Mammoths and Moas at NCBI

The presence of ancient DNA sequences in GenBank was noted in the Spring 1999 issue of the NCBI news. Many of the ancient DNA sequences then available were sequences of extant species from ancient material such as human mummies. Some others were from long-extinct taxa such as the saber toothed cat, ground sloth, mammoth and mastodon. But all were short fragments of mainly mitochondrial sequences, a far cry from what would be needed to garner comprehensive genomic information. Six years later, at the close of 2005, the prospect of reconstructing the genomes of ancient extinct organisms has come tantalizingly close with publications of a large quantity of genomic sequence[1] and a complete mitochondrial genome[2,3] from the extinct woolly mammoth (Mammuthus primigenius). The groups produced the greatest quantity of sequence ever obtained (28 million base pairs) from an extinct organism and the most complete organelle genome from an extinct organism. Both the woolly mammoth genomic sequence and mitochondrial genome have been deposited at NCBI and are available for searching through NCBI's trace archive, the BLAST services, and the integrated Entrez system. This article shows how to retrieve and work with these woolly mammoth sequences. It also provides an update on the extinct organism sequences at the NCBI with tips on using the Entrez system, the taxonomy browser, the Trace Archive, and the BLAST service to access these interesting data.

Extinct Organisms in Entrez: Sequences and Taxa

The mammoth mitochondrial genome is available in GenBank as well as in the Reference Sequence database, Table 1. These sequence data are fully integrated into the Entrez nucleotide, protein, gene, and genome databases. The Entrez system and the taxonomy database can be used to retrieve these and a number of records for other extinct taxa.

A remarkable number of woolly mammoth nucleotide sequences are retrieved with the following organism query, which can be entered in the search box on the NCBI Homepage:

Woolly Mammoth [Organism]

The initial set of nucleotide sequences can be further limited to mitochondrial records by using the 'Limits' tab and limiting to 'Mitochondrion' in the 'Gene location' pull-down list. However, simply adding 'mitochondrion' as a Title search retrieves only the complete mitochondrial genomes from GenBank (accessions DQ188829 and DQ316067 ) and RefSeq (accession NC_007596).

Woolly Mammoth [Organism] AND mitochondrion[Title]

An easy way to access data for all extinct taxa including the woolly mammoth is via the NCBI Taxonomy services.

www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Taxonomy

Here, a list of extinct taxa can be retrieved using the following query:

Extinct[Properties]

An informative view of these organisms is available by selecting 'Common Tree' from the display options pull-down list. This view arranges these taxa according to taxonomic classification and makes it easy to identify the major groups of organisms present (Fig. 1). Several different ages and extinction events are represented by these organisms.

Click on image to view larger

Figure 1. Common trees generated from the Entrez taxonomy service showing the extinct mammals and birds. A. Selected placental mammals from the Pleistocene. B. Extinct birds. All bird species except the great auk (Pinguinus impennis), the dodo (Raphus cucullatus), the Rodrigues Solitaire (Pezophaps solitaria), the elephant bird (Mullerornis agilis), and the imperial woodpecker (Campephilus imperialis) were endemic to the New Zealand or Hawaiian archipelagos.

Most of the placental mammals (Eutheria) including the woolly rhinoceros (Coelodonta antiquitatis), mammoth (Mammuthus primigenius), American mastodon (Mammut americanum), cave bear (Ursus spelaeus), saber-toothed cat (Smilodon fatalis) and ground sloths (Nothrotheriops shastensis, Mylodon darwinii) and even Neanderthal man (Homo sapiens neanderthalensis) are from the ice age fauna of the Northern hemisphere. These organisms disappeared at the end of the Pleistocene, around 10,000 years ago, but are well represented because there are abundant well-preserved bones and even frozen soft tissues that make it possible to obtain relatively intact DNA samples. Increased hunting pressure accompanying the rise and spread of modern humans may have played a role in the disappearance of these mammals although climate and vegetation change may also have been important.

There is another series of extinctions more closely linked to the arrival of humans that is documented in the extinct birds (Aves). Two of the birds listed, the great auk (Pinguinus impennis) and the dodo (Raphus cucullatus), are by now standard icons of the devastating consequences of human predation in historic times. Many of the other extinct bird species are testimony to the arrival of modern humans in New Zealand less than 1,000 years ago. Prior to the arrival of the Maori settlers, there were no terrestrial mammals on the islands. In New Zealand many of the ecological roles occupied by mammals in other places were occupied by the flightless moas (Dinornithiformes). All of the moas became extinct within 200 years of the arrival of mammals in the form of man, his domestic animals, and camp followers such as rats. The giant eagle, Harpagornis moorei, that likely preyed on moas, also disappeared, along with a number of other New Zealand endemics. Twenty one of the 25 extinct birds with sequences in GenBank were former residents of the New Zealand archipelago. While many of the sequences for the extinct birds are short fragments, nearly complete mitochondrial genomes are available for three moas; the little bush moa (Anomalopteryx didiformis)[4], the eastern moa (Emeus crassus)[5] and the giant moa (Dinornis giganteus)[5] (Table 1).

Table 1. GenBank and RefSeq accession numbers of mitochondrial genomes of extinct organisms at NCBI.

A similar set of extinctions to those on the New Zealand archipelago followed the arrival of Polynesians on the Hawaiian Islands 1,600 years ago and resulted in the loss of perhaps half of the endemic species of birds. This extinction event is represented at NCBI by sequences from two of the giant flightless waterfowl species once present on the islands—Thambetochen chauliodous and the Giant Hawaiian Goose (Branta sp.).

Woolly Mammoth found in Trace Archive

The woolly mammoth genomic sequence is made up of short unassembled environmental sequence reads that are accessible through a Trace Archive query or through the Trace Archive BLAST services:

www.ncbi.nlm.nih.gov/Traces/

(see the “Trace Archives” article in this issue.)

The woolly mammoth sequences can be displayed and downloaded by entering the following query in the Trace Archive search box:

species_code='MAMMUTHUS PRIMIGENIUS'

There are 302, 692 traces for woolly mammoth. Only 40 thousand traces at a time can be obtained from the Trace Archive Web service, so a better option for getting all of the mammoth data is the species ftp directory:

ftp.ncbi.nlm.nih.gov/pub/TraceDB/mammuthus_primigenius/

The Trace Archive sequences can be searched using the specialized trace archive megablast services linked to the nucleotide section of BLAST homepage:

www.ncbi.nlm.nih.gov/BLAST

as well as on the Trace Archive homepage. Both a discontiguous megablast service for cross-species comparisons and a standard megablast service for intra-species comparisons are available. The woolly mammoth traces are available in the 'Mammuthus primigenius-other' database through the trace archive megablast services.

Improvements in techniques for handling ancient biological samples and improvements in DNA sequencing technologies will increase the availability of molecular data from the recent as well as the distant past. These data will provide important insights into the phylogenetic relationships of extinct taxa, and details of the make-up of past biological communities. The Entrez system and the NCBI BLAST services will continue to provide rapid and powerfully integrated access to these important data.

[1] Poinar HN, et al. Metagenomics to paleogenomics: large-scale sequencing of mammoth DNA. Science. 2006 Jan 20;311(5759):392-4. Epub 2005 Dec 20. PMID: 16368896.

[2] Rogaev EI, et al. Complete mitochondrial genome and phylogeny of Pleistocene Mammoth Mammuthus primigenius. PLoS Biol. 2006 Mar;4(3):e73. Epub 2006 Feb 7. PMID: 16448217.

[3] Krause J, et al. Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae. Nature. 2006 Feb 9;439(7077):724-7. Epub 2005 Dec 18. PMID: 16362058.

[4] Haddrath O, Baker AJ. Complete mitochondrial DNA genome sequences of extinct birds: ratite phylogenetics and the vicariance biogeography hypothesis. Proc Biol Sci. 2001 May 7;268 (1470):939-45. PMID: 11370967.

[5] Cooper A, et al. Complete mitochondrial genome sequences of two extinct moas clarify ratite evolution. Nature. 2001 Feb 8;409 (6821):704-7. PMID: 11217857.