what's old and new in molecular phylogenetics

AMERICAN JOURNAL OF PHYSICAL ANTHROPO1,OGY 85 207-219 119911

What’s Old and New in Molecular Phylogenetics JONATHAN MARKS Departments of Anthropology and Biology, Yale Uniuersity, New Haven, Connecticut 06520

KEY WORDS noidea

Genetics, Molecular evolution, Phylogeny, Homi-

ABSTRACT While it is fairly easy to devise a phylogenetic tree based on molecular data, it has proven difficult to tell how reliable any such tree is. Thus while the genetic inference that humans, chimpanzees, and gorillas cluster together is widely accepted, the genetic inference that the primary division among Old World human populations is between Asia and EurAfrica is not. A molecular phylogenetic inference linking humans and chimpanzees was pro- posed in the 1980s based on the technique of DNA hybridization. Despite several recent publications in primary and secondary source material, much confusion still exists surrounding the work. This paper tries t o clarify issues that may still be confusing t o physical anthropologists, and proposes criteria upon which to judge the robusticity of a phylogenetic inference based on DNA hybridization, in light of a recent published claim of replication. The claim of replication is considered critically. Interestingly, the original DNA hybridization data may actually show a chimp-gorilla link, in harmony with other phylogenetic results.

Genetic methods can provide an inde en-

concordant results; sometimes they do not. When genetic methods and anatomical methods provide disordant results, the data, analyses, and interpretations of each need to be critically examined to see why they conflict, and which, if either, is to be considered more trustworthy.

A quarter-century ago, molecular methods were not part of the ph logenetic main- stream. Goodman (1962) &owed that insofar as genetic similarity could be estimated, chimpanzee and gorilla clustered with human, not orang-utan. That inference appears to be well-founded (Marks, 1988). Concurrently, Cavalli-Sforza and Edwards (1965) found that aboriginal populations of the Old World clustered in an East-West orientation, with Europe and Africa segregating apart from Asia. Yet this conflicted with the tree the

clustered Euro e and Asia apart from Africa,

(19761.’

dent test of ph lo enetic h potheses P rom anatomical met Xf o s. Usual P y they provide

generated from anthropometric data, whic K also supporte (P craniometrically by Howells

~- ’The Rarnupcthecus controversy concerned molecular evolu-

tionary rates. not molecular phylogenetic inferences

What did a conflict between the genetic and anthropometric trees indicate? To Cav- alli-Sforza (1974: 87) it indicated that the anatomy was not tracking phylogeny, while the genes were.

lThe alnthropometric t ree . . . differs from the evolutionary tree. . . . This suggests that anthropometric characteristics are more affected by climate than genes are, so that the relations shown [by morphology I are due more to similar environments than to similar descents.

The morphologically based tree could not, it may be noted, even be called evolutionar ;

for the genetic tree. Indeed, this is simply one example of the

“semi-circular reasoning’’ noted by Simpson (1964: 1535): “agreement between [mole- cule-based phylogeny and others] has been taken as the re uisite validation of the molecular ap roa& to hylogeny, but non-

greater reliability of the molecular method.” Yet this particular phylogenetic conclusion has not stood up well, for other protein dis-

that adjective would be reserved exclusive 9 y

agreement K as been ta B en as evidence of the

Received December 20,1989; accepted November 14, 1990

@ 1991 WILEY -LISS, INC

208 J. MARKS

tance studies (e.g., Nei and Roychoudhury, 19811, as well as nuclear (Wainscoat et al., 1986) and mitochondria1 (Cann et al., 1987) DNA polymorphisms have shown a EurAsia versus Africa split (Cavalli-Sforza et al., 1988). We can note in retrospect that the phylogenetic inference based on a phenetic clustering of allele frequencies from serum proteins was fairly weak. But the assump- tion that any tree derived from genetic data is a priori better than any tree derived from any other data is a naive one, and indeed foreshadows later polemics over the issue of DNA hybridization.

The point of this prologue is simply that phylogenetic inferences based on genetic data are often hard to jud e. Though they

tered among other classes of data and analyses, genetic data have their own difficulties, which are of a different sort, and which are often technique-specific. Just how to distin- guish a weak ph logenetic inference from a

ses, is a critical and largely unresolved issue, which continually resurfaces. As Penny (1989: 305) writes in reference to phylogenetic reconstruction from DNA sequence data, “collecting sequences is the easy part of any study. The hard part is estimating the accuracy of the results.”

DNA HYBRIDIZATION IN HISTORICAL PERSPECTIVE

transcend many of the dif B iculties encoun-

strong one, base c r on genetic data and analy-

In the 1980s another apparent conflict between some genetic and anatomical data was raised, in the work of Sibley and Ahl- uist (1984twhich inferred a human-

&imp link, rather than a chimp-gorilla link. The technique was DNA hybridization, the results clear (different mean distances between human-chimp and human-gorilla or chimp-gorilla) and replicable (low standard deviations for any set of mean distances). The conclusions were widely reported in the secondary literature (Lewin, 1984; Pilbeam, 1984,1986; Diamond, 1988a).

William King Gregory had written in 1910 that two of the most important criteria in phylogenetic reconstruction are “to keep in touch with all data bearing on the subject,” and to “avoid explaining the little known throu h the less known.” Yet in 1984, DNA h bri ization was among the “less known” of

DNA Iybridization, however, did not take the eclectic a proach to phylogeny advocated by Gregory, ! ut adopted a confrontational,

p i? ylo enetic methods. The advocates of

indeed exclusionary, attitude to their genetic results that echoed the earlier dismissal of anthropometric relations of human groups. Thus Sibley and Ah1 uist (198713: 118) artic- ulated the view that NA hybridization was the best, if not the only, scientific method for inferring phylogeny:

If we are ever to reconstruct phylogeny, it must be done with methods that do not rely on the human eye as the instrument of comparison. It is self-de- luding to assume that what we see is all we need to know to reconstruct phylogenies. Such a procedure is subjective and qualitative, and results only in an opinion-and this is a definition of Art, not of Sci- ence. For a method to qualify as scientific, it must be objective and quantitative. (Sibley and Ahlquist, 1987b:118, emphasis in original.)

Two decades earlier, alternative methods of phylogenetic inference could not be labelled as evolutionary; now they could not even be science.

THE SIBLEY-AHLQUIST WORK

To evaluate the strength of the claims made on behalf of DNA hybridization, it is necessary to review some basic properties of the data. Plotting the denaturation of the hybrid DNA2 into single strands versus temperature yields a bell-shaped curve, which can be transformed into a cumulative sig- moid curve (Fig. 1). The goal of the analysis is to document and track shifts in the DNA peak, which would represent differences in the thermal stability of different hybrid DNA samples. But the roduct of the actual hybridization of the D&s of two s ecies is a

cules and labeled unhybridized DNA. When the thermal stability of the double-stranded molecules is subsequently measured, three options exist for interpreting the curve which comprises the result of any given experiment (Fig. 2). One can measure T,, the temperature correspondin to the higk est point on the curve (Browne B 1,1983; Bled- soe, 1987; Sarich et al., 1989). Alternatively, one can measure a median, the point at which 50% of the DNA has become single-

mixture of both labeled hybrid D Rr A mole-

- stranded.

But 508 of what DNA? One can take 50% ~~

2The hybrid DNA consists of one strand of radiolabelled DNA lacking the most redundant fractions of the genome (the “tracer”) and one strand of unlabelled DNA (the “driver”1. The driver is added in large excess to prevent renaturation of tracer with tracer. If tracer and driver are derived from the same species, the two-stranded hybrid is a homoduplex; if from different species, it is a heteroduplex. Any AT value is the reduction in melting temperature between a heteroduplex and a homoduplex made from the same tracer DNA.

MOLECULAR PHYLOGENY 209

(bount of S i n g l e Stranded A

DWA

Temperature

Cumulative Percent of

DNA si ng* ............................................... I J E

T a p s r a t u r e

Fi 1 Hypothetical melting profile for determining the tfermal stability of a sample of hybrid DNA, assum- ing complete hybridization. Above, the DNA remains double-stranded at low temperatures, denatures into single strands a t high temperatures, until the sample is fully denatured. The highest oint can be taken as the melting temperature. Below, tge same data presented as a cumulative or integrated curve. The 50% point can also be taken as the melting temperature.

of the DNA which formed hybrids, that is, excluding the unhybridized DNA from sub- sequent analysis. The result is T,, used by many workers (e ., Caccone et al., 1987; Bledsoe and Shelfon, 1989). Alternative1 one could take 50% of the DNA which c o d conceivably have formed h brids-that is,

(relative to the control) as part of the total. This measure is T50H, used by Sibley and Ahlquist but few others (e.g., Bonner et al., 1980).

The differences between these measures is critical. T,, measures only the thermal stability of t i e DNA, and is insensitive to anomalies in the shape of the meltin curve, as it only takes the highest point. 9, also measures only the thermal stability of the DNA, but can be affected by variations or anomalies in the shape of the melting curve (Fig. 3). These anomalies in the low-temperature com onent of the melting curve are

(i.e., serial1 homologous) DNA segments,

in that temperature range (Schmid and Marks, 1990). These poorly paired serial ho- molo s generally constitute 20-3096 of the

tions in the extent to which these paralogs have formed duplexes will affect the median (50% point), but not the mode. A difference in T, might therefore be attributable to rela-

including the amount of un II ybridized DNA

probably cp ue to the melting of paralogous

which woul K be poorly bonded, and denature

total a ybridized DNA in a reaction. Fluctua-

Si

Re1 a t i v e amount of

n g l e Stranded DWe ,.,

Temperature

Temperature

Fig. 2. Hypothetical melting profile for determining the thermal stability of a sample of hybrid DNA, assum- ing incomplete hybridization. Above, single stranded DNA is initially present in the denaturation experiment as a result of never having formed duplex structures. This does not affect the determination of Tmodt., but presents two options for the determination of a median value. Below, T,,,H is shifted to the left of T,, because T,,H takes into account the unhybridized DNA, and thus the curve begins with its lowest point already above zero.

tive differences in the sizes of these low- temperature com onents of the melting curve, and not to ifferences in the thermal stability of the main body of (orthologous) DNA at all (Fig. 4).

The measure used by Sibley and Ahlquist, T,,H, however, measures not simply the thermal stability of the hybrid DNA, but combines it with an estimate of the relative extent to which hybridization occurred. Thus, a difference in TSoH may be attributable to more hybrids being formed, rather than to the hybrids being more thermally stable. Further, it is also sensitive to those variations or anomalies in the shape of the curve (Fig. 4):

An anal sis of a fraction of the Sibley-

difference between the T5,Hs of human- chimp and human-gorilla DNA hybrids was attributable to a small mean difference in extent of hybridization, and not to a difference in the thermal stability of the hybrids formed (Marks et al., 1988). A difference in thermal stability was not apparent when either T, or Tmode was used.

The extent of hybridization is highly variable for any pair of taxa. For close relatives it does not discriminate, though for distant relatives it may give phylogenetic information. In the present situation, restricting their analysis to T50H, Sibley and Ahlquist

Ahlquist B ata showed that a small mean

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DNn

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T o d e

-x - T,,H Fig. 4. Differences among the three statistics dis-

cussed in the text, based on the kinds of phenomena encountered in the Sibley-Ahlquist data. T,, <,de measures only the position of the peak; T, measures the position of the peak, but also includes variation in shoulders and secondary peaks; T,,H includes all the phenomena of T, and variation in extent of hybridization as well.

had not analyzed their data comprehen- sively or adequately (Sarich et al., 1989).

In addition, however, Sibley and Ahlquist had covertly shifted a significant proportion of their T5,H values closer to the mean for any given pair of taxa (Fellman, 1988; Lewin, 198813; Horgan, 1989). This was found when a subset of data were compared to published values for the same experiments (Marks et al., 1988; Sibley and Ahl- quist, 1987a).

THE DATA ALTERATIONS These adjustments to the data were not

reported in the Sibley/Ahlquist papers; their magnitude was initial1 minimized (Sibley

concede that over 40% of the AT,,Hs reported in the 1987 paper were altered, and that the alterations may indeed have been the direct cause of the phylogenetic resolu- tion they claimed to have achieved.

The alteration procedures were inferred for specific experiments by Marks et al. (1988) and Sarich et al. (1989). Obviously statistical correction is often called for in science. But one needs objective criteria to determine which data to adjust, and how much to adjust them by. Adjusting only the experiments that gave undesirable results, and adjusting them by the amount that it would take to make the results desirable, would obviously be unacceptable as scientific practice.

Sibley et al. (1990) now list three kinds of adjustments to the data for the hominoids.

in Lewin, 1988b), but Si is ley et al. (1990) now

First, the “linear correction” involved the substitution of a homoduplex in a set of ex eriments, thus affecting all the AT5,H

they determined that the human-chimp and human-gorilla values were smaller than expected. Though the homoduplex melted at 85.4”, well within the range of melting temperatures for homoduplexes, they inferred on other grounds that the driver was imperfect (Sibley et al., 1990: 2151, and simply substituted the homodu lex from experi-

the net effect of adding 1.2 to each AT5,H value in the 1165 experiments. It is still unclear what the criteria for the choice of this value from this experiment were, as opposed to choosing other human-human homoduplexes from other experiments, which melted at other tem eratures. Yet in making this adjustment t K ey skipped the human-orangutan hybrid (1 16-14), which was already close to the mean value from other experiments for those two species, but then made the adjustment to the human-

‘bbon hybrid (1165-15; Marks et al., 1988; Rbley et al., 1990: 231, noteg).

Second, a “proportional correction,” apparently performed very often, involved the inference that a particular heteroduplex h - bridized too extensively, or not extensive 9 y enough, relative to the thermal stability of that heteroduplex (Sible et al., 1990: 232)- i.e., that ercent hybriJization was highly

lished this by plotting normalized ercent hybridization (NPH) versus therma P stability (T,,H) over a wide range of values, and obtaining a regression line. “The correction is made by moving the aberrant point to the linear regression, determining the new NPH value, and calculating the new T,,H value” (Sibley et al., 1990: 232). In other words, after insisting in the first place upon the use of T,,H, which incor orates the extent of

cent hybridization for over 40% of the hetero- duplexes, to bring the data points closer together. This obvious1 reduced the scatter

Thir c f , they now maintain that they made an adjustment for variation in driver len h in four of their hundreds of heterodup T ex experiments (Sibley et al., 1990: Table 7). Though variation in duplex len h can affect the melting temperature, an c f they cite a relationshi between T, (not T50H) and DNA lengtl! (Hall et al., 19801, it is not clear

va P ues of that set. In their experiment 1165,

ment 1154, which melte CQ at 86.6”, and had

variable P or a pair of species. They estab-

hybridization, they t l? en changed the per-

of data oints drastical 9 y.

212 J. MARKS

why only experiments 785A-10, 786-5, 786- 6, and 843-6 would require this adjustment (cf. below). It was “not precise” (1990:232), was “estimated” (1990: 231, note e ) , and involved “relying. . . on our experience to detect and correct for seriously aberrant samples” (1990: 232); yet it was used to reduce the ATSOH values of four human- chimp h brids from at least 2.5,2.6,2.7, and

value), to 1.5, 1.5, 1.6, and 1.7. Odgy, Ahl- quist (pers. comm., 3/21/88) had previously explained the alteration of 843-6 to me as a proportional correction for too little hybridization.

The ap lication of these data alterations

ley et al. (1990), to have been very inconsis- tent. Experiment 785A involved an alteration due t o the inference, based on the position of the chimp-human melting curve, “that the [human] driver was faulty, apparently short-stranded (p. 203). Regardless of whether the mean length of the driver was in fact measured and a correction derived from it, “the same faulty [human] driver used in Exp. 785A was also used in Exp. 785B” (p. 204)-but no adjustment was deemed necessary. Their Table 7 shows that the adjustment to 785A brought it into harmony with their phylogenetic conclusion, by lowering the value of a chimp-human experiment, while the lack of adjustment to 785B also brought that into harmony by not lowering the value of a gorilla-human experiment. Interestingiy, taken at face value, both 785A and 785B show chimp-gorilla to be consider- ably more thermally stable than chimp- human and orilla-human, as does 786.

Sibley an f Ahlquist did not alter experiments 790B and 828, though they now judge them have been “Poor” (p. 205bwhile they did alter experiments 1152 and 1164, though they judge them to have been “Good” (p. 21 1). For experiment 844, the four human-chim “curves were aberrant and were omitte8 from their Figure 11 ( . 2071, but were nev-

of the four having been changed. In experiments 864 and 869, where hu-

man, chimp, and gorilla all hybridized up- wards of 9596, no changes were made for percent hybridization. Yet Sibley and Ahl- quist did change the results for experiments 1163 and 1152 on that basis, even though they a pear to have had the same amount of hybri8zation. They did not change the values in experiments 1151 and 1154, in which

2.7 (we P 1 above the human-chim mean

appears, P rom the information given in Sib-

ertheless included in t Fl eir analysis, only one

hybridization was about 90-95%, but did single out 847-4 for change, which appears to have hybridized equally as well, from their Figure 12. They also chose not to adjust chimp-gorilla hybrid 849-4, though it only hybridized about SO%, and therefore showed a relatively large AT,,H between the two species. They chose not to adjust the chimp- gorilla hybrid 848-4, though it hybridized about 10% less than the corres onding

note that it specifically “was imperfect as shown by its wide separation . . .” (p. 208). In both 848 and 849, the less extensive hybridization of chimp-gorilla relative to chimp- human caused an exa geration of a difference in AT,,H; accorfing to Sibley et al. (1990) Table 1, their ATmodes are very similar, and for 848, the chimp- orilla distance is

Indeed one probably needs to judge these ad‘ustments principally in terms of their

the average hybridization was over 95% for both human and chimpanzee, and the experiment is judged to be ‘ Good ” by Sibley et al. (1990). Nevertheless, according to their Ta- ble 7, they altered 21 of the 24 heterodu-

lexes for aberrant percent hybridization. k he effect of these corrections was to inflate the mean and curtail the scatter. Thus, the mean and standard deviation of the 12 gorilla-human melts went from 2.1 _t 0.6 before alteration, to 2.4 ? 0.2 after; the 12 gorilla- chimp melts went from 2.0 * 0.5 to 2.3 i 0.2. For experiment 1152, also judged “Good” and

over 95% hybridization the scatter was only affected the

means. The 11 chimp-human melts changed from 1.3 2 0.2 to 1.6 i- 0.2, but the 12 chimp-gorilla experiments jumped from 1.5 ? 0.1 to 2.3 ? 0.2.3 Clearly the alterations brought this experiment into accord with the hylogenetic position being advocated. One

mean unadjusted AT5,H for experiment 1152 is probably attributable to the shape of the melting curve, as the ATmodes show no such difference.

Most significantly, however, their Figure 40, adduced to show a correlation between extent of hybridization and melting temper-

chimp-human hybrid, and though t hp ey now

actually smaller than the c a imp-human.

ef f ! ects. For experiment 1153 (Gorilla tracer),

f. inds as well that the small difference in

_ _ “The increase in the standard deviation is actually only from

0.14 to 0.17.


ature, shows clearly no correlation at all in the range of interest for human-chimp-gorilla--85-100% hybridization! There is conse- quently no justification whatsoever for even judgmg values in that range to be aberrant, much less for altering them on such a basis.

It appears that whatever criteria they used were independent of the quality of the experiments, were applied in a largely ad hoc manner, and most importantly, were applied covertly. These unreported changes to the AT,,H measurements had the effect of dras- tically reducing the standard deviations apparent to statisticians (e.g., Felsenstein, 1987), and of renderin the specific values

stantially inaccurate. The resulting small standard deviations

associated with each mean distance between taxa can hardly be sur rising, given the

tivity of the data alterations. The fact t at they were concealed from reviewers and readers raises a number of possible explana- tory scenarios, of which the most charitable invokes extreme scientific naivete combined with statistical naivete combined with acci- dental omission of the vital information.

There are thus two sets of problems: first, the incomplete analysis, which can be ar-

ed about; second, the covert data manipu-

While reviewers could not be expected to have known about the data problems, they probably should have detected and/or noted the analytical problems. The acknowledg- ment of secret data tampering leads to the conclusion that the two papers on hominoids by Sibley and Ahlquist were accepted and

ublished by The Journal of Molecular Evo- rution under false pretenses; that is, having misled the reviewers and readers (whatever the explanation) about the nature and quality of the data. Thus, we now know that Sibley and Ah1 uist's main conclusions (that human-chimp 8NA is more thermally stable than human-gorilla or chimp-gorilla DNA, and that this result is highly replicable) are at best gross exa gerations; at worst, simple

emerge to be, the results are useless to physical anthropologists.

reported by Sibley and hl lquist (1987a) sub-

overall directionality an B apparent sub'ec- h

K" ations, which are harder to argue about.

falsehoods. Whic fl ever they may ultimately

the conc P usions are less significant than the

THEBURDENOFPROOF Clear1 there is an instructive lesson here:

methodologies. One needs to know where the data came from, how they were collected,

and how they were inter reted. To rely on

ondary sources focus on the conclusions and not on the methods; and to focus on the issue of statistical significance in the absence of an understanding of the mechanics of the experiment is inade uate as well. The substance of a DNA hybri ! ization experiment lies in its melting curves, and without examining them, one can do little to evaluate the strength of a phylogenetic claim.

This is not, however, to say that DNA hybridization does not or cannot give phylogenetic information on the base-pair mismatch of DNA samples. If one finds a difference between ATBOHs, which measure a) thermal stability of well-paired orthologous duplexes, b) poor1 paired paralogous duplexes melting at ow tem eratures, and c)

and fails to find a difference between AT,,+, which measure just a and b, then it stands to reason that those differences in ATSOH are attributable to differences in c, the extent of hybridization. Likewise, if one finds a difference between AT,s, which measure a and b, but fails to find a difference between ATmodes, which measure only a, the thermal stability of well-paired orthologous duplexes, then it stands to reason that the differences exist in the low-temperature parts of the curves, corresponding to the paralogs.

Comprehending the source of the difference between two DNA melts, or set of DNA melts, may be facilitated by reference to another generation's controversy in our field, IQ. Though the difference in IQ between two people (or groups) may have a genetic component, there are other causes contributin to any difference in I& as well- such as up ringin , family size, nutrition,

and ex erimental controls.

of an average untreated PKU patient and an average lawyer can be attributed largely to genetics, the small difference in IQs between two lawyers is more likely to have another set of explanations. The burden of proof falls heavily upon any investigator claiming that small I& differences are attributable to different genetic backgrounds, and not to other causes.

The burden of proof likewise falls on the DNA hybridizer to demonstrate that a small difference between human-chimp and chimp- gorilla is attributable to a shift in the posi-

secondary sources is ina (P equate, since sec-

the extent to which L F hybri ization occurred;

etc. This is the 9 prob em of multiple causation

Whi f e the large difference between the IQs

214 J. MARKS

tion of the peak of the meltin curve. Sibley

this, and apparently are unable to do this, since whatever differences between human- chimp and chimp-gorilla median melting temperatures exist in their work are apparently due to a combination of 1) amount of hybrid formed; 2) low-temperature variations in the curves; and 3) data tampering.

In order to be convincing, a DNA hybridization study will have to do three things for its readers. First, it will have to show sample melting curves, to show the readers the en- era1 uality of the experimental results. !his

the Sibley-Ahlquist work. Second, it will have to analyze modes in addition to medians, to show that differences in medians are attributable to shifts in the location of the main peak, and not to average differences in the shape of the melting curves. Third, adjustments to the data will have to be discussed and fully justified; and discrimination among taxa should probably not be largely based on such adjustments.

and Ahlquist (1984, 1987a) a ave not done

was R t e crux of the original argument about

REPLICATION AND CONCORDANCE? Almost immediately upon the heels of the

revelations concernin the Sibley/Ahlquist work, a colleague of Si tis ley’s, Jeffrey Powell, claimed to have obtained the same results, using a different method of DNA hybridization. Like Sible ’s, these conclusions were

cation. In their context (Fellman, 1988; Lewin, 1988b; Diamond, 198813; Lowenstein and Zihlman, 1988; Britten, 19891, the invo- cations have been made in “defense” of the Sibley-Ahlquist work. Most ex licitly, Lewin

char es have been brought against Sibley and wh lquist, their work has passed the acid test of science.” The “acid test” may be con- vincin , but we should recall that it was also passef by the observation of 48 chromosomes in human cells (Kottler, 19741, poly- merized water (Franks, 1980), cold fusion, canals on Mars, and Piltdown Man.

This work has now been published (Cac- cone and Powell, 1989), and therefore merits attention. The rincipal claim is that the authors have o r3 tained virtually the same mean distances as Sibley and Ahlquist. One im lication of this match, since the match is

alterations were proper. But more importantly, the match “increases the confidence

widely cited an a” invoked even before publi-

(1988b: 1759) quoted Powe f 1: “Whatever

on P y to the altered numbers, is that the

in the technique itself independent studies of the same taxa yield nearly identical results” (Caccone and Powell, 1989: 936).

Yet where Sibley and Ahlquist (1984, 1987a) measured T Caccone and Powell (1989) measured fm. These, as discussed above, are different scales. They are related, as are centimeters and inches, but they are not the same scale. One includes extent of hybridization, the other does not (see Cac- cone et al., 1988a). The only way the T, and TSoH distances would be expected to match is if the average extent of hybridization for any pair of taxa in the Sibley data were exactly 100%; and the relative distances among human, chimp, and gorilla should only match if there were no average difference between pairs of taxa in extent of hybridization. Nei- ther of these conditions is known to hold, and the evidence that has been made available su ests that actually neither condition hoffs4 (Marks et al., 1988). The claimed match is therefore spurious, and the chain of reasoning that stems from the match is spurious as well. Indeed, Sibley et al. (1990) have now converted their unaltered data to Tn,s, and enabled the calculation of mean T,’s for the pairs of taxa. These, as would be expected, do not match the Caccone-Powell numbers (Table 1).

The Caccone-Powell aper argues, in ef-

because 2) Sibley and Ahlquist’s hominoid work is intact, because 3) humans and chimps are sister grou s, because the num-

seen) is spurious, this chain of reasoning hangs from a false premise. Any of its conclusions may of course be true, but their truth must be assessed on other grounds.

1. It is true that DNA hybridization is in some sense untainted; Sibley and Ahlquist’s abuse of the technique discredited only that particular application, not the technique itself. All parties agree that the technique has some value in phylogenetic determina- tions-though there are limitations to DNA hybridization as a measure of genetic dis-

fect, that 1) DNA hybri LQ ization is untainted,

bers match. But since t K e match (as we have

‘For the 15 human-chimp experiments we analyzed. mean and standard deviation of the normalized percent hybridization was 96.1 z 2.4; far the 20 human-gorilla experiments it was 95.2 2 1.6. This created a small difference in mean 1T5,H’s that did not inhere in the mean 1T,,s or IT,,,,,,,.s; and unless the heteroduplex hybridizes more extensively than the homoduplex, a lT,c,H should be somewhat larger than a ATml for the same experiment.


TABLE I . Three sets of mean melting temperatures for the “trichotomv”‘

S/A ATsoH C/P AT, S/C/A AT, (1987) (1989) (1990)

Hu-Ch 1.6 i .2 1.59 k .16 1.4 f .8 Ch-Go 2.3 * .2 2.55 * 2 4 1.7 * .4 Hu-Go 2.3 f .2 2.51 i .10 1.8 + .8

‘The first column represents published AT5oH values from Sibley and Ahlquist (1987); the second column represents mean AT,,, values calculated from the appendix to Caccone and Powell (1989); the third column represents mean ATTm values calculated from Table 1 ofSibleyetal.(199O).CacconeandPowell’s(1989)published means and s tandard deviations (second column) match those of Sibley and Ahlquist well (1987; first column); their numbers, however, are more comparable to those given in the third column, and do not match well. The discrepancy between the first and third columns is due to inclusion of percent hybridization, a n d to significant alterations, in the derivation of values in the first column. Hu, human; Ch, chimpanzee; Go gorilla

tances, and to genetic distances as indicators of phylogeny.

2. However, Sibley and Ahlquist’s work is not intact; their methods of data alteration are generally unacceptable as scientific practice, certainly undermine their statistical treatment, were withheld from readers and reviewers, and were only discovered by a highly fortuitous series of circumstances.

3. Humans and chimpanzees may be sister taxa, though there is little presently on which to base that inference. DNA sequences have large amounts of homoplasy; and though some analytical models favor a human-chimp clade from the available se- uence (Holmquist et al., 1988; Williams and 6 oodman, 1989), others find it statistically

unresolvable (Hasegawa et al., 1989; Holmes et al., 1989); and other genetic traits and analyses have linked human-gorilla (e.g., Miller, 1977; Saitou, 1988; Ueda et al., 1985) or chimp-gorilla (e. ., Hixson and Brown, 1986; Bianchi et a f , 1985). Caccone and Powell (1989), it should be noted, have cited the relevant literature very selectively, which ives the appearance in this case of a concor 3 ance of genetic data for human- chimp.

Caccone and Powell (1989) indeed go to considerable lengths to persuade the scientific community of such a enetic concor-

(except for chimp-gorilla and human-gorilla) with the direct estimates of sequence divergence obtained in the pseudo-eta gene re ion

agreement,” they write, “is obtained if one assumes that AT, is converted to percentage

dance. For exam le, they s a ow that their AT,s for pairs o F taxa match very closely

by Miyamoto et al. (1987). “Good abso 7 Ute

base-pair mismatch in a 1: l manner” (Cac- cone and Powell 1989: 937). Yet they fail to cite their own work here, which showed a conversion of 1:1.7 (Caccone et al., 1988b)- and which would undermine the “good abso- lute agreement” noted, if the reader is suffi- ciently familiar with the literature. Again, this highly discriminatory analysis makes a genetic concordance appear much stronger than it should, and when combined with the selective literature citations and the spurious match of AT, and AT5,-,H, makes a super- ficially persuasive case for a genetic concordance exculpating Sibley and Ahlquist (whose work may be then taken as, at worst, overzealous prophecy), and also ma help

from DNA hybridization. Britten (1989) has likewise argued that

the existence of other studies with genetic data linking human and chim (though

human and chim 1 weighs in favor of accept- ing the Sibley-Ahyquist work, despite what is now known about it. But a human-chimp link bears not a t all on the competency or honesty of the Sibley-Ah1 uist work; the

sets of data, and the Sibley/Ahlquist work will have to be judged on its own merits. The specific branching sequence within the hominoids is an interesting, but ultimately mi- nor, issue; the more important issue is what data and analyses can be used by physical anthropologists and biologists to infer phylogenies, and how reliable the results are. Indeed, the Sible -Ahlquist avian work, con-

probably compromised as well (Cracraft, 1985; Houde, 1987; Prum, 1988; Sarich et al., 19891, though some of their conclusions may someday turn out to have been correct.

Caccone and Powell’s data appear to be the strongest set of data a t resent in support of

convincing, however, we have to assume that the quality of the scientific work is incom- mensurate with the poor quality of the rea- sonin . Is it?

presently, since of the three criteria listed above for the satisfactor demonstration of

Caccone and Powell (1989) paper satisfies none. First, no melting curves are shown; second, no modes are analyzed. Third, undue

buoy confidence in any other results 6” erived

again failing to cite those that 2 o not link

phylogeny will have to stan 1 or fall on other

stituting the bu i k of their publications, is

a human-chimp clade. P n order for it to be

Un B ortunately, it is not possible to tell

conclusions based on DN i hybridization, the

216 J. MARKS

phylo enetic wei ht is given to an explicit

based on estimates of their DNA fragment length to the nearest single nucleotide, after sonication, labelling, incubation, and en- zyme digestion. Such a degree of precision is counterintuitive, and reliance on this fragment-length estimate for the ph logenetic

able. The strongest claim that can be made for

the Caccone-Powell paper is that it may afford genetic distances which associate human and ~himpanzee.~ How strong a phylogenetic conclusion may be drawn from distance data is, of course, a lon -standing

cent analyses of the eta-globin DNA sequence (Hasegawa et al., 1989; Holmes et al., 1989; EGshino and Hasegawa, 1989) the X- chromosome pseudoautosomal region (Ellis et al., 1990), and of the involucrin DNA se uence (Djian and Green, 1989) strongly

Powell (1989: 938) “that the problem of the genetic relatedness of the hominoids has been largely solved [in favor of human- chimp]” is premature, if not false.

The conclusions of Caccone and Powell (1989) thus remain anomalous, particularly in light of their claim of numerical concordance to the mean ublished ATSOH values of

these conclusions may reflect upon Sibley and Ahlquist (1987) they are false; insofar as they may reflect upon the phylogeny of the hominoids they are insufficiently documented. If indeed this techni ue permits the

entl tainted series of numbers, then one mi t legitimately question whether the tec nique is actually measuring something biologically real, or ma be, rather, simply an efficient enerator o f t e results being antic-

trans B ormation o P the melting temperatures,

discrimination is therefore probab i y inadvis-

contentious issue in systematics. B ndeed, re-

in 1 icate that the conclusion of Caccone and

the Sibley and A g lquist study. Insofar as

replication of a non-compara B le and appar-

K ipated, li a e the Clever Hans effect.6

Lewin, 1989) discuss the Sib f! ey and Ahlquist

6 WHERE WE STAND

Several recent books, (e. ., Willis, 1989;

study, and properly mention controversy ~ _ _ ~ - ‘Curiously, the Caccone-Powell mean distances do not show the

branching of the gorilla as even being particularly close to the human-chimp divergence. This is notably discordant from other genetic data, particularly phenetic data, on this problem (Marks et al., 1988: 784).

‘Lewin (1988b:1758) quoted Powell in defense of Sibley and Ahlquist’s extraordinary data alterations: “You can argue that this isn’t being objective. but we all make judgment calls.’’

surrounding the interpretation of the data. While this is true, it is only half the story. The other half is that the data-the AT,,H’s-were covertly altered for presenta- tion, resulting in a much lower scatter, and wider mean differences, than the data di- rectly indicate. This has been documented and discussed at length in two pa ers

therefore insufficient to characterize the dispute over the Sibley-Ahlquist work as merely one of interpretation.

In most other fields of scientific inquiry, the alteration of data, shielded by the misre- porting of analytical methods, shielded by the withholding of the data, would not be tolerated. In DNA hybridization, however, the significance of these issues has been minimized by some ractitioners (R. Britten

Lewin, 1988a, 1988133. If this is normal in DNA hybridization, it is oor scientific prac-

it is up to the other practitioners to repudiate it. To date, they have been reluctant to do so. If they fail to repudiate it, readers have no way of knowing whether any or all other studies are doing the same to their data. But it is up to those other practitioners to assure the evolutionary biology and anthro ology

tion, and not representative of DNA hybridization as a whole.

There is clearly a problem in the molecular evolutionary literature concerning the strin- ency of review of papers based on DNA

kybridization (Marks et al., 1989). To outsid- ers, there are simply no available criteria for distinguishing good DNA hybridization work from poor DNA hybridization work. And a broad editorial lapse has made it impossible for interested readers to distin- guish one category from the other. Conse- quently, the researchers who are doing the good work need to be encouraged to prove it. We require criteria for a stron phylogenetic inference based on DNA hybri 5 ization: if the conclusions are solid, researchers must be prepared to show a skeptic how solid they are. This is hardly an extravagant appeal in science.

As discussed above, there are three simple criteria that can be used to judge whether

(Marks et al., 1988; Sarich et al., 1989). P t is

and J. Powell in F ellman, 1988; and in

tice; if it is abnormal in r; NA hybridization,

communities that this example is an a \ erra-

should be attributable to shifts in the peak of the melting curve, rather than to changes in


the shape of the melting curve: comparin Trdes to T,s accomplishes this. And t h i r t a justments to the data should be carried out blindly (i.e., without knowing which taxa are involved) and justified. The greater the reliance upon them for the placement of taxa, the more carefully they must be scruti- nized.

The issue of ATmpde, the measure of relative thermal stability least adulterated by the shape of the melting curve or the extent of hybridization, deserves the final word. Had Sibley and Ahlquist analyzed their data comprehensive1 , or been compelled b The

their mean ATmodes would have been available to the anthropological community long before now. These values have finally now been published (Sibley et al., 19901, and are gwen in Table 2.

Looking at these data, one can note immediately that all the means involving human as tracer DNA are extraordinarily low. Each mean distance using human as tracer DNA is about half the value of its reciprocal; e.g., human-chimp is about half of chimp-human. Indeed, the two lowest values given in the table are not for human-chimp and chimp- human, but for human-chimp and human- gorilla. And the human-orang value is less than half the orang-human (reciprocal) value, and much smaller than all the other values involving orang DNA. Not having seen the melting curves themselves, an hy- pothesis that readily comes to mind is that there is a technical roblem with some or all

a tracer. Let us, therefore, segregate the numbers

derived from experiments using human tracer DNA, and compare the ones that did not use it. From Table 2, it should be readily a parent that chimp-gorilla and gorilla-

and are at east as thermally stable as chimp-human. This would suggest an unresolved genetic trichotomy with, interestingly, chimp and gorilla being slightly, though most likely insignificantly, more ~ i m i l a r . ~ This interpretation would be in

Journal of Mo P ecular Evolution to $0 so,

of the experiments t R at used human DNA as

P c hp imp are ver concordant with one another,

.~ ~

’Independently, Krajewski and Dickerman (1990) performed a bootstrap analysis of Sibley and Ahlquist’s data excluding those identified as “problematic” and including some unpublished hybrids involving the orang-utan. They concluded that these AT s indeed never did indicate human-chimp, and most likely in%- cated chimp-gorilla. This again leaves Caccone and Powell in the awkward position of having derived a strong concordance only to the results they mistakenly believed Sibley and Ahlquist to have obtained.

TABLE 2. .ITn,,,du values given by Sibley et al. (1990)‘

Ch-Go 1.81 Ch-Hu 1.90 Go-Hu 2.11 C h - O r 4.95 Go-Ch 1.86 Hu-Ch 0.97 Hu-Go 1.29 Or-Ch 4.31

Go-Or 3.56 Or-Go 4.39 O r - H u 4.76 HiJ-Or 2 30

‘Hu, human; Ch, chimpanzee; Go, gorilla; and Or, orang-utan. Tracer DNA is the left of each pair. Values with humanas tracer are about half the magnitude of the values of reciprocal experiments; and both chimp-gorilla and gorilla-chimp are a t least as thermally stable as chimp-human.

considerable harmony with a great mass of other data on hominoid phylogeny (Ciochon, 1983; Andrews and Martin, 1987).

CONCLUSIONS

Demonstrating a phylogenetic connection between humans and chimpanzees (exclusive of gorillas) would have implications for various aspects of physical anthropology, including rates of evolutionary change, modelling the evolution of humans (from a chimp- like or non-chimp-like ancestor), modelling the evolution of bipedalism (from a knuckle- walking or non-knuckle-walking ancestor), and general methods of phylogenetic reconstruction. Indeed, if true, it would be one of the most significant revisions to physical anthropological theory in the 20th century.

Yet, contrary to strong claims in the pri- mar literature, and reports in the secondary 8terature, the present DNA hybridization evidence does not clearly demonstrate such a linkage. It is an axiom of science that extraordinar claims require extraordinary

DNA h bridization evidence has not even met orJinary standards. If and when the derivation and analysis of these distances are made explicit, we will then be in a position to debate their phylogenetic significance.

Apparently the human-chimp-gorilla split is genetically so close a call that it is difficult to render a decision even when all the data being considered are reliable. The preemp- tive closure of the problem b advocates of

far more questions than it would resolve, and the generation of any consensus on the issue will require careful examination of the basis for the conclusions of each study. It is difficult to imagine there ever being a substitute for critical thought in science; and it does not look presently as if DNA hybridization can

standards o i! documentation; to date, the

DNA hybridization’s depen dr ability raises

218 J. MARKS

form part of whatever consensus ultimately emerges.

It is important to bear in mind, however, that if it was actually a three-way split, or if there is a real conceptual gap between molecular data and phylo eny (in terms of what patterns are discerni 8 le, understanding how they come about, and knowing how to treat them to reconstruct every divergence reli- ably), then the “trichotomy” may actually be unresolvable. And it is not difficult to imagine that in the foreseeable future, additional genetic sets of data will continue to emerge linking humans, chimps, and gorillas, in various pairwise combinations. Each will have to be evaluated on its own merits.

The methods of phylogenetic reconstruction are diverse, and genetic methods are powerful; but they augment, rather than supersede, other methods. This is a t least as true now as it was at the dawn of the molecular era. The apes and humans provide a challenge to those who wish to relate organ- ismal and molecular evolution to one another, the rationale for which is “the balanc- ing of points of view and the achievement of more complete explanations” (Simpson, 1964: 1535). But it remains to be seen what balance will ultimately be achieved.

ACKNOWLEDGMENTS

I thank Clifford Brunk and Everett Olson for inviting me to express my views at the CSEOL Conference on DNA-DNA Hybrid- ization and Evolution, May 11-14, 1989, which are the ultimate basis for this paper. The written version of this paper benefited from comments received on the oral version from Francisco Ayala, Tony Bledsoe, and Fred Sheldon. The written version benefit- ted from the comments of Andrew Hill, Rob DeSalle, Ken Korey, Matt Cartmill, and anonymous reviewers. Much of what in- sights I have gotten into DNA hybridization I owe to paying attention to Carl Schmid and Vince Sarich. This manuscri t was prepared

ant P rom the Social Science Research F und, Yale University.

with su port from NSF BN f -8819047 and a

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