Schrodinger's scat: a critical review of the currently available tiger (Panthera Tigris) and leopard (Panthera pardus) specific primers in India, and a novel leopard specific primer

Abstract

Background: Non-invasive sampling has opened avenues for the genetic study of elusive species, which has contributed significantly to their conservation. Where field based identity of non-invasive sample is ambiguous (e.g. carnivore scats), it is essential to establish identity of the species through molecular approaches. A cost effective procedure to ascertain species identity is to use species specific primers (SSP) for PCR amplification and subsequent resolution through agarose gel electrophoresis. However, SSPs if ill designed can often cross amplify non-target sympatric species. Herein we report the problem of cross amplification with currently published SSPs, which have been used in several recent scientific articles on tigers (Panthera tigris) and leopards (Panthera pardus) in India. Since these papers form pioneering research on which future work will be based, an early rectification is required so as to not propagate this error further.

Results: We conclusively show cross amplification of three of the four SSPs, in sympatric non-target species like tiger SSP amplifying leopard and striped hyena (Hyaena hyaena), and leopard SSP amplifying tiger, lion (Panthera leo persica) and clouded leopard (Neofelis nebulosa), with the same product size. We develop and test a non-cross-amplifying leopard specific primer pair within the mitochondrial cytochrome b region. We also standardize a duplex PCR method to screen tiger and leopard samples simultaneously in one PCR reaction to reduce cost and time.

Conclusions: These findings suggest the importance of an often overlooked preliminary protocol of conclusive identification of species from non-invasive samples. The cross amplification of published primers in conspecifics suggests the need to revisit inferences drawn by earlier work.

Fig. 1

Electrophoresis picture showing amplifciation of various sympatric carnovores and prey (X-axis lanes(with published tiger and leopard specific primers (Y-axis). Lane1-15: 100 bp ladder, tiger, leopard, lion (Panthera leo persica), striped hyena (Hyaena hyaena), clouded leopard (Neofelis nebulosa), caracal (Caracal caracal), jungle cat (Felis chaus), wolf (Canis lupus pallipes), golden jackal (Canis aureus), dog (Canis familiaris), fox (Vulpes bengalensis), sloth bear (Melursus ursinus), extraction control, PCR control. Note TIG 509 AND TIG 490, both tiger specific primers amplify leopards and at the same product size. NADH4 a leopard specific primer amplifies tigers, lions and clouded leopards and at the same product size as leopards