Non-invasive diet analysis based on DNA Barcoding: the Himalayan Brown Bears (Ursus arctos isabellinus) as a case study
Author(s)
Valentini, Alice
Date Issued
March 7, 2008
Type
Doctoral Thesis
Abstract
The study of food webs and their dynamics is fundamental to understand how the
feeding habits of the different species can affect the community, thus improving our
understanding of the functioning of the ecosystem as a whole. Furthermore, the study of
feeding ecology becomes crucial when it concerns endangered species since a precise
knowledge of their diet is to be gathered when designing reliable conservation
strategies. A wide range of methodologies have been proposed for diet analysis,
including simple ones, as visual observation of foraging behavior, and more complex
ones such as Near Infrared Reflectance Spectroscopy and DNA based methods.
DNA barcoding, i.e. species identification using a standardized DNA region or
markers, has recently received much attention and is being further developed through an
international initiative called "Consortium for the Barcode of Life". When using DNA
barcoding for diet analysis, the choice of the markers is crucial. The ideal DNA
barcoding marker should meet several criteria. It should be variable among species,
standardized, with enough phylogenetic information, extremely robust, and short
enough to allow amplification of degraded DNA.
In this study we propose the trnL (UAA) intron as marker for plant DNA
barcoding. The power and the limitations of this system were evaluated as well as the
possibility of species identification with highly degraded DNA. The main limitation of
this system is its relatively low resolution in discriminating closely related species.
Despite the relatively low resolution, it has many advantages: the primers are highly
conserved, the amplification system is very robust and it is able to work with much
degraded DNA samples. This system has been coupled with massively parallel
pyrosequencing technique. We demonstrate the efficiency of this new approach by
analyzing the diet of various herbivorous species. The whole chloroplast trnL (UAA)
intron (254–767 bp) and a shorter fragment of this intron (the P6 loop, 10–143 bp) were
used in this study. For the whole trnL intron 67.3% of the species retrieved from
GenBank were unambiguously identified and 19.5% for the P6 loop. The resolution is
much higher after calibration of specific contexts using species originating from the
same ecosystem.
Furthermore, the trnL approach was coupled with individual and sex identification
using microsatellites polymorphism in the Himalayan brown bear (Ursus arctos
isabellinus). Among world brown bears populations; those in Asia are the most
ii
endangered and least studied. Here, populations have declined by more than half in the
past century owing to habitat loss and fragmentation and human activity. Presently in
Pakistan brown bear occur sparsely in seven small populations, with the largest isolate
in the Deosai National Park. We examined this population using a combination of fecal
DNA analysis and field data for which geographical location and date of sampling were
available, with the aim to study individual and sexual differentiation in the diet, and also
temporal and geographical variations. Twenty-eight individuals (16 male, 10 females
and 2 unknown sex) were identified in this study with microsatellites markers. Only
eight plant species were found represented in more than 50% of individual feces.
Temporal differences were found with more energetic food detected before the
hibernation periods.
Additional information
Dottorato di ricerca in Ecologia e gestione delle risorse biologiche
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