Revealed by Mass Spectrometry

Comment on "Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry" Mike Buckley, et al. Science 319, 33c (2008); DOI: 10.1126/science.1147046 The following resources related to this article are available online at www.sciencemag.org (this information is current as of February 14, 2008 ):

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TECHNICAL COMMENT

Mike Buckley,1 Angela Walker,2 Simon Y. W. Ho,3 Yue Yang,1 Colin Smith,4 Peter Ashton,1 Jane Thomas Oates,1 Enrico Cappellini,1 Hannah Koon,1 Kirsty Penkman,1 Ben Elsworth,1 Dave Ashford,1 Caroline Solazzo,1 Phillip Andrews,2 John Strahler,2 Beth Shapiro,6 Peggy Ostrom,5 Hasand Gandhi,5 Webb Miller,6 Brian Raney,7 Maria Ines Zylber,8 M. Thomas P. Gilbert,9 Richard V. Prigodich,10 Michael Ryan,11 Kenneth F. Rijsdijk,12 Anwar Janoo,13 Matthew J. Collins1* We used authentication tests developed for ancient DNA to evaluate claims by Asara et al. (Reports, 13 April 2007, p. 280) of collagen peptide sequences recovered from mastodon and Tyrannosaurus rex fossils. Although the mastodon samples pass these tests, absence of amino acid composition data, lack of evidence for peptide deamidation, and association of a1(I) collagen sequences with amphibians rather than birds suggest that T. rex does not. arly reports of DNA preservation in multimillion-year-old bones (i.e., from dinosaurs) have been largely dismissed (1, 2) (table S1), but reports of protein recovery are persistent [see (3) for review]. Most of these studies used secondary methods of detection, but Asara et al. (2) recently reported the direct identification of protein sequences, arguably the gold standard for molecular palaeontology, from fossil bones of an extinct mastodon and Tyrannosaurus rex. After initial optimism generated by reports of dinosaur DNA, there has been increasing awareness of the problems and pitfalls that bedevil analysis of ancient samples (1), leading to a series of recommendations for future analysis (1, 4). As yet, there are no equivalent standards for fossil protein, so here we apply the recommended tests for DNA (4) to the

E

1

BioArch, Departments of Biology, Archaeology, Chemistry and Technology Facility, University of York, Post Office Box 373, York YO10 5YW, UK. 2Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109–0404, USA. 3Evolutionary Biology Group, Department of Zoology, University of Oxford, OX1 3PS, UK. 4Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103, Leipzig, Germany. 5 Department of Zoology, Michigan State University, East Lansing, MI 48824, USA. 6Department of Biology, Pennsylvania State University, University Park, PA 16802, USA. 7Center for Biomolecular Science and Engineering, University of California–Santa Cruz, CA 95064, USA. 8Department of Parasitology, Kuvin Center, Hebrew University of Jerusalem, Israel. 9Biological Institute, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark. 10Chemistry Department, Trinity College, 300 Summit Street, Hartford, CT 06106, USA. 11Cleveland Museum of Natural History, 1 Wade Oval Drive, University Circle, Cleveland, OH 44106, USA. 12 National Museum of Natural History “Naturalis,” P.O. Box 9517, 2300 RA Leiden, Netherlands. 13National Heritage Trust Fund Mauritius, Mauritius Institute, La Chaussée Street Port Louis, Mauritius.

authentication of the reported mastodon and T. rex protein sequences (2) (Table 1). First, the likelihood of collagen survival needs to be considered. The extremely hierar-

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Comment on “Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry”

chical structure of collagen results in unusual, catastrophic degradation (5) as a consequence of fibril collapse. The rate of collagen degradation in bone is slow because the mineral “locks” the components of the matrix together, preventing helical expansion, which is a prerequisite of fibril collapse (6). The packing that stabilizes collagen fibrils (6) also increases the temperature sensitivity of degradation (Ea 173 kJ mol−1) (Fig. 1). Collagen decomposition would be much faster in the T. rex buried in the then-megathermal (>20°C) (7) environment of the Hell Creek formation [collagen half-life (T½) = ~ 2 thousand years (ky] than it would have been in the mastodon lying within the Doeden Gravel Beds (present-day mean temperature, 7.5°C; collagen T½ = 130 ky) (Fig. 1). This risk of contamination also needs to be evaluated. Collagen is an ideal molecular target for this assessment because the protein has a highly characteristic motif that is also sufficiently variable to enable meaningful comparison between distant taxa if enough sequence is obtained (Fig. 2). Compared with ancient DNA amplification, contamination by collagen is inherently less likely. Furthermore, because the bones sampled in (2) were excavated by the

Table 1. Key questions to ask about ancient biomolecular investigations [adapted from (4)]. Test

Sample

Pass

Observation

Mastodon, 300 to 600 ky old

Yes

Collagen T½ at 7.5°C = 130 ky

T. rex, 65 million years old Biomolecular preservation

No

Collagen T½ at 20°C = 2 ky

?

Range of evidence presented (8) but no amino acid compositional data

Macromolecular preservation

Yes

Macromolecular preservation is not the equivalent of biomolecular preservation (9)

Molecular target Handling history

Yes Yes?

Do the data suggest that the sequence is authentic, rather than the result of damage and contamination?

Mastodon and T. rex

No

Do the results make sense, and are there enough data to make the study useful and/or to support the conclusions?

Mastodon

Yes

T. rex

No

Do the age, environmental history, and preservation of the sample suggest collagen survival?

Do the biomolecular and/or macromolecular preservation of the sample, the molecular target, the innate nature of the sample, and its handling history suggest that contamination is a risk?

*To whom correspondence should be addressed. E-mail: [email protected]

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Large (2.5 g) samples increase risk of contamination? Errors in interpretation of spectra [see table S1 and (13)]? Damage-induced errors in sequence Weak affinity to mammals

Affinity of a1(I) peptides to amphibians, not birds or reptiles

33c

TECHNICAL COMMENT signal of the a1(I) fragments of mastodon and T. rex using NeighborNet analysis and uncorrected genetic distances. 10% 50000 meat and –100% 100% Using the sequences bone meal reported in (13), both 45000 the T. rex and masto–10% –100% 40000 don signal display an ar affinity with amphibians dia 35000 (Fig. 2A). Our reinterge ne –30% pretation of the spectra sis 30000 (12) changes the affinity of mastodon but not of 25000 axis 1 (25%) T. rex (Fig. 2B). In –40% 14C dates on addition to the a1(I) 20000 bone collagen peptides used in the Neighbor-Net analysis, 15000 Asara et al. reported two other peptides from 10000 T. rex (13); we question 5000 the interpretation of the a1(II) spectra (identical 0 to frog) but not the a2(I) –5.0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 spectra (identical to Effective Collagen Degradation Temperature (°C) chicken). We require more data Fig. 1. Plot of radiocarbon age versus estimated effective collagen degradation temperature for radiocarbon-dated bones from laboratory databases (principally Oxford and Groningen). The line represents the expected calendar age at which 1% of the original to be convinced of the collagen remains following a zero-order reaction; almost no bone collagen survives beyond this predicted limit. (Inset) The 99% authenticity of the T. rex confidence intervals of amino acid compositions by first two principal component analyses (48% of total variance) for bones from collagen sequences reNW Europe aged 20 °C) (7) environment of the Hell Creek formation (collagen t ½ ~ 2 ka) than it would have been in the mastodon lying within the Doeden Gravel Beds (present day mean temperature 7.5 °C; collagen t ½ 130 ka; Fig. 1). Risk of contamination The molecular target (collagen) is ideal for this investigation; the protein has a highly characteristic motif and yet also is sufficiently variable to enable meaningful comparison between distant taxa if sufficient sequence is obtained (Fig. 2). In comparison with ancient DNA amplification, contamination by collagen is inherently less likely. Furthermore because the bones were excavated by the authors, obvious contamination sources such as animal glue (used in conservation) can be excluded. Concentrating protein from the large amounts of bone used (2.5 g) may have heightened the risk of extraneous proteins entering the sample during extraction, but there have been no systematic studies of this phenomenon. Independent extraction and analyses would have strengthened claims for the authenticity of the origin of the peptides (and potentially ameliorated the original problems of data interpretation) (4).

UKPMC Funders Group Author Manuscript

The remarkable soft-tissue preservation of the investigated T. rex specimen (MOR 1125) has been documented (8); however microscopic preservation does not equal molecular preservation (9). Immunohistochemistry provides support for collagen preservation, although no data regarding inhibition assays with collagen from different species or cross-reactivity with likely contaminants (e.g. fungi 10) are presented. Curiously no amino acid compositional analysis was conducted (c.f. 11) although immonium ions were identified (by TOF-SIMS). In our experience, collagen-like amino acid profiles have been obtained in all bones from which we could obtain collagen sequence (Fig. 1, inset). Proof of sequence authenticity The spectra (12) (SOM of 12) are inconsistent with many of the original sequence assignments (13,14) (SOM Table 1 and 13). A common diagenetic modification, deamidation, not considered in the original publication, may shed light on authenticity. The facile succinimide mediated deamidation of N1156G (14) occurred in peptide Ost 5 (see Table 1 of 13 for nomenclature) from ostrich, presumably during sample preparation. Direct hydrolytic deamidation is slower (14) and an expectation of elevated levels of such products is reasonable for old samples. We agree with the new interpretation (13) of the spectrum illustrated in Fig. 2b of 3 as α1(I) G362SEGPEGVR370, the deamidated (Q→E367) form of the sequence found in most mammals (12). By way of contrast, none of the three glutamine residues in “T. rex” peptides are deamidated (Table S1 SOM). Only time will tell if Q→E is a useful marker for authentically old collagen, but from the evidence presented the mastodon sequence looks more diagenetically altered than T. rex. The unusual, fragmented nature of the reported T. rex sequence does not make it amenable to standard, model-based phylogenetic analysis. Instead, we examined the phylogenetic signal of the α1(I) fragments of mastodon and T. rex using Neighbor-Net analysis and uncorrected genetic distances. Using the originally reported sequences (2), both the T. rex and mastodon signal displayed an affinity with amphibians (Fig. 2a) (12). Our re-interpretation of the spectra changes the affinity of mastodon but not of T. rex (Fig. 2b). Two other peptides are reported Science. Author manuscript; available in PMC 2009 June 11.

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from T. rex (3); we question the interpretation of the (frog) α1(II) spectrum, but not the α2(I) assignment which is identical to chicken.


We require more data to be convinced of the authenticity of the T. rex collagen. Nevertheless, the handful of spectra reported for the temperate Pleistocene mastodon fail neither phylogenetic nor diagenetic tests, highlighting the potential of protein mass spectrometry to bridge the present gulf in our understanding between the fate of archaeological and fossil proteins (Fig 1.). In order to avoid past mistakes of ancient DNA research(1), we would recommend that future fossil protein claims are considered in the light of tests for authenticity such as those we have used.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgments This work was supported by NSF (EAR-0309467), NERC (NE511148, GR9/01656), NCRR (P41-18627), EU (MESTCT-2004-007909, MEST-CT-2005-020601), Wellcome Trust Bioarchaeology Fellowships (KP, HK) Analytical Chemistry Trust Fund, the RSC Analytical Division, EPSRC, and the Michigan Proteome Consortium.

References


1. Willerslev E, Cooper A. Proceedings of the Royal Society of London Series B-Biological Sciences Jan 7;2005 272:3. 2. Asara JM, Schweitzer MH, Freimark LM, Phillips M, Cantley LC. Science April 13;2007 316:280. [PubMed: 17431180]2007 3. Schweitzer MH. Palaeontologia Electronica 2002;5 4. Gilbert MTP, Bandelt H-J, Hofreiter M, Barnes I. Trends in Ecology and Evolution Oct;2005 20:541. [PubMed: 16701432]2005 5. Collins MJ, Riley MS, Child AM, Turner-Walker G. Journal of Archaeological Science 1995;22:175. 6. Miles CA, Ghelashvili M. Biophysical Journal 1999;76:3243. [PubMed: 10354449] 7. Johnson KR, Nichols DJ, Hartman JH. The Hell Creek Formation and the Cretaceous-Tertiary Boundary in the northern Great Plains: Geological Society of America Special Paper 2002;361:503– 510. 8. Schweitzer MH, et al. Science April 13;2007 316:277. [PubMed: 17431179]2007 9. Gupta NS, Briggs DEG, Pancost RD. Journal of the Geological Society Nov;2006 163:897. 10. Sepulveda P, et al. Infect. Immun June 1;1995 63:2173. [PubMed: 7768595] 11. Schweitzer M, Hill CL, Asara JM, Lane WS, Pincus SH. Journal of Molecular Evolution Dec 01;2002 55:696. [PubMed: 12486528]2002 12. Supplementary, Material. 13. Asara JM, et al. Science September 7;2007 317:1324. [PubMed: 17823333] 14. Robinson NE, et al. Journal of Peptide Research 2004;63:426. [PubMed: 15140160]

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UKPMC Funders Group Author Manuscript Figure 1.


Plot of radiocarbon age versus estimated effective collagen degradation temperature for radiocarbon dated bones from laboratory databases (principally Oxford and Groningen). The line represents the expected calendar age at which 1% of the original collagen remains following a zero-order reaction; almost no bone collagen survives beyond this predicted limit. Inset. 99% confidence intervals of amino acid compositions by first two principal component analyses (48% of total variance) for < 11 ka (n = 324), 11-110 ka (n = 210), 110-130 ka (n = 26) and 130-700 ka (n = 31) bones from NW Europe. Pliocene samples are not plotted, as their composition (n = 8) is highly variable and yields of amino acids are low. The orange line indicates a compositional trend observed when compact bone is heated for 32 days at 95 °C, which reduces collagen to 1% of initial concentration, each inflection representing a separate analysis (n = 32). The composition becomes more similar to mixed tissues samples (meat and bone meal, n = 32) principally due to the depletion of Gly. An amino acid profile for mammoth (m) is consistent with collagen, unlike the associated sediment sample (s) (data from 11).


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UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscript

Figure. 2.

Phylogenetic networks of α1(I) sequences using Neighbor-Net analysis (A) with the original assignments (2) and (B) following reinterpretation of the mass spectrometric data (12). T. rex does not group with bird/reptile using either set of sequence alignments. More sequence is required for a full, model-based phylogenetic analysis


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Table 1

Key questions to ask about ancient biomolecular investigations (taken from 4)


Test

Sample

Does the age, environmental history and preservation of the sample suggest collagen survival?

Mastodon 300-600 ka

Collagen t½ @ 7.5 °C = 130 ka

T. rex 65 Ma

Collagen t½ @ 20 °C = 2 ka

Does the biomolecular and/or macromolecular preservation of the sample, the molecular target, the innate nature of the sample and its handling history suggest that contamination is a risk?

Biomolecular preservation

Macromolecular preservation

Pass

?

Observation

Range of evidence presented (8) but no amino acid compositional data. ...but macromolecular ≠ biomolecular preservation (9).

Molecular target Handling history Do the data suggest that the sequence is authentic, rather than the result of damage, and contamination?

Do the results make sense, and are there enough data to make the study useful and/or support the conclusions?

...but large (2.5 g) samples processed Errors in interpretation of spectra (see SOM Table 1 and 13)? Damage induced errors in sequence.

Mastodon

Weak affinity to mammals

T. rex

Affinity of α1(I) peptides amphibians, not birds or reptiles

UKPMC Funders Group Author Manuscript Science. Author manuscript; available in PMC 2009 June 11.

www.sciencemag.org/cgi/content/full/319/5859/33c/DC1

Supporting Online Material for Comment on “Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry” Mike Buckley, Angela Walker, Simon Y. W. Ho, Yue Yang, Colin Smith, Peter Ashton, Jane Thomas Oates, Enrico Cappellini, Hannah Koon, Kirsty Penkman, Ben Elsworth, Dave Ashford, Caroline Solazzo, Phil Andrews, John Strahler, Beth Shapiro, Peggy Ostrom, Hasand Gandhi, Webb Miller, Brian Raney, Maria Ines Zylber, M. Thomas P. Gilbert, Richard V. Prigodich, Michael Ryan, Kenneth F. Rijsdijk, Anwar Janoo, Matthew J. Collins* *To whom correspondence should be addressed. E-mail: [email protected] Published 4 January 2008, Science 319, 33c (2008) DOI: 10.1126/science.1147046

This PDF file includes: SOM Text References Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/319/5859/33c/DC1 Table S1 Collagen a1 Sequences

Buckley et al., Technical Comment on Asara et al., (Reports 13 April 2007, p. 280) “Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry” Supporting online material.

Comment on “Protein Sequences from Mastodon and Tyrannosaurus rex Revealed by Mass Spectrometry” Supporting Online Material Mike Buckley1, Angela Walker2, Simon Y. W. Ho3, Yue Yang 1, Colin Smith4 Peter Ashton1, Jane Thomas Oates1, Enrico Cappellini1, Hannah Koon1, Kirsty Penkman1, Ben Elsworth1, Dave Ashford1, Caroline Solazzo1, Phillip Andrews2, John Strahler2, Beth Shapiro6, Peggy Ostrom5, Hasand Gandhi5, Webb Miller6, Brian Raney7, Maria Ines Zylber8, M. Thomas P. Gilbert9, Richard V. Prigodich10, Michael Ryan11, Kenneth F. Rijsdijk12, Anwar Janoo13, Matthew J. Collins1* 1

BioArch, Departments of Biology, Archaeology, Chemistry and Technology Facility, University of York, York, UK. PO Box 373, York YO10 5YW, UK 2 Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0404, USA 3 Evolutionary Biology Group, Department of Zoology, University of Oxford, OX1 3PS, United Kingdom 4 Department of Human Evolution, Max Planck Institute for Evolutionary, Anthropology, Deutscher Platz 6, D-04103, Leipzig, Germany 5 Department of Zoology, Michigan State University, East Lansing, Michigan 48824, USA 6 Department of Biology, Pennsylvania State University, University Park, PA 16802, USA. 7 Center for Biomolecular Science and Engineering, University of California Santa Cruz, CA 95064, USA 8 Dept. of Parasitology, Kuvin Center, The Hebrew University of Jerusalem, Israel 9 Biological Institute, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark 10 Chemistry Department, Trinity College, 300 Summit Street, Hartford, CT 06106 11 Cleveland Museum of Natural History, 1 Wade Oval Dr., University Circle Cleveland, Ohio 44106, USA 12 National Museum of Natural History 'Naturalis', P.O. Box 9517, 2300 RA Leiden, the Netherlands 13 National Heritage Trust Fund Mauritius, Mauritius Institute, La Chaussée Street Port Louis, Mauritius *To whom correspondence should be addressed. E-mail: [email protected]


Thermal age estimates The effective annual temperature experienced by buried mineralized collagen reflects both the mean annual air temperature (MAT) and seasonal fluctuations. Using climatology data compiled in the years 1987 and 1988, the International Satellite Land Surface Climatology Project (ISLSCP) (1) made global estimates of monthly average soil temperature, at depths of 0 - 49 cms and 49 - 100cms, at a 1° by 1° resolution Estimates were calculated from surface temperature, using the expression T(d)n = (1-c)T(o) + c(a T(s)n + b T(s)n-1)

Equation 1

Where T(o) denotes the mean annual surface temperature, T(s)n and T(s)n-1 are the surface temperature for month n and previous month n-1, a and b are constants defining the temperature phase lag (a = b = 0.5) and c is a constant describing the amplitude damping (c = 0.77). Using this data (and given A = 2.11 x1019, Ea = 173.2kJmol-1 for collagen degradation) we estimated the total reaction over the 24 month cycle to make an estimate of the effective collagen degradation temperature (Teff) at each grid point. The Teff represents the worst-case scenario, shallow buried, poorly thermally buffered bone. An effective collagen degradation temperature could be assigned for each bone in the radiocarbon database for which location data was available. No correction is made for altitude, beyond the resolution of the ISLSCP data-set. This analysis does not take into account palaeoclimatic changes during the burial history of the material but clearly displays the influence of burial temperature on collagen preservation. Based upon the thermal age analysis the mastodon MOR 605 (Doeden Gravel Beds, Eastern Montana, Nr Miles City (mean annual temperature 7.5oC; WS 7266612 MILES CITY, 801m Latitude: 46.41 N, Longitude: 105.84 W.) is predicted (within error) to contain collagen. Amino acid analysis Amino acid analysis was conducted upon the whole bone, soluble and insoluble fractions of samples from (i) Holocene - Pleistocene bones, (ii) meat and bone meal (supplied by Prosper de Mulder) and (iii) bone heated in water at 95ºC for 32 days. The three fractions were prepared as follows; whole bone samples were hydrolysed in 7 M HCl for 18 h @ 110°C, the soluble fractions from 4 h demineralisation with 0.6 M HCl, and concentrated by evaporation were hydrolyzed with 6 M HCl for 18 h @110ºC; insoluble fractions from the same 4 h demineralisation were washed 5 times with deionised water, lyophilised and hydrolyzed with 6 M HCl for 18 h @110ºC. Samples were analysed for amino acid composition using rHPLC with fluorescence detection (as detailed in 2) Imino acids (Pro, Hyp) were not detected by this method. Collagen Alignments We added to sequences available from public databases, by extracting and conceptually translating the α1(I) collagen genes from a whole-genome alignment of 30 vertebrate species that will soon be made available at the UCSC Human Genome Browser; these additional sequences are included as a FASTA file. These underwent minor editing to remove small sections in which the sequence lacked the characteristic Gly-Xxx-Yyy motif, but otherwise did not alter the conceptual translations. These sequences were used for creation of the Hypothetical Collagens database and in Neighbor-Net analysis.


Interpretation of published spectrum The starting points for manual interpretation of spectra (Supporting Online Material from 3)were the sequences originally reported by Asara et.al. The bias was for the collagen GXY motif and the reassignment of all G* calls. For the isobaric I/L/P*, fragment ion intensity (N-terminal side of P*; C-terminal side of I/L) was the prime determinant. Partial de novo sequencing was done where possible. Delta masses were typically +/- 0.2 Da and permitted distinction between amide vs acid side chains. In the text we have used the numbering system of the α1(I) chain of human type I collagen (accession number P02452). The mature chain of α1(I) collagen is from positions 162 to 1217 (1056 residues). Detailed interpretations of the published spectra are presented in Supplementary Online Materials, Table S1. Neighbor-Net Analysis The collagen α1(I) sequences were aligned with orthologues from 32 other species. The alignment was investigated using Neighbor-Net analysis (4) as implemented by the software SplitsTree 4 (5). We compared published sequences from (3) with our assignments of the same spectra obtained by manual or partial de novo interpretation (Table S1). In our assignments we used the sequences with the non-deamidated residue if we believed that deamidation arose diagenetically and was not part of the original sequence. Neighbor-Net is a statistically consistent method closely related to split decomposition. It produces a splits graph, which is a representation of the phylogenetic split signals present in an alignment (4). Our analysis, based on uncorrected genetic distances, revealed that the phylogenetic signal was ambiguous for amphibians, reptiles, and birds. However, the T. rex sequence showed a closer affinity to the amphibian sequences, particularly newt. We did not align the solitary α2(I) or α1(II) peptides reported for T. rex which had identity to bird and amphibian respectively.

References 1.

B. W. Meeson et al. (Published on CD NASA (USA_NASA_GDAAC_ISLSCP_001-USA_NASA_GDDAC_ISLSCP_005). 1995), vol. 1-5.

2.

S. A. Parfitt et al., Nature 438, 1008 (2005).

3.

J. M. Asara, M. H. Schweitzer, L. M. Freimark, M. Phillips, L. C. Cantley, Science 316, 280 (2007).

4.

D. Bryant, Molecular Biology and Evolution 21, 255 (2003).

5.

D. H. Huson, D. Bryant, Mol Biol Evol 23, 254 (2006).