Case report
Application of STR markers in wildlife forensic casework involving Australian black-cockatoos (Calyptorhynchus spp.)

https://doi.org/10.1016/j.fsigen.2011.10.003Get rights and content

Abstract

Parrots and cockatoos are highly prized aviary birds and the demands for such species has fuelled their illegal trade and harvest from the wild. Here we report on three forensic case studies involving black-cockatoos (Calyptorhynchus spp.) endemic to Australia. These cases involve suspected poaching and illegal killing of endangered red- and white-tailed black-cockatoos. Through the prior development of 20 polymorphic microsatellite loci and population databases for white- and red-tailed black-cockatoos, the tools are available to conduct high-resolution paternity and individual identity testing. In one case, we matched a red-tailed black-cockatoo nestling to a tree hollow from which it was poached through the use of DNA from eggshell recovered from the nest. For the second case, we utilized our provenance population database (nest sites), and identified the kinship and geographic origin of a white-tailed black-cockatoo, which was illegally harvested from the wild. The third case determined the number individual white-tailed black-cockatoos allegedly shot at a fruit grower's orchard from body part remains. These genetic investigations highlight the significance and statistical confidence of DNA profiling and associated databases for endangered taxa, such as exotic birds. Our cockatoo population databases are the first of their kind in Australia, and demonstrate the efficacy of such approaches to identify such illegal activity. With a robust set of genetic markers and methodologies in place, we aim to broaden our population databases to include other cockatoo species of conservation concern.

Introduction

Parrots and cockatoos (order Psittaciformes) are recognized globally for their extraordinary plumage, mimicry ability and charismatic character. It is these features that have fuelled their exploitation and capture from the wild [1]. Estimates of the number of birds extracted from the wild are difficult to quantify – the international estimates ranged from ∼7.5 million birds per annum during the peak of trade in the 1970s to around 2 to 5 million individuals per annum in the 1980s [2], with parrots accounting for half of the 519 species of birds traded from 1991 to 1996 [2]. Captive breeding is a major source of individuals to supply aviculture markets, although only a small number of species are sourced from this industry [2]. The total impact of the illegal trade in birds is particularly challenging to quantify in numbers due to its clandestine nature, and the cumulative impact of the mortality rates during capture, transport and pre- and post-sale [2], [3], [4]. The detrimental impacts of such activities are having a measurable effect on cockatoos and parrots in the wild, both in terms of biodiversity and population number [2], [3]. Spix's macaw (Cyanopsitta spixii) is one of the most recent cases of extinction in the wild, caused by illegal harvest [1], [3]. The rarity of this bird in captivity, fuelled by demand by collectors has caused prices to rise to ∼AUD$20,000 per bird [1]. Clearly, there are considerable monetary motivations for taking part in the illegal bird trade, especially considering that prosecutions are rare and the penalties are not particularly harsh [5].

Psittaciformes contain over 370 species, which are mainly concentrated in the Southern Hemisphere [6]. Combinations of anthropogenic induced habitat loss and modification, poaching and illegal trade are significant threats to these birds. Over 20% of parrots and cockatoos are of conservation concern, 85 species are listed as critically endangered, endangered or vulnerable, and 19 species as extinct by the International Union for the Conservation of Nature [7]. In Australia there are five endemic species of black-cockatoo (Calyptorhynchus spp.), and the two endangered white-tailed black-cockatoo (WTBCs) of Western Australia have been significantly affected by anthropogenic activities [7]. White-tailed black-cockatoos have declined ∼50% in population number as a result of habitat loss [8], [9], and they show evidence of population substructure and gene exchange between the two forms (short- and long-billed), possibly as a result of severe land clearing for agriculture [6], [10]. Taxonomic amendment is currently under review based on recent genetic data [6], [10]; therefore the long- (Calyptorhynchus baudinii) and short-billed (Calyptorhynchus latirostris) forms, currently accepted in the Australian checklist of birds [11], will be collectively referred to as WTBCs.

A dichotomy exists for WTBCs amongst some of the West Australian community. On one hand, illegal shooting of WTBCs regularly occurs, with estimates of the number of WTBCs shot annually in the hundreds (P. Mawson, personal communication) despite the fact that WTBCs have had protected species status since 1989. Conflicts with humans occur due to the birds feeding on pome fruit and nut crops [12], which have in select areas replaced native foraging habitat. Illegal collection of eggs and nestlings from the wild to supply aviculture demand was historically common [3], [13], [14], as breeding in captivity is notoriously difficult and has been recorded infrequently [14], [15]. On the other hand, in 1996 a government-run WTBC captive-rearing program was established to increase the supply of the short-billed form in captivity. The program involved the sustainable harvest of eggs and nestlings from the wild [13]. The aim of this program was to provide the short-billed form of WTBC for the Australian domestic aviculture market in order to reduce the value of these birds on the legal market, and curtail the financial incentives for illegal take from the wild. All captive-reared birds were micro-chipped, and biological samples taken for inventory of genetic diversity maintained in captivity [13], [16].

One area of conservation genetics that has long been recognized, is the development of analytical techniques capable of providing DNA evidence to assist in conservation law enforcement, commonly termed ‘wildlife DNA forensics’ [17]. There is an increasing forensic use of animal DNA to determine either; (1) the identity of samples to a genus and/or species level; or (2) assigning samples to an individual or to kin [18]. Species identification with the use of DNA is now widely applied [4], [18], [19], [20], [21], [22], [23], [24], and assigning samples to an individual, population, or geographic origin is gaining momentum [25], [26], [27], [28], [29].

Unlike human forensic DNA profiling and analysis, wildlife DNA forensic markers used to profile avian samples, target microsatellite (STR) loci that are either developed for the species of interest, or taken from an open access database such as GenBank, and examined for specificity (cross-species amplification) [18], [30]. This is followed by the development of a DNA profile or population database for the species of interest is conducted, and when practicable, should contain an adequate number of samples and be representative of the population(s) in the wild. Often 200 members are the de facto standard for a population database [18], although the size of databases is dependent on the number of potential contributors and locus diversity levels [18]. There are numerous advantages and applications of such databases, although there are few such databases in existence for wild animals [18], [29], [31]. From a forensic genetic perspective, identifying the geographic origin of a sample is equivalent to identifying its reproductive population of origin [18], [28]. Often it is necessary to demonstrate from a body part (e.g. horn, tusk, bone, skin) or by-product, such as eggshell, if the sample(s) originated from a specific individual, or from similar body parts, if they were derived from a number of individuals, such as shark fins in prepared food. Of equal importance is the regulation of the legal trade and harvest of species, to ensure that illegally obtained wildlife cannot be laundered into a legitimate supply chain. In Western Australia there are many suspected instances of illegal activity involving cockatoos. Together with the assistance of the Western Australian Department of Environment and Conservation (DEC) we are now able to successfully implement DNA profiling technologies with the aim of increasing the probative value of evidence presented in wildlife DNA forensic cases.

Section snippets

Suspected poaching from the wild, Property A

On December 9th of 2008 an alert member of the public photographed a man on road reserve vested in the Local Government Authority in Morawa (310 km NNE of Perth), Western Australia climbing a tree with a ladder (Fig. 1a). The licence plate number of the man's vehicle was included in the photographs. The photographs were sent to the Nature Protection Branch, DEC. A search warrant was issued for the address (Property A) listed under the vehicle registration via details sourced from the licence

Materials and methods

Feathers (approx., 4–8) were collected from four black-cockatoos held in captive-care and 13 deceased black-cockatoos (Table 1), and stored in 20% dimethylsulphoxide saturated with sodium chloride. Eggshell from a tree hollow was stored in a clean, dry sample container. DNA from the feathers was extracted using Chelex®100 (supplementary information), and DNA from the eggshell was extracted following the protocol of Oskam et al. [33]. DNA extracts, prior to genotyping, were quantified (qPCR) for

Red-tailed black-cockatoo case 1

DNA was successfully recovered from samples B08-750 and B08-765 (Table 1). Complete 17-locus genotypes were obtained (Table S1). Multilocus genotypes of B08-750 and B08-765 were found to be identical, except for locus pClD122, in which a homozygous profile was obtained for B08-765 (Table S1) – we suspect this was a single incidence of allelic dropout. Probability of identity (PID) in the RTBC population was calculated using the allele frequencies in the database (Table 2). PID values for each

Conclusions

Two of the three cases described above indicate that it is possible to definitively determine illegal trade or harvest of live animals from the wild. This is hard, or even impossible to achieve, without the assistance of molecular techniques. DNA profiling offers a non-invasive means to monitor wild and aviary populations. We advocate, as part of the licensing agreement to maintain some protected species in captivity that DNA samples should be taken with the explicit intention that they may be

Acknowledgments

This work was supported by funding from the Robert Hammond Research Studentship (to N.E. White), Murdoch University, Department of Environment and Conservation, Department of Environment, Water, Heritage and the Arts, Birds Australia, and ARC Future Fellowship FT0991741 (M.B.). The authors wish to thank P.B.S. Spencer for use of laboratory facilities, iVEC Informatics Facility, Centre for Comparative Genomics (iVEC; D. Schibeci) and the State Agricultural and Biotechnology Centre (F. Brigg) for

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