*Division of Vertebrate Zoology, American Museum of Natural History, New York, New York;
Aaron Diamond AIDS Research Center, New York, New York;
Istituto di Medicina Legale, Universitá Cattolica del Sacro Cuore, Rome, Italy;
Division of Invertebrate Zoology, American Museum of Natural History, New York, New York;
||Zoological Institute, Russian Academy of Science, St. Petersburg, Russia; and
¶Aaron Diamond AIDS Research Center at Tulane Regional Primate Research Center, Covington, Louisiana
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Abstract |
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Introduction |
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In the absence of a "virological fossil record" for any mammalian taxon, it is unknown how ERVs have managed to establish their distributions (at the individual, population, and species levels) or how ERV complements have varied across time and host ranges. With respect to ERV-Ls, among major mammalian clades studied to date, only primates and rodents exhibit marked expansion of genomic ERV-L content and diversification of sequences (Bénit et al. 1999
). In both primates and rodents, ERV-L copy number and number of unique sequences are high compared with those of carnivores, lagomorphs, and ungulates. For example, Southern blot data indicate that most placental mammals exhibit 1030 ERV-L copies per genome, whereas primates and rodents have at least 100200 copies (Bénit et al. 1999
).
Not all mammalian orders have been examined for ERV-L incidence, but it is already clear that there are some significant interordinal differences. As noted, most primates and rodents possess a large number of distinct ERV-L elements in their genomes, but artiodactyls have very few (e.g., the cow has two reported unique sequences). Marsupials and monotremes appear to lack this element class altogether (Bénit et al. 1999
). Why such differences should exist at all is not obvious, and to make any headway in understanding the evolution of ERV-Ls, it will be necessary to collect empirical dataespecially data that permit comparisons between individuals within species and across appreciable lengths of time.
In undertaking this study, we attempted to collect both kinds of information simultaneously to capitalize on the fact that, despite the technical problems that attend their use, fossils are potentially the best empirical source of temporally distributed data on ERV-L prevalenceassuming, of course, that the relevant information can be reliably collected. Among mammals, the ideal group for exploring ERV-L paleovirology is Elephantidae (Proboscidea). In addition to living Loxodonta africana (African elephant) and Elephas maximus (Asian elephant), this family includes Mammuthus primigenius, the extinct woolly mammoth of late Quaternary Europe, northern Asia, and North America. Given the record of low ERV-L incidence in investigated ungulates, it might be expected that proboscideans would exhibit low copy numbers and few unique sequences in their genomes. This assumption implies that it should be comparatively easy to fully characterize the incidence and relative diversity of ERV-L sequences among individuals of proboscidean species over various time depths. Fortunately, fossils of woolly mammoths are abundant in many parts of this species' former range. Previous studies have shown that genetic material from high-latitude mammoth sites is often exceptionally well preserved (Johnson, Olson, and Goodman 1985
; Yang, Golenberg, and Shoshani 1996
; Greenwood et al. 1999
), indicating that temporal examination of ERV-L evolution should be possible in principle. To provide a relevant context for interpreting the results of these experiments, equivalent data should be collected for members of the larger taxonomic group of which Proboscidea is a memberUranotheria, the taxon which includes Asian and African elephants, sirenians (manatees and dugongs), and hyraxes (McKenna and Bell 1997
).
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Materials and Methods |
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DNA extraction from samples of living elephantids presented no special problems. However, investigation of mammoth material required the use of appropriate ancient DNA techniques (table 1 ) (Pääbo 1989
; Janczewski et al. 1992
; Greenwood et al. 1999
). Ancient DNA extractions were performed in an American Museum of Natural History (AMNH) facility dedicated solely to ancient DNA extraction and PCR setup, in which elephant investigations had previously never been undertaken. Our protocols for avoidance of contamination are described in Greenwood et al. (1999)
. In addition to the precautions mentioned therein, bacterial transformation and PCR using bacterial colonies as template "colony PCR" for all products were carried out (by F.L.) at an institute separate from the ancient DNA laboratory (ADARC) (see Greenwood et al. [1999
] for procedures). Modern DNA PCR and cloning was carried out in a separate AMNH laboratory to avoid contamination of the facility in which ancient extractions and PCR setup were carried out. A portion of each sample was sent to the Istituto di Medicina Legale, Universita Cattolica di Sacro Cuore, where extraction and PCR of a nuclear DNA locus were performed independently.
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PCR, Cloning, and Sequencing
PCR conditions used for ancient samples were performed as described in Greenwood et al. (1999)
. PCRs of extant taxa included 30 cycles performed in a 50-µl volume with 1 µg DNA using Taq polymerase and the standard buffer supplied by Boehringer Mannheim. PCR primers and the annealing temperature used for the elephant-specific microsatellite are described in Nyakaana and Arctander (1999)
. Primers and PCR conditions for cytochrome b and 28S rDNA are described in Greenwood et al. (1999)
. ERV-L primers and the annealing temperature for the larger pol fragment are described in Bénit et al. (1999)
. Primers for the shorter product applied to mammoth extracts combined the 3' primer described in Bénit et al. (1999)
with a primer designed based on initial elephant ERV-L sequences (5'-CAGCAATACACCTTCACTTG-3') at a 60°C annealing temperature. PCR product cloning and colony sequencing was done as in Greenwood et al. (1999)
except that (1) colony PCR products were purified with QIAquick columns (Qiagen), and (2) sequencing with plasmid-specific primers T7 and SP6 was executed with an ABI 377 sequencer and the manufacturer's protocol. Both strands were sequenced for all ERV-L clones. Prior to attempting proviral sequence amplification in the mammoth specimens, we established that each sample contained endogenous mammoth DNA by demonstrating the presence of specific mitochondrial, multicopy nuclear DNA sequences and a single-copy microsatellite sequence reported here for the first time for mammoth material (fig. 1
) (Nyakaana and Arctander 1999
). Facilities, extraction of nucleic acids, and PCR amplification are described in detail elsewhere (Greenwood et al. 1999
).
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Results and Discussion |
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A 28S rDNA fragment with a characteristic amplification product size for elephants and mammoths (179 bp in elephantids versus 150 bp in humans; Greenwood et al. 1999
) was successfully amplified for all three mammoths, thereby demonstrating the presence of intact multicopy nuclear DNA (data not shown).
To demonstrate the presence of intact endogenous single-copy sequences, a microsatellite sequence described for elephants was amplified (Nyakaana and Arctander 1999
). The expected product, approximately 160 bp, was recovered for all three mammoths, although products were generally much weaker than observed for mitochondrial or multicopy nuclear DNA amplifications (fig. 1 and other data not shown). Again, negative controls were always devoid of PCR product. A portion of each sample was sent to another institute (Istituto di Medicina Legale [IML]) where the DNA was independently extracted and the same microsatellite was amplified (by C.C.). Clones from the two products for each mammoth are shown in figure 2
(for ACDs, see table 1
). A greater ACD for the Engineer Creek mammoth is consistent with previous DNA analysis from this sample and may reflect greater postmortem DNA damage and modification (Greenwood et al. 1999
). The Wrangel Island mammoth at one position exhibited G in three clones of the first amplification and C at six (
in fig. 2
). Similar results were obtained for clones from the IML extract (G in three clones, C in two). This difference, which has been observed before in mammoth nuclear DNA sequences, is most likely due to allelic variation. The mammoths differed from the published African elephant sequence by two to three substitutions and by a 2-bp deletion (Nyakaana and Arctander 1999
). The mammoths differed from the two Asian elephant alleles by zero to two substitutions. Both Asian elephant alleles shared the same 2-bp deletion observed in the mammoth sequences. Subsequent sequencing of additional African elephants by our group has shown that the African elephant microsatellite-flanking regions also differ from mammoths by zero to two differences and do not exhibit the 2-bp insertion observed in the published sequence (fig. 2 , La1 and La2). The same sequences were retrieved for each mammoth at both the Rome and the New York labs, thus providing confirmation of the presence of endogenous mammoth DNA in these samples.
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For the mammoth specimens, data had to be acquired from the shorter ERV-L fragment because experience has shown that long nuclear DNA fragments extracted from mammoth material will not amplify with existing techniques (Greenwood et al. 1999
). The shorter primer combination excludes humans and cows, two potential contamination sources (Taylor [1996
] and other data not shown). As noted above, all elephant amplifications, cloning, and sequencing were done in facilities removed from the ancient DNA laboratory that have never held uranothere collections in order to exclude the possibility of contamination of mammoth ERV-L sequences.
Using the shorter ERV primer combination, products of expected size were obtained from the DNA of all three mammoth specimens, while all negative controls were always devoid of product. Each mammoth sample was independently amplified three times by PCR, and 10 individual clones per PCR product were sequenced to evaluate ERV-L diversity. Thus, 30 clones were generated from three independent PCR reactions for each mammoth. For the Engineer Creek mammoth, the third amplification was derived from an extract that was different from the first two. Subsequent to the mammoth work, the same PCR sampling protocol was applied to DNA extracted from an Asian elephant and an African elephant. For comparative purposes, the same fragment was amplified from DNA derived from the manatee and the hyrax; 10 clones were sequenced for each PCR product.
Again, contrary to expectations, PCR reactions with both mammoth and elephant DNA yielded multiple distinct ERV-Ls (alignment available from GenBank, accession numbers AF312038AF312207). The average among-clones absolute difference, including substitutions and insertions/deletions, for the entire elephantid data set was 20.42 (table 2 ). Each PCR revealed approximately the same average level of ACDs, with the mammoth from Engineer Creek having the highest average (table 2 ). For this latter specimen, first- and second-extract clones did not differ in diversity (table 2 ).
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At the beginning of this study, it was not known how many ERV-L sequences might exist in different uranothere lineages, even to an order of magnitude (in some vertebrate species, ERV sequences may comprise up to 1% of the genome) (Coffin 1996
). The results of Bénit et al. (1999)
in sequencing clones from ERV-L amplifications from several ungulate groups suggested that proboscideans would probably display only a few different elements. Thus, it is of great interest to report that novel ERV-L sequences were found in each replicate in our study, suggesting that many more unique sequences could be obtained if sampling were to continue. Uranotheres evidently genomically maintain a large pool of ERV-L sequences, unlike virtually all other mammalian orders sampled to date with the exception of primates and rodents (Bénit et al. 1999
). In order to compare results across species and major taxa, we assumed that detection was related to abundance, such that the sequences exhibiting the highest copy numbers in any individual's genome were the ones most likely to be detected by PCR. This assumption was justified by the fact that our primers, although doubtlessly excluding some sequences, were nevertheless able to amplify a great diversity of sequences across all selected uranothere taxa.
Although diverse, not all recovered ERV-L sequences were unique (table 3
). The Wrangel Island and Chekurovka mammoths and the Asian elephant each exhibited sequences that were individually unique but appeared more than once among clones, with the Wrangel Island mammoth showing four groups of multiply occurring sequences (table 3
). Among the mammoths, five sequences were shared (table 3 ). The Engineer Creek and Wrangel Island mammoths exhibited more sequences in common than either did with the Chekurovka mammoth. Thus, it is arguable that the same elements were present with little or no change in mammoth populations ranging temporally from 26,000 to 4,500 years ago and geographically from north-central Siberia to central Alaska. Interestingly, the mammoths shared four sequences with Elephas and four different ones with Loxodonta, while the Asian elephant and the African elephant exclusively shared three sequences. Additionally, three sequences were shared by mammoths and both elephant species. Since these three lineages probably diverged from one another during the Early Pliocene 45 MYA (Todd and Roth 1996
), it is evident that some elephantid ERV-Ls have been maintained in descendant taxa over a considerable time span.
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Five manatee sequences had 711 differences from three of the modern elephant sequences (table 3
). All of these sequences were rare in extant elephants; interestingly, none occurred in the mammoths. It is likely that the sequences were shared by the last common ancestor of manatees and elephants. This is consistent with the view that sirenians and proboscideans are related, albeit distantly (their last common ancestor probably lived in Middle to Late Paleocene, 5660 MYA; Fischer 1996
). No comparable sequences were observed in the hyrax, indicating that the possibility of persistence of a given ERV-L is not unlimited.
In cases where sequences are identical or nearly so (i.e., among mammoths and elephants), the simplest explanation is that the shared sequences represent orthologs. However, it is formally possible that more divergent sequences (such as those of the manatee) are paralogous. Yet, even if descriptively identical loci are not identical by virtue of actual descent, paralogy is nevertheless consistent with the proposition that both taxa shared a common progenitor element. Lack of a high degree of sequence sharing is also consistent with observations utilizing the longer pol gene fragment. Burst sequences within monophyletic groups showing significant ERV-L activity, such as primates, tend to be much more similar within a group than between unrelated groups (Bénit et al. [1999]
and other data not shown).
The 110-bp product retrieved is insufficient to draw any strong phylogenetic conclusions regarding uranothere interrelationships. However, preliminary phylogenetic analysis of a subset of uranothere sequences and those of other mammalian orders revealed that the primate and rodent sequences tend to cluster as monophyletic groups, while ERV-Ls from ungulates, lagomorphs, and carnivores are randomly distributed, a finding which is consistent with previous analysis (Bénit et al. 1999
). In addition, with few exceptions, the uranothere sequences also formed a monophyletic group. This is consistent with observations and interpretations regarding rodent and primate ERV-L expansion events (Bénit et al. 1999
). However, as tree support was not robust, wider sampling of mammalian orders is necessary, and accumulation of longer sequences will be required to draw any further conclusions.
Explanation for ERV Expansion
Horizontal transfer and repeated novel infection are unlikely sources of overall diversity of ERV-Ls, as these entities generally lack an env gene, tend to cluster phylogenetically by vertebrate class, and are noninfectious (Herniou et al. 1998
). Lack of an abundance of shared sequences within higher-level taxa (e.g., uranotheres) also argues against horizontal transmission as the main force generating diversity (Bénit et al. 1999
). More generally, ERVs activate during fertilization and embryogenesis (Löwer 1999
). During gametogenesis and development, the normal suppression of ERVs is released. The effects can be dramatic, with the appearance of RNA transcripts from multiple ERVs and ERV-induced chromosomal rearrangements in offspring of interspecific hybrids (Waugh O'Neill, O'Neill, and Marshall Graves 1998
; Löwer 1999
). In the present case, the sequences that appear most often among individual mammoths are a combination of elements that have expanded successfully in this species. Some elements, many with apparent coding potential, have been retained within specific elephantid lineages for millions of years, presumably having generated enough copies of themselves to avoid removal by selection, recombination, or genetic drift (Bénit et al. 1999
).
The fact that primates, rodents, and proboscideans each display unique expanded elements suggests that ERV-Ls, which are generally nonfunctional, may have acquired functionality to expand. An alternative explanation is that lineages were infected with functional exogenous viruses that subsequently expanded endogenously. It is also possible that ERVs could recombine in the genome or during reverse transcription to form novel elements, as do HERV-K elements (Berkhout, Jebbink, and Zsíros 1999
). These combinations of events could lead to remarkable lineage-specific bursts of transposition, formation of novel elements, and expansion of ERV-Ls. Although more evidence is required, if such events do occur, the significance of ERVs for phylogenetic investigations could be great indeed.
In summary, the existence of high-quality mammoth remains from Late Quaternary paleontological sites provides a unique opportunity for examining different levels of sequence evolution within an unquestionably monophyletic group of mammals. In particular, our data demonstrate that a high proportion of elephantid ERV-Ls have been able to successfully persist despite the effects of time, geographical distance, and speciation. Finally, recovery of numerous ERV-L sequences from mammoth material suggests that it should be possible to use molecular probes to search for evidence of exogenous viruses in well-preserved fossil remains. Eventually, this may lead to important insights into the epizootiological history of now-extinct populations and species (cf. MacPhee and Marx 1997
).
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Acknowledgements |
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Footnotes |
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3 Abbreviation: ERV-L, endogenous retrovirus-like element with tRNA leucine primer.
1 Keywords: endogenous retrovirus
mammoth
ancient DNA
evolution
elephant
microsatellite
2 Address for correspondence and reprints: Alex D. Greenwood, Division of Vertebrate Zoology, American Museum of Natural History, Central Park West at 79th Street, New York, New York 10024-5192. alexgr{at}amnh.org
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