assigning pathogenicity to mitochondrial trna mutations: when ‘definitely maybe’ is not good...
TRANSCRIPT
Update TRENDS in Genetics Vol.20 No.12 December 2004 591
species (belonging to all the bilaterian embranchments[9,10]). To be complete, such a reconstruction should alsoinclude bilaterian outgroup phyla, for example, theCnidarians [11] (Figure 2). From such an ancestral-genome deduction and from knowledge that will emergeduring the post-genomic era [12], we can dream ofreconstructing the genome of Urbilateria – our mostdistant bilaterian ancestor.
AcknowledgementsWe thank Nathalie Balandraud, Alexandre Vienne and Michael F.McDermott for helpful discussions and their critical review of ourmanuscript. A special thanks for editorial comments that helped inmaking the message more clear, concise and direct.
Supplementary data
Supplementary data associated with this article can befound at doi:10.1016/j.tig.2004.09.009
References
1 Kasahara, M. et al. (1997) Chromosomal duplication and theemergence of the adaptive immune system. Trends Genet. 13, 90–92
2 Abi-Rached, L. et al. (2002) Evidence of en bloc duplication invertebrate genomes. Nat. Genet. 31, 100–105
3 Vienne, A. et al. (2003) Evolution of the proto-MHC ancestral region:more evidence for the plesiomorphic organisation of human chromo-some 9q34 region. Immunogenetics 55, 429–436
4 Castro, L.F. et al. (2004) An antecedent of the MHC-linked genomicregion in amphioxus. Immunogenetics 55, 782–784
5 Postlethwait, J.H. et al. (2000) Zebrafish comparative genomics andthe origins of vertebrate chromosomes. Genome Res. 10, 1890–1902
6 Dehal, P. et al. (2002) The draft genome of Ciona intestinalis: insightsinto chordate and vertebrate origins. Science 298, 2157–2167
Corresponding author: Douglass M. Turnbull ([email protected]).
www.sciencedirect.com
7 Azumi, K. et al. (2003) Genomic analysis of immunity in a Urochordateand the emergence of the vertebrate immune system: ‘waiting forGodot’. Immunogenetics 55, 570–581
8 Danchin, E.G. et al. (2003) Conservation of the MHC-like regionthroughout evolution. Immunogenetics 55, 141–148
9 Nielsen, C. (2001) Animal Evolution: Interrelationships of the LivingPhyla., Oxford University Press
10 Tessmar-Raible, K. and Arendt, D. (2003) Emerging systems: betweenvertebrates and arthropods, the Lophotrochozoa. Curr. Opin. Genet.Dev. 13, 331–340
11 Kortschak, R.D. et al. (2003) EST analysis of the cnidarian Acroporamillepora reveals extensive gene loss and rapid sequence divergencein the model invertebrates. Curr. Biol. 13, 2190–2195
12 Eisenberg, D. et al. (2000) Protein function in the post-genomic era.Nature 405, 823–826
13 Morris, S.C. (1998) The Crucible of Creation, Oxford University Press14 De Robertis, E.M. and Sasai, Y. (1996) A common plan for dorsoventral
patterning in Bilateria. Nature 380, 37–4015 Boutanaev, A.M. et al. (2002) Large clusters of co-expressed genes in
the Drosophila genome. Nature 420, 666–66916 Lehner, B. et al. (2004) Analysis of a high-throughput yeast two-
hybrid system and its use to predict the function of intracellularproteins encoded within the human MHC class III region. Genomics83, 153–167
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18 Vienne, A. et al. (2003) Systematic phylogenomic evidence of en blocduplication of the ancestral 8p11.21–8p21.3-like region. Mol. Biol.Evol. 20, 1290–1298
19 Danchin, E. and Pontarotti, P. (2004) Stastistical evidence for a morethan 800 million years old evolutionary conserved genomic region inour genome. J. Mol. Evol. (in press)
20 McLysaght, A. et al. (2002) Extensive genomic duplication duringearly chordate evolution. Nat. Genet. 31, 200–204
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doi:10.1016/j.tig.2004.09.009
Assigning pathogenicity to mitochondrial tRNAmutations: when ‘definitely maybe’ is not good enough
Robert McFarland1, Joanna L. Elson1, Robert W. Taylor1, Neil Howell2,3
and Douglass M. Turnbull1
1Mitochondrial Research Group, School of Neurology, Neurobiology and Psychiatry, The Medical School,
University of Newcastle-upon-Tyne NE2 4HH, UK2MitoKor, San Diego, CA 92121, USA3Department of Radiation Oncology, University of Texas Medical Branch, Galveston, TX 77550-2780, USA
Some mutations in mitochondrial tRNA (mt-tRNA)
genes cause devastating disease, whereas others have
no clinical consequences. We understand little of the
factors determining the pathogenicity of specific
mt-tRNA mutations, making prediction of clinical out-
come extremely difficult. Using extensive sequence
databases, we compared the characteristics of neutral
variations with those of pathogenic mutations. We
recommend that the location of the proposed mutation
within the secondary structure of the mt-tRNA molecule
and the disruption it causes to Watson-Crick base pair-
ing should be considered when assessing the patho-
logical significance of a novel mt-tRNA mutation.
The mitochondrial tRNA (mt-tRNA) genes comprise only asmall fraction of the mitochondrial genome (Figure 1a,Table 1), but contribute disproportionately to the aetiologyof mitochondrial DNA (mtDNA) disease [1]. At present, asmall number of ‘common’ mutations are thought to beresponsible for the majority of mitochondrial disease due
TRENDS in Genetics
D-Loop
ND1
ND2
COI
COII
ND6
12srRNA
ATP8
ND4L
ATP6
16srRNA
Pro
Thr
Glu
Leu(CUN)Ser(AGY)His
Arg
Gly
LysAsp
Ser(UCN)
TyrCys
AsnAla
Trp
IleGln
Met
Leu(UUR)
Val
Phe
Cyt b
ND3
COIII
ND5
ND4
0 neutral variants1–2 neutral variants3–4 neutral variants>4 neutral variants
3–9bases0–10
bases DHU
0 mutations1–2 mutations3–4 mutations>4 mutations
0–10bases
1–2 bases
1–2 bases 1–2 bases
1–2 bases
3–9bases
3–5 bases 3–5 bases
7 bases 7 bases
DHUTψC
(a)
(b) (c)
TψC
Figure 1. The structure of the mitochondrial (mt) genome. (a) The relative positions and strand-of-origin of the 22 mt-tRNA genes in the mitochondrial genome.
(b) A composite of all 22 mt-tRNA secondary structures, demonstrating the location of pathogenic mutations and (c) neutral variants. Hotspots for pathogenic mutation (O4)
are coloured purple (b), whereas regions of multiple neutral variations (O4) are coloured dark blue (c).
Update TRENDS in Genetics Vol.20 No.12 December 2004592
to mt-tRNA mutations [2–6]. However, the advent ofrelatively inexpensive sequencing technology has resultedin the frequent detection of novel variants in mt-tRNAgenes. Determining the pathological significance of thesemt-tRNA variants is challenging but important forproviding accurate genetic advice.
DiMauro and Schon succinctly described the canonicalpathogenic criteria for mtDNA point mutations (Box 1) [7].Inevitably, some pathogenic mtDNA mutations fail to meetthese criteria, particularly those that are homoplasmic
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and therefore do not demonstrate the required segregationwith either biochemical deficiency or clinical disease [8–11].Other genuine mutations might ‘escape’ identificationbecause of poor evolutionary conservation in the region, orbecause there is difficulty demonstrating segregation withdisease in small families. Conversely, neutral variants canbe mistaken for pathogenic mutations when they arelimited to a few related individuals (‘private polymorph-isms’) or where heteroplasmy has been detected (althoughneutral polymorphism heteroplasmy is well described,
Table 1. Size and location of human mitochondrial tRNA genes
Mitochondrial tRNA Location in genome Size (bp)
Phenylalanine 577–647 71
Valine 1602–1670 69
Leucine (UUR) 3230–3304 75
Isoleucine 4263–4331 69
Glutamine 4329–4400 72
Methionine 4402–4469 68
Tryptophan 5512–5579 68
Alanine 5587–5655 69
Asparagine 5657–5729 73
Cysteine 5761–5826 66
Tyrosine 5826–5891 66
Serine (UCN) 7445–7516 72
Aspartate 7518–7585 68
Lysine 8295–8364 70
Glycine 9991–10 058 68
Arginine 10 405–10 469 65
Histidine 12 138–12 206 69
Serine (AGY) 12 207–12 265 59
Leucine (CUN) 12 266–12 336 71
Glutamine 14 674–14 742 69
Threonine 15 888–15 953 66
Proline 15 955–16 023 69
Update TRENDS in Genetics Vol.20 No.12 December 2004 593
particularly in the D-Loop, heteroplasmy of variants in mt-tRNA has generally been regarded as direct evidence forpathogenicity). Occasionally, pathogenicity of mt-tRNAmutations can be established through demonstrating directfunctional consequences, such as a substantial decrease insteady-state levels oraberrantaminoacylation of the specificmt-tRNA, both of which are technically challenging.
We have evaluated all mt-tRNA mutations listed onMITOMAP, the largest publicly available compendium ofmtDNA polymorphisms and mutations [1], using a scoringsystem (Box 2) based on functional evidence, conservation,frequency of reports and the presence of heteroplasmy(Table 2). Numerical values have been attributed to thevarious criteria to reflect the strength of the evidence theypresent for pathogenicity. The rank order of the changeswould vary if different criteria were used. However, thechanges we have placed in the ‘definite’ and ‘neutral’categories are robust to variations of the scoring system,and are consistent with what is known about the naturalhistory of disease.
In addition, by incorporating neutral variants from theMitoKordatabase [12]andMITOMAP-listed ‘polymorphisms’in our analysis, we have been able to compare the natureand location of neutral variations with those of pathogenic
Box 1. Canonical criteria defining the pathogenicity of a
mitochondrial (mt)-tRNA mutation
† The mutation must be heteroplasmic (i.e. co-existent wild-type and
mutated mitochondrial genomes in the same tissue).
† The proportion of mutated mtDNA should be higher in tissue from
affected individuals than in the same tissue from their unaffected
relatives.
† The proportion of mutated mtDNA should be higher in clinically
affected tissues.
† The proportion of mutated mtDNA should segregate with a
biochemical defect.
† The mutation should occur at an evolutionary conserved site in the
mitochondrial genome.
† The mutation should be absent from healthy controls.
For more information, see Ref. [7].
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mutations to a much greater extent than has beenpreviously possible [13,14].
Identification of mitochondrial mutations
mt-tRNA mutations
In total, 91 different mt-tRNA mutations were listed aspathogenic on MITOMAP, but 66 (73%) of these werelisted as ‘provisional’. The pathogenic status of fourmutations was listed as ‘unclear,’ whereas another fourmutations were not clearly described as either pathogenicor polymorphic. A dual entry on both the list of pathogenicmutations and the list of polymorphic variants occurred onsix occasions, whereas two mutations that were provi-sionally identified as pathogenic also have a correspond-ing neutral variation at the same site. We have recentlyreassigned the 606(A/G) substitution as a neutralvariant [15] and have not included it in the analysis, norhave we included variants that were listed as ‘unclear’ orwhere the pathogenic status was not defined (supplemen-tary material online; Table 1).
After scoring the published evidence for pathogenicity, itbecame clear that a large proportion (26%) of the pathogenicmt-tRNA mutations listed on MITOMAP are not supportedby sufficient evidence to be considered pathogenic and only16% of the total were pathogenic beyond doubt.
mt-tRNA neutral variants
There are 64 different neutral variants in mt-tRNA geneslisted on MITOMAP, six of which also appear on the list ofpathogenic mutations. On the MitoKor database, weobserved 131 distinct neutral mt-tRNA polymorphisms,32 of which either define haplogroups or are haplogroup-associated markers. MITOMAP listed only 44 (34%) of the131 changes observed on the MitoKor database as poly-morphisms, thus indicating that many of the mt-tRNAgene variants in the MitoKor database are ‘private’.Conversely, 39 (61%) of the polymorphic mt-tRNA genevariants listed on MITOMAP were detected in the MitoKordatabase, indicating that themajorityof those listed aretruepolymorphisms. In total, including mutations that score sixor less, we recorded 163 different neutral variations on theMitoKor and MITOMAP databases.
Distribution of mutations and neutral variants
The greatest sequence variability (23 different changes)and highest pathogenic mutation to neutral variant ratiowas seen in the mt-tRNALeu(UUR) gene. The mt-tRNAPro
gene had the least variability with only four changes, oneof which was pathogenic. Approximately 73% of mt-tRNAmutations occurred in the stem structures, and Figure 1bdemonstrates mutation ‘hotspots’ in both the anticodonand acceptor stems. Furthermore, almost 94% of thepathogenic mutations that occurred in stem structuresdisrupted Watson–Crick base pairing with only threeexceptions, all of which are likely to be pathogenic; both5537insT and 5874(A/G) score highly and although7445(A/G) scores only nine, it is a homoplasmicmutation with an organ-specific phenotype and there isgood evidence of a functional defect [16]. By contrast, asmaller proportion (44%) of the 163 neutral variationsoccurred in stem structures (Figure 1c), and only 57%
Box 2. Selection and scoring of pathogenic mutations and neutral variants
The analysis included mitochondrial tRNA (mt-tRNA) mutations that
were clearly listed on MITOMAP as pathogenic (as of 1 Dec 2003). For
each mutation, the listed references were examined and a score
compiled that was based on the published evidence for pathogenicity.
Beginning with the earliest reference, this ranking process was
performed until either the references were exhausted or the mutation
had achieved the maximum score of 20 points (see Table 2).
Transmitochondrial cybrid (cell fusion) experiments, measurement
of mt-tRNA steady-state levels and segregation of mutation with a
biochemical defect at the level of single cells were weighted heavily
in the scoring system to reflect the quality of such evidence in
demonstrating pathogenicity. ‘Polymorphic’ or neutral variations in
mt-tRNAs were identified on MITOMAP and from the MitoKor
database of complete mitochondrial genome sequences. The MitoKor
database has been compiled from high quality sequencing of 1341
ethnically diverse control subjects and 560 of these genomes are
publically available.
None of the mutations listed exclusively as ‘polymorphisms’ on
MITOMAP achieved a score of more than six points, thus providing a
‘bright line’ distinction between clinically benign and pathogenic
substitutions. We assumed that the site of a pathogenic mutation had
to exhibit at least moderate evolutionary conservation (one point).
Therefore, the minimum score for pathogenic mutations that occurred
at sites of moderate evolutionary conservation, which satisfied the
other canonical criteria, and that had been reported on more than one
occasion, was 11 points. Those mt-tRNA mutations with additional
evidence thus scored a minimum of fourteen points (11 points plus
three points for supportive single-fibre studies). Overall, mutations
were categorized into four distinct groups:
(i) definitely pathogenic (R14 points); (ii) probably pathogenic
(11–13 points); (iii) possibly pathogenic (7–10 points); and (iv) neutral
variants (%6 points).
All of the mutations that scored more than six points were further
analyzed regarding the nature of the mutation and its location within
the tRNA structure. The same analysis was then also performed for
neutral variants and those mutations scoring less than six points.
Update TRENDS in Genetics Vol.20 No.12 December 2004594
disrupted Watson–Crick base pairing. In addition, patho-genic mutations disrupted A–T and C–G base pairs (22:23)almost equally, whereas neutral variants disrupted A–Tpairing three times more often than C–G pairing (32:9).
Consequences of classifying mitochondrial mutations
Unsubstantiated claims
Although the canonical criteria of DiMauro and Schon arenow generally accepted, they have been neither rigorouslynor uniformly applied to suspected novel pathogenic
Table 2. Scoring criteria applied to mitochondrial-tRNA
mutations listed on MITOMAP
Pathogenic criteria Score
Evolutionary conservation
of the base
No change 2
Single change 1
More than one change 0
More than one
independent report
Yes 2
No 0
Presence of heteroplasmy Yes 2
No 0
Histochemical evidence of
mitochondrial disease
Strong evidence 2
Weak evidence 1
No evidence 0
Biochemical defect in
complexes I, III or IV
Yes 2
No 0
Segregation of the
mutation with disease
Within an affected individual
or family
2
No segregation 0
Single-fibre studies,
demonstrating higher
levels of mutation in COX
negative fibresa
Yes 3
No 0
Steady-state levels of
mutated mt-tRNAb or
evidence of pathogenicity
from cybrid cellsc
Yes 5
No 0
Maximum score 20aHeteroplasmic mitochondrial (mt)-tRNA mutations are a cause of skeletal muscle
pathology and are often associated with a mosaic distribution of COX negative
(deficient) muscle fibres. If the levels of the mutation are higher in COX negative
fibres than in neighbouring COX positive fibres, it means that there is a direct
correlation between the levels of mutation and biochemical dysfunction.bSome mt-tRNA mutations have been shown to associate with a reduction in the
steady-state level of that particular mt-tRNA; a useful indicator of pathogenicity for
homoplasmic mt-tRNA mutations.cCybrids are immortalized cells that have been depleted of their own mtDNA and
repopulated with exogenous mitochondria carrying a specific mtDNA mutation by
fusion with patient-derived enucleated cells (cytoplasts). The phenotypic differ-
ences amongst cybrid lines are thus a direct consequence of the patient mtDNA.
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mt-tRNA mutations before publication. Several mt-tRNAmutations were reported before the precepts of thecanonical criteria were conceived but others, publishedmore recently, simply lack the required evidence. In ourstudy, 26% of the ‘pathogenic’ mt-tRNA mutations listedon MITOMAP fulfilled three or fewer basic criteriathat would confirm pathogenicity and, on this basis, webelieve they should be considered as neutral variants. It ispossible that, for a few of these changes, circumstancesprecluded more extensive investigation and their categ-orization might have to be revised in the light of newevidence.
Advice, understanding and prediction
Clinically, misattribution of pathogenicity is an import-ant issue because of the consequences for geneticcounselling that is provided to individuals and familieswith mitochondrial disease. In addition, from amechanistic perspective, accurately mapping sites ofpathogenic and neutral variation within the secondarystructure of mt-tRNAs is vital to our understanding ofthe fundamental structure-function relationships thatgovern the biological activity of these molecules.Furthermore, the different distribution patterns ofpathogenic and neutral variants might prove usefulin devising algorithms to predict the pathogenic effectsof novel mt-tRNA changes. Although given our currentunderstanding of mt-tRNAs, it is unlikely that, evenwith extensive ‘training’, such algorithms will identifyevery pathogenic mutation.
Bias
The considerable variation in frequency of pathogenicmutations between the different mt-tRNA genes isexplained in part by the intense investigation of somewith a ‘track record’ of pathogenicity. For example, thecategorization of patients into groups with clinicalsyndromes has directed diagnostic investigation towardsparticular mt-tRNA genes where genotype–phenotyperelationships have previously been established. Anotherpotential source of bias that must be considered is‘survivor bias’, where sporadic pathogenic mutations that
Update TRENDS in Genetics Vol.20 No.12 December 2004 595
affect the loops or stem structures of certain mt-tRNAs arelethal at an early stage of in utero development. The rangeand location of pathogenic mutations thus observedreflects those who survive to be investigated. Finally, itis also possible that those mutations in mt-tRNA genescausing a severe biochemical disturbance behave in asimilar manner to mtDNA deletions, which are not easilytransmitted through the maternal germ line [17]. Theabsence of a maternal-inheritance pattern might dissuadeclinicians from pursuing extensive investigation formitochondrial disease in these patients.
Location is important
Figure 1b demonstrates hotspots for pathogenicmutations in both the acceptor and anticodon stems.Few of the mt-tRNA genes have pathogenic mutationsthat occur in the loop structures. The exception is themt-tRNALeu(UUR) gene where five mutations [includingthe mutation 3243(A/G), which occurs in those withmitochondrial myopathy, encephalopathy, lactic acidosisand stroke-like episodes (MELAS)] occur in the dihydro-uridine (DHU) loop and a further two in the TJC loop.Interestingly, mt-tRNALeu(UUR) also has the largest DHUloop (ten bases) and a large TJC loop (seven bases).Recent in vitro work by Sohm et al. suggests that thetertiary L-shaped structure of mt-tRNALeu(UUR) is some-what ‘relaxed’ in comparison with cytosolic tRNAs [18].This relaxed tertiary structure is probably adapted bypost-transcriptional modifications in vivo (illustrating thedifficulties in relating structure to function in mt-tRNAmolecules); however, it might also represent a structurethat is particularly vulnerable to an alteration in the basecomposition of the large DHU and TJC loops, wheretertiary hydrogen bonding is thought to be important inmaintaining the structural integrity of the tRNA molecule[19]. Furthermore, Sohm et al. also noted potentialstructural anomalies in the folding of the DHU-arm ofmt-tRNALeu(UUR) caused by mutations in the loop[3243(A/G) and 3250(T/C)], but not in the stemstructures [3303(C/T) and 3271(T/C)] [18]. Mutationsthat occur in other mt-tRNAs also occur in loops that arelarge [8344(A/G) in the largest TJC loop of nine bases]or, in the case of the anticodon loop, in a location wherethey are anticipated to exert an enormous influence on thefunction of the mt-tRNA.
Disruption and tolerance
The disruption of Watson–Crick base pairing is an import-ant determinant of pathogenicity [13]. In our analysis,almost 94% of the pathogenic mutations occurring in stemstructures disturb Watson–Crick base pairing. By compari-son, only 57% of neutral variants affect Watson–Crick basepairing in the stems. Approximately 22% of the neutralvariants affect cytosine–guanine (C–G) interactions,whereas 51% of pathogenic mutations disrupt this C–Gtype of Watson–Crick base pairing. Adenine–thymine (A–T)bonding has a lower thermodynamic energy than C–Gbonding and it is possible that disruption of A–T bondsexerts less of an effect on the tertiary structure of the mt-tRNA than a similar disruption of a C–G bond. Conse-quently, disruption of A–T bonds could be ‘tolerated’ by the
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mt-tRNA without adverse functional sequelae, and par-ticularly so, if there were adjacent stabilizing influences inthe tertiary structure.
Concluding remarks
Our analysis of the published mt-tRNA mutationslisted on MITOMAP indicates that, at present, there isinsufficient evidence to classify a large proportion of thereported mutations as pathogenic. Furthermore, throughour analysis we have been able to arrive at the followingconclusions: (i) w73% of pathogenic mutations occur inthe stems of mt-tRNA secondary structure; (ii) ‘hotspots’for pathogenic mutations occur in both the acceptor andanticodon stems; (iii) the disruption of Watson–Crick basepairing is an almost universal feature of pathogenicmutations occurring in the stem structures; (iv) thedisruption of C-G base pairing is a significantly morecommon feature of pathogenic mutations than neutralvariants; and (v) when pathogenic mutations do occur inloop structures, they tend to occur in loops that areunusual in their size and/or influence on the tertiaryfolding or function of the mt-tRNA.
Acknowledgements
The research reported here was supported in part by the Wellcome Trust(D.M.T), The Newcastle upon Tyne Hospitals NHS Trust (R.W.T) and theNational Science Foundation (N.H). R.M. and J.L.E. are supported by anMRC (UK) Clinician Scientist Fellowship and an MRC (UK) Bioinfor-matics Training Fellowship, respectively.
Supplementary data
Supplementary data associated with this article can befound at doi:10.1016/j.tig.2004.09.014
References
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0168-9525/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tig.2004.09.014
Letters
The comparative method for evaluating fossilcalibration dates: a reply to Hedges and Kumar
Robert R. Reisz and Johannes Muller
Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Road N., Mississauga, ON L5L 1C6, Canada
Hedges and Kumar [1] (referred to as H&K hereafter)have responded to an article that we published recently inTrends in Genetics [2]. In that paper, we presented amodern perspective on what can and can not be inter-preted from the fossil record, particularly in view of theadded information that rigorous phylogenetic analyseshave provided for evaluating the nature of vertebratehistorical evidence. In the process of defending their use ofthe fossil record, they have made certain statements thatwe feel obligated to correct.
H&K state categorically in their abstract and in the textthat we have not provided robust fossil evidence for ourarguments. We disagree with these statements. We do notwant to reintroduce our evidence here but rather emphasizethat, in contrast to their research or the paleontologicalreview papers that they cite, the research group led byRobert Reisz has published extensively on the fossils thatare related to the bird–mammal split. Our arguments arebased on these primary research publications, and ourconclusion is that the bird–mammal split is not a scientifi-cally sound choice for a paleontologically based calibrationdate. None of the papers that they have cited, to counter ourarguments, address the quality of the fossil record for thiscalibration date or use comparative methods to determinewhich part of the fossil record is best suited for this type ofstudy. We proposed in our original article [2] a novel,comparative method for testing the quality of the fossilrecord for use as calibration dates. On that basis, ourargument that the fossil record of terrestrial communities,prior to the first appearance of amniotes, is grosslyinadequate for any scientific evaluation of the possibletime of the bird–mammal split still stands.
Contrary to H&K’s claim, we did not present alterna-tive calibrations dates as if they were novel, and we arewell aware of Benton’s excellent review paper [3] where hepresented minimum age estimates for 22 major events invertebrate evolutionary history. Instead, our novel, rigor-ous method for evaluating the quality of the fossil recordwas used to demonstrate that another major evolutionaryevent (i.e. the crocodile (or bird)–lizard split; Figure 2 inRef. [2]) is preferable as a calibration date to the bird–mammal split.
Contrary to their repeated use of the term in theirarticle, modern cladistics and phylogenetics can notassume the existence of transitional taxa. Unfortu-nately, the hypothetical phylogeny shown in H&K’srebuttal (Figure 2 in Ref. [1]) has no correspondence tothe fossil record, and no modern phylogeny incorporat-ing the fossil record possesses their so-called transi-tional forms (more correctly termed internodal taxa).The basics of this concept have been known fordecades [4], and the use of the term ‘transitionalforms’ should be avoided in this context. Our illus-tration (Figure 1 in Ref. [2]) corresponds to the widelyaccepted phylogeny and to the known temporal rangesof particular taxa as indicated by the fossil record.There are not only no ‘transitional forms’ in thisrepresentation of early amniote history but also thereare numerous, huge gaps that make the timing of thebird–mammal split difficult to assess.
Finally, it should be obvious that H&K’s argument thatthe mammal–bird split is preferable because there are notenough sequence data available for the taxa of crocodilesand lizards, which we propose as better alternatives, is adefense of past work and, therefore, should not be adeterrent for the future. Databases such as GenBank