1. IntroductionBirds of prey, like many predatory species, have been subject to human persecution for centuries and this continues to be a global concern [[31]Madden K.K. Rozhon G.C. Dwyer J.F. Conservation letter: raptor persecution.]. In the United Kingdom persecution has taken many forms including shooting, trapping, and poisoning by landowners and gamekeepers who consider them a threat to livestock and gamebirds (RSPB 2015). In addition, many were illegally taken from the wild as eggs or nestlings and ‘laundered’ into the captive-bred population to meet the national and international demand for falconry birds ([[16]Fleming L.V. Douse A.F. Williams N.P. Captive breeding of peregrine and other falcons in Great Britain and implications for conservation of wild populations.], Willcox et al. 2019). The Wildlife and Countryside Act 1981 introduced a Bird Registration Scheme (BRS) for certain rare birds held in captivity in Great Britain. This was intended to make keepers accountable for birds in their possession and to prevent wild-taken birds being laundered into the captive market. The scheme required declaration of the identity of the parent birds when captive-bred nestlings were fitted with a uniquely numbered permanent ring within the first month of life [[16]Fleming L.V. Douse A.F. Williams N.P. Captive breeding of peregrine and other falcons in Great Britain and implications for conservation of wild populations.]. For people with experience in the keeping and breeding of certain species, the BRS was relatively simple to bypass. Species like peregrine falcon (Falco peregrinus) and goshawk (Accipiter gentilis), were highly sought after for falconry, difficult to breed in captivity, and commanded high prices. By obtaining wild-taken eggs or young chicks the birds could be declared as captive bred, registered and effectively legitimised, then sold. The fact that some keepers involved were also successfully breeding these birds in captivity and used wild stock to boost claimed productivity provided a further cover for illegal activities. However, there was little that could be done to check the legitimacy of claimed relationships prior to the advent of DNA testing.In 1991, the Royal Society for the Protection of Birds (RSPB) initiated the first ever use of DNA testing in the UK to investigate a wildlife crime relating to the alleged captive breeding of goshawks. A human-derived DNA fingerprinting probe indicated four sibling chicks were highly unlikely to be related to the putative female parent. The accused later pleaded guilty to unlawful possession of the chicks; the first wildlife conviction based on DNA testing [[41]The success of DNA profiling in wildlife law enforcement.]. Whilst revolutionary, DNA fingerprinting was poorly suited to large-scale applications as the complex multi-locus profiles comprising alleles from tens of loci were difficult to compare reliably [22Jeffreys A.J. Wilson V. Thein S.L. Hypervariable ‘minisatellite’ regions in human DNA., 23DNA typing: approaches and applications.]. This led to the development of single-locus profiling (SLP) in which a succession of radioisotope-labelled probes was used to detect the alleles at a number of defined minisatellite loci [[59]Wong Z. Wilson V. Patel I. Povey S. Jeffreys A.J. Characterization of a panel of highly variable minisatellites cloned from human DNA.]. This approach required the cloning of a panel of minisatellites from birds of prey so that the homologous locus alone would be detected [[55]A suite of falcon single‐locus minisatellite probes: a powerful alternative to DNA fingerprinting.]. Between 1993 and 1997 SLP probes were used in cases involving more than 350 birds of prey resulting in twelve convictions for a variety of offences involving the laundering of wild birds. The SLP technique required specialist facilities for handling radioisotopes and was extremely labour-intensive, leading to its rapid replacement in human forensics by capillary electrophoretic (CE) screening of panels of microsatellites, also known as short tandem repeats (STRs).Recent advances in DNA sequencing technology have begun to impact the field of human DNA forensics. In particular, the use of massively parallel sequencing (MPS) in place of CE for the analysis of STRs opens up further advantages [[14]From next generation sequencing to now generation sequencing in forensics.]. Since MPS is not reliant on discrimination by length, there is greater multiplexing capability (e.g. validated human forensic MPS kits can simultaneously interrogate 27 autosomal, 24 Y-chromosomal and 7 X-chromosomal STRs, along with 172 SNPs (Jӓger et al. 2017)). This multi-target approach in a single reaction further conserves DNA which may be very limited in forensic cases, and as the method is not reliant on differentiating both alleles and loci by length shorter amplicons with overlapping size ranges can be analysed which benefits the analysis of degraded DNA. By establishing the sequences of STR alleles rather than simply determining their overall length, MPS exploits all the existing variation, for example allowing discrimination between isometric alleles [20Huszar T.I. Jobling M.A. Wetton J.H. A phylogenetic framework facilitates Y-STR variant discovery and classification via massively parallel sequencing., 60Evaluation of the Illumina ForenSeq™ DNA Signature Prep Kit–MPS forensic application for the MiSeq FGx™ benchtop sequencer.] and can thus reduce random match probabilities. Additionally, MPS has the potential to provide more accurate information on mixed DNA samples - a stutter from a major contributor and a real allele from a minor contributor could appear as one peak in CE analysis but they may be distinguishable by MPS [[36]Exploring new short tandem repeat markers for DNA mixture deconvolution.].In this study we describe the identification and characterization of a panel of novel tetra- to hexanucleotide repeat STRs for falcons to replace the dinucleotide markers widely used in falcon population studies (e.g. [[11]Dawnay N. Ogden R. Wetton J.H. Thorpe R.S. McEwing R. Genetic data from 28 STR loci for forensic individual identification and parentage analyses in 6 bird of prey species.]) which are now considered unsuitable for UK casework. We demonstrate how they can be implemented using either conventional fluorescent multiplexing and CE, or MPS approaches, by applying them to historical casework samples previously typed with SLP markers. The STR markers provide fully concordant results with equivalent statistical power but with much greater sensitivity, ease of use and potential for application in a wider range of investigations. We highlight the advantages of each approach and provide the first example of how MPS could be used in the investigation of wildlife crime.4. DiscussionThe past thirty years have seen great advances in forensic genetics with clear applications to the investigation of wildlife crime, illustrated here by reanalysis of one the earliest cases with the first application of massively parallel sequencing of STRs for relationship testing in non-human vertebrates. Human DNA fingerprinting protocols which could be directly applied to animal DNA were rapidly replaced in the early 1990s by single-locus minisatellite profiling more suited to pairwise comparisons among large numbers of individuals. In 1993 the first SLP tests were carried out on birds of prey held in captivity. In that year, the BRS showed there were 154 goshawks and 360 peregrines declared as captive bred, though a small unknown proportion of the peregrines were hybrids (probably ≤60 birds). Though less than 20% of all declared 1993 progeny were tested, this work showed 39 peregrines (~13% allowing for hybrids) and 18 goshawks (11.6%) were not related to their declared parents, clearly indicating that significant numbers of birds declared as captive bred were actually taken from wild populations [41The success of DNA profiling in wildlife law enforcement., 54

Wetton , J.H. and Parkin , D.T. , 1994 . Genetic variation in birds of prey. Department of the Environment, Bristol, UK. 50pp. Genetic Variation in Birds of Prey, Phase IV Final Report

]. There were a number of high-profile successful prosecutions as a result, with this case, and another large-scale peregrine laundering investigation, leading to custodial sentences. In 1994 the number of peregrine and goshawk offspring declared as captive-bred under the BRS fell by approximately 20%. This was suspected to be due to the deterrent impact of DNA profiling and the increased likelihood of detection and prosecution [16Fleming L.V. Douse A.F. Williams N.P. Captive breeding of peregrine and other falcons in Great Britain and implications for conservation of wild populations., 41The success of DNA profiling in wildlife law enforcement.]. Indeed, by 1996 SLP DNA profiling on samples from 69 registered goshawks and peregrines collected during announced inspections confirmed that all the offspring were bred from the claimed parent birds [16Fleming L.V. Douse A.F. Williams N.P. Captive breeding of peregrine and other falcons in Great Britain and implications for conservation of wild populations., 57The application of DNA technology to enforce raptor conservation legislation within Great Britain.]. Whilst the impact of SLP testing on theft from nests was significant, it was recognized that a more cost-effective method would be needed if a registration scheme were to be accompanied by routine DNA profiling.An STR multiplexing system, mirroring that developed for human forensics, would be cheaper, faster, more sensitive, and straightforward for databasing. Initial attempts to apply such systems were limited by the STRs cloned and sequenced at the time [11Dawnay N. Ogden R. Wetton J.H. Thorpe R.S. McEwing R. Genetic data from 28 STR loci for forensic individual identification and parentage analyses in 6 bird of prey species., 34Nesje M. Røed K.H. Lifjeld J.T. Lindberg P. Steen O.F. Genetic relationships in the peregrine falcon (Falco peregrinus) analysed by microsatellite DNA markers.] which were predominantly dinucleotide repeats rather than the tetranucleotide repeat units favoured in human forensics and now also championed for non-human DNA forensic investigations ([25Johnson R.N. Wilson-Wilde L. Linacre A. Current and future directions of DNA in wildlife forensic science., 30Linacre A. Gusmao L. Hecht W. Hellmann A.P. Mayr W.R. Parson W. Prinz M. Schneider P.M. Morling N. ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations., 47van Hoppe M.J. Dy M.A. van den Einden M. Iyengar A. SkydancerPlex: A novel STR multiplex validated for forensic use in the hen harrier (Circus cyaneus).], Ciavagli & Linacre 2018) as well as molecular ecology (e.g. [[9]Caballero I.C. Bates J.M. Hennen M. Ashley M.V. Sex in the city: breeding behavior of urban peregrine falcons in the midwestern US.]). Tetra-, and indeed penta- and hexanucleotide repeats show the ideal combination of characteristics - many alleles and high heterozygosity (resulting in high discrimination power), reasonably short total allele length (typically [1]Andreassen R. Schregel J. Kopatz A. Tobiassen C. Knappskog P.M. Hagen S.B. Kleven O. Schneider M. Kojola I. Aspi J. Rykov A. A forensic DNA profiling system for Northern European brown bears (Ursus arctos).]. In recent years, the need to select STRs from cloned libraries of fragmented DNA has diminished as they can now be identified directly by data-mining of genomic sequences that are becoming available. Screening genomes returns many more STRs than are required to provide robust forensic statistics and so a subset of loci which will amplify under similar conditions can be chosen and assayed together in a single multiplex. Constraints on multiplex size imposed by the limited number of fluorescent labels that could be used in CE to differentiate between loci with overlapping size ranges, are significantly eased by adopting the MPS approach.Inadvertent selection of linked loci will also become rarer as improved sequencing approaches enables the many genome sequences that still comprise hundreds or thousands of short scaffolds to be linked into chromosome level assemblies [[38]Peona V. Weissensteiner M.H. Suh A. How Complete Are" Complete" Genome assemblies?-An Avian Perspective.]. STRs widely separated on the same chromosome may not necessarily be a concern as even quite small family groups can reveal evidence of frequent recombination between physically linked loci but examination of many, or larger, pedigrees is still desirable to determine whether closer associations might adversely affect forensic interpretation. Alternative, albeit less direct means of evaluating independence of markers used for wildlife crime investigations, such as determining population level associations via measures of LD have been proposed [[52]Statistics for wildlife forensic DNA.], though this requires adequate numbers of unrelated individuals all from the same and relevant population which may still be challenging to obtain. Defining the relevant population depends upon the case circumstances, and ideally both the captive and wild populations should be extensively sampled in order to establish the degree of differentiation. The 1990s UK captive peregrine population had been largely established from UK wild stock, including birds taken under licence from the wild (permitted until 1988), ongoing legal integration of wild disabled birds and a significant number of laundered wild birds. Further diversity came from smaller numbers of imported birds and limited hybridization with other falcon species [[16]Fleming L.V. Douse A.F. Williams N.P. Captive breeding of peregrine and other falcons in Great Britain and implications for conservation of wild populations.]. The use of individuals sampled during casework in this study provided a representative panel of unrelated individuals comprising birds drawn both from the wild populations being illegally exploited (largely Scottish in this instance) and the captive population into which they are being laundered.For some forensic applications, such as testing the legitimacy of claimed parent/offspring relationships, even close linkage will not risk false prosecution as the absence of shared alleles across several loci is all that is needed to refute a false claim and this can only arise through multiple mutation events (with associated low probabilities), the inheritance of null alleles at several loci or misassignment of parentage. Questions of common origin are more seriously impacted due to the increased likelihood of sharing a suite of linked alleles. In this study the linked STR loci are all separated by at least 10.5 Mb, greater than the 6.3 Mb between vWA and D12S391, two loci in the standard European human DNA profiling multiplex, which are usually treated as unlinked except in cases (e.g. incest) where discrimination between very close kin is required [[37]O’Connor K.L. Tillmar A.O. Effect of linkage between vWA and D12S391 in kinship analysis.]. While greater physical distance is likely to increase the likelihood of recombination the rate varies throughout the genome. For the two human loci the recombination fraction in multigeneration pedigrees was estimated as 0.108 [[8]Budowle B. Ge J. Chakraborty R. Eisenberg A.J. Green R. Mulero J. Lagace R. Hennessy L. Population genetic analyses of the NGM STR loci.]: from our very limited data we see a similar recombination fraction of 0.083 or more between all but one pair of loci and so in combination with the absence of detectable LD among unrelated birds an assumption of independence is reasonable if the likelihood of close relationship is low. As seen within the legitimate sibling group in this case physical linkage can result in anomalously high or low estimates of the coefficient of relatedness but this will diminish with each generation as recombination breaks up linkage groups. To account for the potential persistence of association between alleles among birds who are believed to be unrelated, the use of extremely conservative measures of the probability of identity based on the probability of siblings sharing identical profiles has been proposed [[50]Waits L.P. Luikart G. Taberlet P. Estimating the probability of identity among genotypes in natural populations: cautions and guidelines.]. In this case our marker sets still yield combined probabilities of non-exclusion of sib identity (NE-SI) of

The STR markers described here offer similar discrimination power to the minisatellite loci that were used in the 1990s but with greater ease of analysis and reduced sample requirements (~30 ng, fragment length 100-500 bp). Although CE analysis of the multiplexes required several independent amplifications to split loci with similar size distributions into distinguishable sets, the MPS approach, for which they were ultimately intended, allows them to be combined in a single amplification and demultiplexed bioinformatically into individual loci whilst keeping amplicon lengths as short as possible. Furthermore, we have demonstrated here that additional discrimination amongst predominantly UK-derived peregrines can be gained using MPS, either by using loci with compound repeat structures and/or including polymorphism information in the flanking DNA. The MPS approach is likely to be most suited to complex persecution casework where limited amounts of degraded and/or mixed DNA may have to be analysed to link a suspect or item to a shot, trapped or poisoned bird. This has increasingly been the focus of UK raptor investigations (RSPB 2019).

The nature of raptor crime within the UK has also changed in other ways over the intervening period following a shift towards the production of hybrid falcons by artificial insemination that accelerated dramatically from the mid-1990s as the take from the wild in the UK declined. Prior to 1994, hybrid falcons had made up a negligible proportion of UK captive breeding claims but by 2003 they represented more than 70% of submissions, leading to growing concern about their escape and potential genetic introgression into the wild population [[16]Fleming L.V. Douse A.F. Williams N.P. Captive breeding of peregrine and other falcons in Great Britain and implications for conservation of wild populations.]. This has led to an additional requirement of distinguishing between pure and hybrid birds. The STRs described in this study were chosen with this in mind and were tested across a range of falcon species to ensure that they were polymorphic in all. Initial data suggest that whilst the allele size ranges overlap between species the additional information in the repeat structures and flanking polymorphisms detectable by MPS may prove useful in determining a bird’s origins. Furthermore, these markers, and the parallel set developed for accipiters using similar approaches and selection criteria [[3]Beasley J. Wetton J.H. May C.A. Bird of prey CE and MPS multiplexes: high discrimination for forensic and conservation applications.], could be applied globally in the study of wild populations to explore interaction between subpopulations and sympatric species, and recovery following population bottlenecks [17Groombridge J.J. Dawson D.A. Burke T. Prys-Jones R. Brooke M.D.L. Shah N. Evaluating the demographic history of the Seychelles kestrel (Falco araea): genetic evidence for recovery from a population bottleneck following minimal conservation management., 24Johnson J.A. Talbot S.L. Sage G.K. Burnham K.K. Brown J.W. Maechtle T.L. Seegar W.S. Yates M.A. Anderson B. Mindell D.P. The use of genetics for the management of a recovering population: temporal assessment of migratory peregrine falcons in North America., 45Talbot S.L. Palmer A.G. Sage G.K. Sonsthagen S.A. Swem T. Brimm D.J. White C.M. Lack of genetic polymorphism among peregrine falcons Falco peregrinus of Fiji., 58Phylogeny of Falconidae and phylogeography of Peregrine Falcons., 7Brown J.W. Van Coeverden de Groot P.J. Birt T.P. Seutin G. Boag P.T. Friesen V.L. Appraisal of the consequences of the DDT‐induced bottleneck on the level and geographic distribution of neutral genetic variation in Canadian peregrine falcons, Falco peregrinus.].

The application of SLP profiling to tackle the illegal laundering of birds of prey during the 1990s is widely regarded as one of the most successful examples of the use of a forensic technique to investigate UK wildlife crime. It also led to new legislation being introduced to facilitate the collection of samples to undertake identity or ancestry checks. The success of DNA profiling was dependent on three key aspects of the BRS; birds were individually and uniquely identifiable by numbered leg-rings or microchips, the physical location of individual birds was known, and information on all the declared familial relationships was available. Unfortunately, in 2008 (2009 in Scotland and Wales) the government reduced registration controls on peregrines kept in captivity in Great Britain. This effectively made it substantially more difficult for the statutory agencies to locate birds suspected of being taken from the wild once they had been sold or moved on by the breeder. Without this audit trail the value of DNA profiling in enforcement has been severely hampered. Concerningly, the last two decades have seen a significant rise in the sport of falcon racing in the Middle East which has again created an increased demand for peregrines and other falcons. Unsurprisingly, this has led to a substantial increase in UK prices, with female peregrines fetching £5,000 or more.

Around 2008, about 350 peregrines were declared captive bred each year, about the same number as the 1990s. Some ten years on this had more than doubled, and hundreds of these birds were being exported outside the EU. In the UK, eggs and chicks continue to be taken from some wild peregrine nests, so there seems little doubt that an unknown percentage of birds declared as captive bred are in reality illegally taken from the wild. Consequently, there is a need for DNA testing on captive breeding claims of those suspected of illegally dealing in wild taken birds, in conjunction with an annual program of random checks, akin to the work undertaken in 1996. This work needs to be focussed on the period shortly after birds are hatched and before they are sold or moved on. This approach would provide a strong deterrent for those illegally dealing in falcons and allow some assessment of the scale of the problem. Having validated, highly discriminating and cost-effective DNA profiling methods to assess the legitimacy of captive breeding claims will be a vital component of any such work and the STR markers reported for the first time here will provide the basis of such tests.