Abstract
Trade in ivory from extant elephant species namely Asian elephant (Elephas maximus), African savanna elephant (Loxodonta africana) and African forest elephant (Loxodonta cyclotis) is regulated internationally, while the trade in ivory from extinct species of Elephantidae, including woolly mammoth, is unregulated. This distinction creates opportunity for laundering and trading elephant ivory as mammoth ivory. The existing morphological and molecular genetics methods do not reliably distinguish the source of ivory items that lack clear identification characteristics or for which the quality of extracted DNA cannot support amplification of large gene fragments. We present a PCR-sequencing method based on 116 bp target sequence of the cytochrome b gene to specifically amplify elephantid DNA while simultaneously excluding non-elephantid species and ivory substitutes, and while avoiding contamination by human DNA. The partial Cytochrome b gene sequence enabled accurate association of ivory samples with their species of origin for all three extant elephants and from mammoth. The detection limit of the PCR system was as low as 10 copy numbers of target DNA. The amplification and sequencing success reached 96.7% for woolly mammoth ivory and 100% for African savanna elephant and African forest elephant ivory. This is the first validated method for distinguishing elephant from mammoth ivory and it provides forensic support for investigation of ivory laundering cases.
Highlights
Trade in ivory from extant elephant species namely Asian elephant (Elephas maximus), African savanna elephant (Loxodonta africana) and African forest elephant (Loxodonta cyclotis) is regulated internationally, while the trade in ivory from extinct species of Elephantidae, including woolly mammoth, is unregulated
All PCR amplicons (160 bp, with flanking primer sequences incorporated) were successfully sequenced and the primer sequences subsequently trimmed to obtain actual gene fragment sequences of 116 bp, which confirmed their correctness for all elephantid species with percent nucleotide similarity (%NS) of 100% for Asian elephant, and 100% for African savanna elephant, African forest elephant and woolly mammoth (Supplementary Table S2)
The top 250 hits are highly dependent on the number of GenBank database sequences represented by closely matching species, 80% of the total top 250 hits matched the target elephantid species sequences (i.e. 46.8% M. primigenius, 7.6% L. cyclotis, 0.8% E. maximus and 24.8% L. africana halpotypes) in the database, which could vary upwards depending on the true identity of the sequence hit matches identified as Mammuthus sp
Summary
Trade in ivory from extant elephant species namely Asian elephant (Elephas maximus), African savanna elephant (Loxodonta africana) and African forest elephant (Loxodonta cyclotis) is regulated internationally, while the trade in ivory from extinct species of Elephantidae, including woolly mammoth, is unregulated. We present a PCR-sequencing method based on 116 bp target sequence of the cytochrome b gene to amplify elephantid DNA while simultaneously excluding non-elephantid species and ivory substitutes, and while avoiding contamination by human DNA. It is difficult to satisfy the identification needs for carvings on which diagnostic characteristics cannot be detected Another issue for ivory DNA analysis is human contamination because ivory specimens are repeatedly handled by humans from the time they are obtained to processing and trading. Practitioners are advised to use great caution during DNA isolation[28]
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