DNA Surveillance

The Science

This web-based tool is composed of three alignments (ATP6, COI, and cytb) that can be used as references to assign unknown samples to species. The alignments were assembled mainly from available sequences from GenBank and BOLD (Barcode of Life Data Systems, Ratnasingham return false;"href="https://files.nyu.edu/pbc227/public/Table.html">here.

Taxonomy

The DNA Surveillance - Carnivora follows the species-level taxonomy proposed by Wozencraft 2005, which recognizes 286 carnivoran species. The alignments also include extinct species whenever sequences for those are available. All species are grouped into one of the 16 families and their names are color-coded according to Family-level affinity. When multiple subspecies exist, their sequences were merged into the species group (e.g. 'Canis familiarissequences are grouped into Canis lupus).
[[Image:Tree|center]]

A phylogenetic tree of the 16 extant families in the order Carnivora (after Eizirik et al.2010). The blue and orange phylogenetic groups represent the sub-orders Feliformia and Caniformia, respectively. The species within each family in the DNA Surveillance database are color-coded in the same way as the family names are in this tree.

DNA Segments

The three segments used here are from the mitochondrial DNA. Excluding the primers, all segments are shorter than 200 base pairs (bp). For this reason, many of the reference sequences in the DNA Surveillance - Carnivora database are not found in GenBank, which does not archive sequences that are shorter than 200 bp.
[[Image:mtDNA|center]]

A linearized mtDNA molecule showing the relative size and position of the three segments. Using the dogs mtDNA genome (NC_002008.4, 16.7 Kb) as reference, the cytbsegment (110 bp) lies between bases 14475 and 14584. It is at the end of the widely used 307 bp segment from Kocher et al. 1989. The COIsegment (187 bp) lies at the 5867-6053 bp interval, which is the end of the 658 bp segment proposed by Hebert et al. 2003as a standard DNA barcode for animals. The ATP6segment (126 bp) lies between bases 7988 and 8113. The hatched square shows the overlapping region at the end of ATP8 and the beginning of ATP6 genes (modified from Chaves et al. in press).

ATP6

According to a sliding-window analysis, the ATP6segment has more variable sites per nucleotide than the COIand cytbsegments. It is 126 bp long (172 bp including primers) and as of September 2011, there were 128 species represented by at least one haplotype in the alignment. The complete ATP6alignment comprises 244 sequences.

Suggested PCR primers:

ATP6-DF3: 5'-AACGAAAATCTATTCGCCTCT-3'(L24)
ATP6-DR1: 5'-CCAGTATTTGTTTTGATGTTAGTTG-3'(H151)

COI

The COIsegment is 187 bp long (239 bp including primers) and has 133 species represented in the alignment. The COIalignment also contains several sequences from carnivores not yet assigned to species. These unassigned sequences were downloaded from GenBank (produced by the Barcode of Life Data Systems - BOLD) and are displayed in black in the tree output of DNA Surveillance. Currently, these sequences are identified as "Carnivora_sp_Accession#_Voucher#"and they will be updated as improved information on them becomes available. The current alignment has a total of 356 sequences, of which 88 are unidentified sequences from BOLD.

Suggested PCR primers:

BC-F3: 5'-CCCCTATTCGTATGATCAGTATTAATTAC-3'(H572)
BC-R2: 5'-TAAACCTCAGGATGTCCGAAGAATCA-3'(H706)
BC-F2*: 5'-ATCACCACTATTGTTAATATAAAACC-3'(L518)

(*) The COIreference alignment was originally assembled with the primer pair BC-F2 and BC-R2. However, this combination tended to amplify prey DNA in scat samples. The primer pair BC-F3 and BC-R2 produces a shorter COIsegment (133 bp, or 188 bp including primers) and works well with noninvasive samples.

cytb

The cytbsegment is 110 bp long and has 218 species represented in its reference alignment. This segment is derived from Farrell et al.2000, whose primers produce a PCR amplicon that is 171 bp long. The current cytbreference alignment contains 585 sequences.

Suggested PCR primers:

Forward: 5'-TATTCTTTATCTGCCTATACATRCACG-3'(L292)
Reverse: 5'-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3'(H403)

The numbers in parenthesis after the primer sequences correspond to the 3'base annealing position relative to the length of the gene (ATP6: 681 bp, COI: 1545 bp, cytb: 1140 bp).

The primers listed above were tested on a number of samples from a diverse set of carnivores. Although they work well both in tissue and noninvasive samples, it is likely that additional (more specific) primers will be needed to cover the order Carnivora more broadly. For additional information please see Chaves et al.(in press) or contact us.

Last Update

The last update of the reference alignments was performed in September 2011.

References

1. Chaves PB, Graeff VG, Lion MB, Oliveira LR, Eizirik E (2012) DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples.Molecular Ecology Resources12, 18-35.
   
   
2. Eizirik E, Murphy WJ, Koepfli KP, Johnson WE, Dragoo JW, Wayne RK, O'Brien SJ (2010). Pattern and timing of diversification of the mammalian order Carnivora inferred from multiple nuclear gene sequences.Molecular Phylogenetics and Evolution56, 49-63.
   
   
3. Farrell LE, Roman J, Sunquist ME (2000). Dietary separation of sympatric carnivores identified by molecular analysis of scats.Molecular Ecology9, 1583-1590.
   
   
4. Hebert PD, Cywinska A, Ball SL, deWaard JR (2003). Biological identifications through DNA barcodes.Proceedings of the Royal Society B: Biological Sciences270, 313-321.
   
   
5. Kocher TD, Thomas WK, Meyer A, Edwards SV, Pbo S, Villablanca FX, Wilson AC (1989). Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers.Proceedings of the National Academy of Sciences of the U.S.A86, 6196-6200.
   
   
6. Ratnasingham S, Hebert PD (2007) BOLD: The Barcode of Life Data System (http://www.barcodinglife.org).Molecular Ecology Notes7, 355-364.
   
   
7. Wozencraft WC (2005) Order Carnivora. In: Mammal Species of the World, 3rd edn (eds Wilson DE Reeder DM), pp 532-628. The Johns Hopkins University Press, Maryland.