Highlights
- •Validated mitochondrial DNA toolkit for Shingleback lizard trafficking casework.
- •Established Shingleback phylogeographic genetic database.
- •Identified five Shingleback phylogeographic lineages.
- •Identified lineages of Shinglebacks confiscated from the illegal wildlife trade.
Abstract
Shingleback lizards (Tiliqua rugosa) are among the most trafficked native fauna from Australia in the illegal pet trade.
There are four morphologically recognised subspecies of shinglebacks, all with differing
overseas market values. Shinglebacks from different geographic locales are often trafficked
and housed together, which may complicate identifying the State jurisdiction where
the poaching event occurred. Additionally, shinglebacks can be housed and trafficked
with other species within the same genus, which may complicate DNA analysis, especially
in scenarios where indirect evidence (e.g. swabs, faeces) is taken for analysis. In this study, a forensic genetic toolkit was
designed and validated to target shingleback DNA for species identification and geographic
origin. To do this, field sampling across Australia was conducted to expand the phylogeographic
sampling of shinglebacks across their species range and include populations suspected
to be poaching hotspots. A commonly used universal reptile primer set (ND4/LEU) was
then validated for use in forensic casework related to the genus Tiliqua. Two additional ND4 primer sets were designed and validated. The first primer set
was designed and demonstrated to preferentially amplify an ∼510 bp region of the genus
Tiliqua over other reptiles and builds on existing data to expand the available phylogeographic
database. The second primer set was designed and demonstrated to solely amplify an
∼220 bp region of T. rugosa ND4 over any other reptile species. Through the validation process, all primers were
demonstrated to amplify T. rugosa DNA from a variety of sample types (e.g. degraded, low quality and mixed). Two of the primer sets were able to distinguish
the genetic lineage of T. rugosa from the phylogeographic database. This work provides the first forensically validated
toolkit and phylogeographic genetic database for Squatmate lizards.
Keywords
To read this article in full you will need to make a payment
Purchase one-time access:
Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online accessOne-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:
Subscribe to Forensic Science International: GeneticsAlready a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
References
United Nations Office on Drugs and Crime. (2020). World Wildlife Crime Report. Available at: 〈https://www.unodc.org/unodc/en/data-and-analysis/wildlife.html〉.
- Wildlife across our borders: a review of the illegal trade in Australia.Aust. J. Forensic Sci. 2008; 40: 147-160
- Strengthening protection of endemic wildlife threatened by the international pet trade: the case of the Australian shingleback lizard.Anim. Conserv. 2022; 25: 91-100
- Using Google trends to determine current, past, and future trends in the reptile pet trade.Animals. 2021; 11: 676
Orr, A. (2013). Shingleback lizards found in luggage at Perth Airport. WA today. Retrieved from: 〈https://www.watoday.com.au/national/western-australia/shingleback-lizards-found-in-luggage-at-perth-airport-20131003–2uut2.html〉.
Titmus, A. (2007). Husbandry manual for the shingleback lizard. University of Western Sydney, Hawksbury. Retrieved from: 〈https://aszk.org.au/wp-content/uploads/2020/04/Reptiles.-Shingleback-Lizard-2007AT.pdf〉.
- The trade in nationally protected lizards from Australia, Cuba, and Mexico and the EU’s role as a main destination.TRAFFIC Bull. 2019; 31: 59
ProWildlife. (2016). Stolen Wildlife II. Retrieved from: 〈https://www.prowildlife.de/wp-content/uploads/2022/01/stolen-wildlife-ii-2016.pdf〉.
Convention on International Trade in Endangered Species of Wild Fauna and Flora ,2022. Notification to the Parties: Amendments to Appendix III. Retrieved from: 〈https://cites.org/sites/default/files/notifications/E-Notif-2022–019.pdf〉.
R. v Qui, Lei. (2021). NSWDC Retrieved from: 〈https://www.cdpp.gov.au/case-reports/couple-jailed-over-illegal-attempted-export-exotic-australian-animals〉.
R. v Kennedy ,2016. NSWDC. Retrieved from: 〈https://sherloc.unodc.org/cld/case-law-doc/wildlifecrimetype/aus/2019/r_v_kennedy.html〉.
- Wildlife Legislation in Australia: Trafficking Provisions.Australian Institute of Criminology, Canberra1994
- The official status of frogs and reptiles in New South Wales.Herpetol. Aust. Divers. Discip. 1993; : 404-408
Shea, G.M. (1992). The systematics and reproduction of bluetongue lizards of the genus Tiliqua (Squamata: Scinidae) (Doctoral dissertation, University of Sydney).
- Comparative phylogeography of woodland reptiles in California: repeated patterns of cladogenesis and population expansion.Mol. Ecol. 2006; 15: 2201-2222
- A molecular phylogeny of the Australian monitor lizards (Squamata: Varanidae) inferred from mitochondrial DNA sequences.Aust. J. Zool. 2006; 54: 253-269
- Variations in mitochondrial tRNA gene organization of reptiles as phylogenetic markers.Mol. Biol. Evol. 1995; 12: 759-772
- Plio-Pleistocene diversification and biogeographic barriers in southern Australia reflected in the phylogeography of a widespread and common lizard species.Mol. Phylogenetics Evol. 2019; 133: 107-119
- Current issues with the investigation of wildlife crime in Australia: problems and opportunities for improvement.J. Int. Wildl. Law Policy. 2015; 18: 244-263
- Population ecology of the sleepy lizard, Tiliqua rugosa, at Mt Mary, South Australia.Aust. J. Ecol. 1995; 20: 393-402
- Social monogamy and extra-pair fertilization in an Australian lizard, Tiliqua rugosa.Behav. Ecol. Sociobiol. 1998; 44: 63-72
- Home-range fidelity in the Australian sleepy lizard, Tiliqua rugosa.Aust. J. Zool. 1999; 47: 125-132
- Home range overlap of mothers and their offspring in the sleepy lizard, Tiliqua rugosa.Behav. Ecol. Sociobiol. 1998; 42: 357-362
- DNA based method for determining source country of the short beaked echidna (Tachyglossus aculeatus) in the illegal wildlife trade.Forensic Sci. Int. 2019; 295: 46-53
- Wildlife forensic science: a review of genetic geographic origin assignment.For. Sci. Int. Genet. 2015; 18: 152-159
- ‘ForCyt’DNA database of wildlife species.Forensic Sci. Int.: Genet. Suppl. Ser. 2017; 6: e466-e468
- Wildlife crime in Australia.Emerg. Top. Life Sci. 2021; 5: 487
- RhODIS® (The Rhinoceros DNA Index System): the application of simple forensic and genetic tools help conserve african rhinoceros.Wildlife Biodiversity Conservation. Springer, Cham2021: 463-485
- Elephant genotypes reveal the size and connectivity of transnational ivory traffickers.Nat. Hum. Behaviour. 2022; : 1-12
- An overview of risk investment in the transnational illegal wildlife trade from stakeholder perspectives.Wiley Interdiscip. Rev. Forensic Sci. 2021; 3e1397
- Recovering trace reptile DNA from the illegal wildlife trade.Forensic Sci. Int. Anim. Environ. 2022; 100040
- Low-cost direct PCR for aged and processed wildlife sample analysis.Forensic Sci. Int.: Genet. Suppl. Ser. 2013; 4: e71-e72
- Forensic DNA analysis for animal protection and biodiversity conservation: a review.J. Nat. Conserv. 2014; 22: 195-205
Australian Government Department of Agriculture, Water and the Environment ,2021). Wildlife smuggling operation ringleader jailed. Retrieved from: 〈https://www.awe.gov.au/news/media-releases/wildlife-smuggling-operation-ringleader-jailed〉. August 25, 2021.
- Mitochondrial DNA sequence divergence and phylogenetic relationships among eight chromosome races of the Sceloporus grammicus complex (Phrynosomatidae) in central Mexico.Syst. Biol. 1994; 43: 387-418
- Numerous transposed sequences of mitochondrial cytochrome oxidase i–ii in aphids of the genus Sitobion (Hemiptera: Aphididae).Mol. Biol. Evol. 1996; 13: 510-524
- Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data.Bioinformatics. 2012; 28: 1647-1649
- Database resources of the National Center for Biotechnology Information.Nucleic Acids Res. 2018; 4: 46
- IDT SciTools: A suite for analysis and design of nucleic acid oligomers.Nucleic Acids Res. 2008; 36: W163-W169
ANSI/ASB Standard 047, First Edition. (2019). Wildlife Forensics Validation Standard – Validating New Primers for Sequencing. Retrieved from 〈http://www.asbstandardsboard.org/wp-content/uploads/2019/05/047_Std_e1.pdf〉.
- Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega.Mol. Syst. Biol. 2011; 7: 539
- Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees.Mol. Biol. Evol. 1993; 10: 512-526
- Dating of the human-ape splitting by a molecular clock of mitochondrial DNA.J. Mol. Evol. 1985; 22: 160-174
- A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.J. Mol. Evol. 1980; 16: 111-120
- Median-joining networks for inferring intraspecific phylogenies.Mol. Biol. Evol. 1999; 16: 37-48
- PopART: Full-feature software for haplotype network construction.Methods Ecol. Evol. 2015; 6: 1110-1116
- Decline of a biome: evolution, contraction, fragmentation, extinction and invasion of the Australian mesic zone biota.J. Biogeogr. 2011; 38: 1635-1656
- Loxodonta Localizer: a software tool for inferring the provenance of African elephants and their ivory using mitochondrial DNA.J. Hered. 2019; 110: 761-768
- Phylogeography of the iconic Australian red-tailed black-cockatoo (Calyptorhynchus banksii) and implications for its conservation.Heredity. 2020; 125: 85-100
- Wildlife forensics: a boon for species identification and conservation implications.Forensic Sci. Int. 2020; 317110530
- Current and future directions of DNA in wildlife forensic science.Forensic Sci. Int. Genet. 2014; 10: 1-11
- Current methods, future directions and considerations of DNA-based taxonomic identification in wildlife forensics.Forensic Sci. Int. Anim. Environ. 2021; 1100030
- Illegal wildlife trade: scale, processes, and governance.Annu. Rev. Environ. Resour. 2019; 44: 201-228
Article info
Publication history
Published online: September 29, 2022
Accepted:
September 28,
2022
Received in revised form:
September 27,
2022
Received:
June 6,
2022
Identification
Copyright
© 2022 Elsevier B.V. All rights reserved.
