeniyurt, J. M. Lopez, J. F. Orueta, D. Frigerio, and M. Unsöld. 2021. The northern Bald Ibis Geronticus eremita: history, current status and future perspectives. Oryx 55(6):934-946. https://doi.org/10.1017/S0030605320000198

Böhm, C., K. Pegoraro. 2011. Der Waldrapp Geronticus eremita: Ein Glatzkopf in Turbulenzen. Neue Brehm Bücherei, Bd. 659. VerlagsKG Wolf, Magdeburg, Germany.

Böhm, C., K. Schad, E. Fienieg, and M. Voorham. 2018. Long-term Management Plan for the Northern Bald Ibis (Geronticus eremita) European Endangered Species Programme (EEP). Alpenzoo Innsbruck-Tirol, Innsbruck, Austria, and EAZA Executive Office, Amsterdam, The Netherlands.

Botstein, D., R. L. White, M. Skolnick, and R. W. Davis. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. American Journal of Human Genetics 32(3):314.

Bowden, C. G. R., K. W. Smith, M. El Bekkay, W. Oubrou, A. Aghnaj, and M. Jimenez- Armesto. 2008. Contribution of research to conservation action for the Northern Bald Ibis Geronticus eremita in Morocco. Bird Conservation International 18:S74-S90. https://doi.org/10.1017/S0959270908000403

Brzeski, K. E., D. R. Rabon, M. J. Chamberlain, L. P. Waits, and S. S. Taylor. 2014. Inbreeding and inbreeding depression in endangered Red Wolves (Canis rufus). Molecular Ecology 23(17):4241-4255. https://doi.org/10.1111/mec.12871

Chapuis, M. P. and A. Estoup. 2007. Microsatellite null alleles and estimation of population differentiation. Molecular Biology and Evolution 24(3):621-631. https://doi.org/10.1093/molbev/msl191

Choi, E. H., G. Kim, S. Y. Baek, S. J. Kim, J. Hwang, J. Jun, K. H. Jang, S. H. Ryu, and U. W. Hwang. 2020. Development and characterization, and application of ten polymorphic microsatellite markers in the crested ibis Nipponia nippon from South Korea. Animal Systematics, Evolution and Diversity 36(2):154-158.

Cornuet, J. M. and G. Luikart. 1996. Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144(4):2001-2014. https://doi.org/10.1093/genetics/144.4.2001

Cramp, S. and K. E. L. Simmons. 1998. The Complete Birds of the Western Palearctic. CD-ROM. Oxford University Press, Oxford, UK.

Fritz J. 2021. The European LIFE Northern Bald Ibis reintroduction project. Oryx 55(6):809-810. https://doi.org/10.1017/S003060532100123X

Ganapathi, P., R. Rajendran, and P. Kathiravan. 2012. Detection of occurrence of a recent genetic bottleneck event in Indian hill cattle breed Bargur using microsatellite markers. Tropical Animal Health and Production 44:2007-2013. https://doi.org/10.1007/s11250-012-0171-8

Goudet, J. 2001. FSTAT: a program to estimate and test gene diversities and fixation indices (version 2.9.3). https://www2.unil.ch/popgen/softwares/fstat.htm

Hatipoğlu, T. 2016. Conservation project, Birecik, Turkey. Pre-meeting materials for circulation to participants of the 4th IAGNBI meeting 5^th-6^th August 2016, 34-39. Alpenzoo Innsbruck-Tirol, Innsbruck, Austria, and Royal Society for the Protection of Birds, Sandy, UK.

Hoffman, J. I., J. Forcada, P. N. Trathan, and W. Amos. 2007. Female fur seals show active choice for males that are heterozygous and unrelated. Nature 445:912-914. https://doi.org/10.1038/nature05558

Johnson, J. A., A. Stock, P. Juergens, B. Mutch, and C. J. McClure. 2021. Temporal genetic diversity and effective population size of the reintroduced Aplomado falcon (Falco femoralis) population in coastal South Texas. Journal of Raptor Research 55(2):169-180. https://doi.org/10.3356/0892-1016-55.2.169

Kalinowski, S. T., A. P. Wagner, and M. L. Taper. 2006. ML‐Relate: a computer program for maximum likelihood estimation of relatedness and relationship. Molecular Ecology Notes 6(2):576-579. https://doi.org/10.1111/j.1471-8286.2006.01256.x

Kalinowski, S. T., M. L. Taper, and T. C. Marshall. 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 16(5):1099-1106. https://doi.org/10.1111/j.1365-294X.2007.03089.x

Kumar, S., G. Stecher, M. Li, C. Knyaz, and K. Tamura. 2018. MEGAX: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35(6):1547-1549. https://doi.org/10.1093/molbev/msy096

Linnaeus, C. 1758. Systema Naturae per Regna Tria Nature, secundum classes, ordines, genera, species cum characteribus, differentitis, synonymis, locis. Tomus I. Editio Decima Reformatum. Laurentio Salvii, Holmiae.

Luikart, G., F. Allendorf, J. Cornuet, W. Sherwin. 1998. Distortion of allele frequency distributions provides a test for recent population bottlenecks. Journal of Heredity 89(3):238-247. https://doi.org/10.1093/jhered/89.3.238

Marshall, T., J. Slate, L. Kruuk, and J. Pemberton. 1998. Statistical confidence for likelihood-based paternity inference in natural populations. Molecular Ecology 7(5):639-655. https://doi.org/10.1046/j.1365-294x.1998.00374.x

Matheu, E., J. del Hoyo, G.M. Kirwan, and E. F. J. Garcia. 2020. Northern Bald Ibis (Geronticus eremita), version 1.0. Pages in J. del Hoyo, A. Elliott, J. Sargatal, D. A. Christie, E. de Juana, editors. Birds of the World, Cornell Lab of Ornithology, Ithaca, New York, USA. https://doi.org/10.2173/bow.waldra1.01

Miño, C. I. and S. N. Del Lama. 2007. Genetic structure in Brazilian breeding colonies of the Roseate Spoonbill (Platalea ajaja, Aves: Threskiornithidae). Genetics and Molecular Research 6(2):338-347.

Oubrou, W., M. El Bekkay. 2018. Rapport sur la reproduction de l'Ibis Chauve Geronticus eremita dans la région de Souss-Massa. Saison 2018. HCEFLCD. https://www.grepom.org/wp-content/uploads/Rapport-de-reproduction-IC-2018-.pdf

Peakall, R., P. E. Smouse. 2012. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics 28(19):2537-2539. https://doi.org/10.1093/bioinformatics/bts460

Piry, S., G. Luikart, and J. M. Cornuet. 1999. Computer note. BOTTLENECK: a computer program for detecting recent reductions in the effective size using allele frequency data. Journal of Heredity 90(4):502-503. https://doi.org/10.1093/jhered/90.4.502

Rozas, J., A. Ferrer-Mata, J. C. Sánchez-DelBarrio, S. Guirao-Rico, P. Librado, S. E. Ramos-Onsins, and A. Sánchez-Gracia. 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Molecular Biology and Evolution 34(12):3299-3302. https://doi.org/10.1093/molbev/msx248

Saura, M., A. Perez-Figueroa, J. Fernandez, M. A. Toro, and A. Caballero. 2008. Preserving population allele frequencies in ex situ conservation programs. Conservation Biology 22(5):1277-1287. https://doi.org/10.1111/j.1523-1739.2008.00992.x

Schenker A. and A. Erhardt. 2023. Assessment of suitable habitats using satellite imagery: example of the Northern Bald Ibis Geronticus eremita in south-eastern Turkey. Bird Conservation International 33(e75):1-8 https://doi.org/10.1017/S0959270923000242

Serra, G., J. Lindsell, L. Peske, J. Fritz, C. Bowden, C. Bruschini, G. Welch, J. Tavares, and M. Wondafrash. 2015. Accounting for the low survival of the critically endangered northern Bald Ibis Geronticus eremita on a major migratory flyway. Oryx 49(2):312-320. https://doi.org/10.1017/S0030605313000665

Smith, D., T. Meier, E. Geffen, L. D. Mech, J. W. Burch, L. G. Adams, and R. K. Wayne. 1997. Is incest common in gray wolf packs? Behavioral Ecology 8(4):384-391. https://doi.org/10.1093/beheco/8.4.384

Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25(24):4876-4882. https://doi.org/10.1093/nar/25.24.4876

Tracy, L. N., G. P. Wallis, M. G. Efford, and I. G. Jamieson. 2011. Preserving genetic diversity in threatened species reintroductions: how many individuals should be released? Animal Conservation 14(4):439-446. https://doi.org/10.1111/j.1469-1795.2011.00448.x

van Oosterhout, C., W. F. Hutchinson, D. M. Wills, and P. Shipley. 2004. MICROCHECKER: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes 4(3):535-538 https://doi.org/10.1111/j.1471-8286.2004.00684.x

Willoughby, J. R., N. B. Fernandez, M. C. Lamb, J. A. Ivy, R. C. Lacy, and J. A. DeWoody. 2015. The impacts of inbreeding, drift and selection on genetic diversity in captive breeding populations. Molecular Ecology 24(1):98-110. https://doi.org/10.1111/mec.13020

Wirtz, S., C. Böhm, J. Fritz, T. Hankeln, and A. Hochkirch. 2016. Isolation of microsatellite loci by next-generation sequencing of the critically endangered Northern Bald Ibis Geronticus eremita. Journal of Heredity 107(1):363-366. https://doi.org/10.1093/jhered/esw013

Wirtz, S., C. Böhm, J. Fritz, K. Kotrschal, M. Veith, and A. Hochkirch. 2018. Optimizing the genetic management of reintroduction projects: genetic population structure of the captive Northern Bald Ibis population. Conservation Genetics 19(4):853-864. https://doi.org/10.1007/s10592-018-1059-6

Witzenberger, K. A. and A. Hochkirch. 2011. Ex situ conservation genetics: a review of molecular studies on the genetic consequences of captive breeding programmes for endangered animal species. Biodiversity and Conservation 20:1843-1861. https://doi.org/10.1007/s10531-011-0074-4

Yeniyurt, C., S. Oppel, S. Isfendiyaroğlu, G. Özkinaci, L. I. Erkol, and C. G. R. Bowden. 2016. Influence of feeding ecology on breeding success of a semi-wild population of the Critically Endangered northern Bald Ibis Geronticus eremita in southern Turkey. Bird Conservation International 27(4):537-549. https://doi.org/10.1017/S0959270916000253