Anthropoid affinities of Tarsius supported by lens αA-crystallin sequences

Wilfried W. de Jong”

Anthropoid affinities of Tarsius supported by lens ccA-crystallin sequences

Department ofBiochemistry University of Nijmegen, P.O. Box 9101, 6500 HB Nijmqen, The Netherlands

The amino acid sequences of the eye lens protein ori\-crystallin of Tursius sytichta and Aotus triuirgatus have been determined and compared with those of 3 lemuriform and 2 catharrhine primates and 40 other mammalian species. The Aotus olA sequence is clearly grouped with those of rhesus monkey and human on the basis of 3 shared derived amino acid replacements. Tarsier CYAhas one residue, 146 Ile, in common with the Anthropoidea, where the Lemuriformes have 146 Val. Ancestral sequence reconstructions revealed that 146 Val must have been the ancestral residue in primates, indicating that the replacement 146 VakIle is a shared derived character supporting the anthropoid affinities of Tarsius. While the tarsier QUAchain has apparently not changed during the 50 million years since its divergence from the anthropoid stem, the CYA chains of the Anthropoidea have evolved at an increased rate and with a directional trend towards replacing serines by threonines. This suggests positive selection for advantageous replacements in the evolution of orA-crystallin in higher primates.

Morris Goodman Department of Anatomy, Wayne State UniversiQ School of Medicine, Detroit, MI 48201, U.S.A. Received 6 January 1988 Revision received 17 May 1988 and accepted 25 May 1988 Publication

date September

1988

Keywords: Tarsius, crystallins, lens proteins, Haplorhini, molecular evolution, primate phylogeny, Aotus.

eye

Journal of Human Evolution (1988) 17, 575-582

Introduction The phylogenetic

position

issue for many decades. the other prosimian humans).

Some

of the tarsiers

primates

authors

(lemurs and lorises) and the anthropoids

considered

1959; Gingerich,

an independent

origin for Tarsius(Romer,

accumulating

Anthropoidea Gebo,

(Luckett

1986; Aiello,

accepted

1981; Schwartz

& Tattersall,

1966; Schwartz,

1978; Rosenberger

1986; Pollock & Mullin, into Strepsirhini

and this arrangement (Martin,

position between

(monkeys,

1945;

1987) and others favoured

1984; Mai, 1985), but evidence

1985).

necessarily

implies a close phylogenetic

1979; Cartmill

increasingly,

but certainly

about the relationships

by the fact that classification

& Sarich,

(tarsiers and

not universally, of Tursiusmay

into the same category

not

relationship.

Comparative biochemical data have generally tended to support division, but rather marginally (Goodman et al., 1978), and contradictory have been given (Cronin

the

et al., 1981;

1987). Pocock (19 18) has already suggested a

In fact, the discussions

have been troubled

& Szalay,

(lemurs and lorises) and Haplorhini

is indeed

sometimes

therefore

apes and

(Simpson,

that tarsiers are the closest relatives of the simian primates, & Szalay,

division of the primates simians),

has been a disputed

them to be closer to the Prosimii

Le Gros Clark, is gradually

among the other primates

In many respects tarsiers occupy an intermediate

1980; Baba et al., 1982). Additional

the haplorhine interpretations

molecular

data are

still highly desirable.

Comparative amino acid sequence analyses of the eye lens protein aA-crystallin have previously provided useful information about mammalian phylogeny (de Jong & Goodman, abundant

1982; Miyamoto component

& Goodman,

of the mammalian

1986; McKenna,

1987).

aA-Crystallin

eye lens, making up approximately

is an

20% of the

total protein, and has a length of 173 amino acid residues (Bloemendal, 1981). Previous studies of aA-crystallin included the primate species man, rhesus monkey, brown lemur, potto and galago

(de Jong

* To whom correspondence 0047~2484/88/050575

et al., 1984) and the observed

and requests

+ 08 $03.00/O

for reprints

amino acid replacements

clearly

should be addressed. fQ 1988 Academic

Press Limited

576

W. W.

distinguished

the anthropoid

analyse the aA sequence thereby the significance

DE JONG

AND

and prosimian

of a tarsier.

M.

branches.

To increase

of the findings,

GOODMAN

It therefore

seemed promising

the density of the primate

we also studied the CXAsequence

to

data set, and

of the New World

monkey Aotus piuirgatus.

Materials

and methods Turks

Two eye lenses (O-41 g wet weight each) of an adult tarsier, on dry ice from the Duke University of the University

a-Crystallin lens

and

chromatography urea. These

the

over Ultrogel

aA-crystallin

subunits

over carboxymethylcellulose

and all further

chains have been described were separated

chromatography.

yichta,

were obtained

N.C. Five lenses (0,30 g Animal

procedures

obtained CM-52)

for the structural

in detail elsewhere

by high-voltage

The “core” peptides,

AcA-34 of the total water-soluble

were

(Whatman (de Jong

The isolated aA chains were S-P-aminoethylated peptides

Durham,

of Nijmegen.

was isolated by gel filtration

proteins,

Center,

Aotus trivirgatus, were provided by the Central

each) from three adult owl monkeys, Facilities

Primate

analysis

ion-exchange of 7M

of the aA-crystallin

et al., 1984).

and digested with trypsin. The soluble

paper electrophoresis insoluble

by

in the presence

at pH 6.5 and descending

at pH 6.5, were separated

by gel filtration

Larger peptides were subdigested with over Sephadex GSO-sf in 0.1 M ammonia. thermolysin. Amino acid analyses were performed on all peptides, and the compositions were compared

with the corresponding

bovine and hamster

the amino acid replacements

peptides of the completely

determined

sequences

of

(de Jong et al., 1984; van den Heuvel et al., 1985) to infer

olA-crystallin

in the primate sequences.

Amino acid compositions

were only

used when values per residue did not deviate more than 20% from integral values, except for threonine, accepted.

serine and tyrosine,

The presence

from ultraviolet

where up to 30% loss due to hydrolytic

of the single tryptophan

fluorescence

of the N-terminal

in the investigated

destruction

was

chains was inferred

tryptic peptide on the peptide map.

Results and discussion Figure

1 summarizes

the evidence

that led to the proposed

chain of Tarsius and Aotus. The phylogenetically

aA-crystallin

amino acid sequences informative

positions

of the of the

oA sequences of these two primates are aligned in Figure 2 with the corresponding positions in the aA chains of the 45 previously published mammalian species, representing 16 Eutherian

orders.

replacements

in the primate

variability

among

This alignment

allows for an objective

EA-crystallin

mammalian

aA chains.

sequences, The

comparison

of the observed

in the light of the prevalent

alignment

reveals

sequence

that the primate

aA

sequences do not have a single synapomorphous replacement which would distinguish them as a group from the other mammals. This lack of resolving power of the aA sequences at the ordinal level is not surprising since tllA is a slowly evolving protein, accumulating replacements at an average rate of three per 100 residues in 100 million years (de Jong et al., 1984), and the radiation of the placental mammalian orders has been completed in a relatively short period of time, between 80 and 50 million years ago (Novacek, 1982). Fortunately, several amino acid replacements have occurred in aA-crystallin in the different primate lineages, permitting a clear distinction between a clade composed of lemur, potto and galago on one hand and the simian primates on the other (Figure 2). The former are characterized by the unique residue 13 Pro and the replacement 61 Ile -+ Val,

._____..____

_____

_____ -

Thr

_-...___---.

________..

--------.

----.....

____ -

______

---

----

____ _________

-Gin ___________ __----

__________

.___

-+--*..>-..

___

_____

__

T$;Thr

---.___.._______________--

-__

Ile-Thr _________ --*-_)+ -x-+-z

Thr-

150 160 -Lys-Val-Gln-Ser-Gly-Leu-Asp-Ala-Gly-His-Ser-Glu-Arg-Ala-Ile-Pro-Val-Ser-Arg-Glu-Glu-Lys-Pro-Ser-Ser-Ala-Pro-Ser-Ser-OH Ile --. .________ _____________________

_____

____________

---.______

120 130 -Arg-Arg-Tyr-Arg-Leu-Pro-Ser-Asn-Val-Asp-Gln-Ser-Ala-Leu-Ser-Cys-Ser-Leu-Ser-Ala-Asp-Gly-Met-Leu-Thr-Phe-Ser-Gly-Pro-

___

90 110 100 -Val-Lys-Val-Leu-Glu-Asp-Phe-Val-Glu-Ile-His-Gly-Lys-His-Asn-Glu-Arg-Gln-Asp-Asp-His-Gly-Tyr-Ilc-Scr-Arg-Glu-Phe-His~ -Gin ------..___ __..__ __________

-.___

______

._______

60 70 80 -Asp-Ser-Gly-Ile-Ser-Glu-Val-Arg-Ser-Asp-Arg-Asp-Lys-Phe-Val-Ile-Phe-Leu-Asp-Val-I,ys-His-Phe-Ser-Pro-Glu-Asp-Leu-Thr-

--._

170

140

____.________

30 40 50 -Glu-Gly-Leu-Phe-G1u-Tyr-Asp-Leu-I,eu-Pro-Phe-Leu-Ser-Ser-Thr-Ile-Ser-Pro-Tyr-Tyr-Arg-Gln-Ser-Leu-Phe-Arg-Thr-Val-I~eu-

____

.________...

10 20 Ac-Mct-Asp-Val-‘~hr-Ile-Gln-His-Pro-Trp-Ph~-Lys-Arg-Ala-Leu-Gly-Pro-Phc-Tyr-Pro-Ser-Arg-Lcu-Phe-Asp-Gln-Phc-Phe-Gly-

___________

Sk-------

- - . _ _ __

. ..--

Figure 1. Evidence for the proposed sequences of Tmius and Aotus oli\-crystallins. The hamster olA sequence (top line), which has been well-determined at the protein (de-Jon? et al., 1984) and DNA level (van den Heuvel etal., 1985), is used as a reference. The 0rA sequence of Tarsius (second line) and Aotus (third line) are deduced from amino acid compositions of tryptic peptides (drawn lines) and from thermolytic subdigcstions (interrupted lines) of some larger tryptic peptides. Observed differences with the hamster sequence are indicated in the Tarsius and Aotus sequences. A few positions in the Aotus sequence were determined by dansyl-Edman degradation (+). The replacement 134 Ser + Thr in A&s aA may also be located at position 142. The fact that the proposed 0rA sequences of Tarsius and Aotus arc inferred by homology with the fully determined hamster arA sequence introduces some uncertainty. The main cause of error would be the overlooking ofreciprocal replacements in peptides ofwhich the compositions are compared. However, the chances to overlook replacements by this approach, where the sequence differences among the compared olA chains are less than 5%, have been caicuiated to be very smali indeed (van Druten etal., 1978).

Tarsius Aotus

Hamster

578

Position NR in Alpha-A-chain Minke Whale Porpoise Horse Tapir Rhinoceros Pig Giraffe, Hippo OX Camel Dog, Cat Bear Mink Ring-tailed cat Seal, Sea-lion Pangolin Bat Hedgehog Tupaia Rat, Mouse, Hamster Squirrel, Gerbil Mole rat Guinea-pig Springhaas Gundi Beaver Pika Rabbit

W. W. DE JONG AND M. GOODMAN

34

11111111111111111111 155567779902223334444455555667 ‘315610240112372342678902358282

*

IA

AST

IK

FQEN

S NS

*

S T T

I

‘i

S

V V V V

I T

i V V VTS VG L L L L L L L L L L

PS T VT VT VT VT VT VT

SPSA

S T

;; S VT

TI

Lemur Galago, Potto FTarsier Owl monkey Rhesus monkey Human

VT VT VT VT VT VT

P P

Elephant Hyrax Manatee Aardvark 2-toed Sloth 3-toed Sloth Tamandua

VT VT VT VT VT

“TQ VT

*

S LSSVPSGMAGSASSS

I

L6

V V

1 T T T If

TTIQTL D D

i

I

I

T

IQT CIQT

L L

-T -T

A IT

Q/LID QLLLD QLLLD QLLLD

I

A A

LD LG R‘1’ LLD

N4 TA TA TA

.

I---VPS I---VPS IL---VPSGTI

T P

2 1 1

Figure 2. Phylogenetically inform: ntive positions in e utheris m aA-crystallin sequences (de Jon&re t al., . . . 1984; de Jong, 1986; Hendriks etal., 1987). Only those positions are shown at whrch replacements occur in two or more species. In addition the autapomorphous replacements (*) (occurring in a single species only) are specified for the primate aA sequences, but not for the others. The numbers of autapomorphous replacements in the non-primate sequences are given in the last column. The vertical lines indicate where residues are identical to the topmost sequence. Species with identical olA sequences, such as seal and sea-lion, are placed on one line. The one-letter notation for amino acids is used; a dash (-) denotes a deletion, and a dot 1.) denotes that the identity has not been determined.

which has also occurred in parallel in some carnivores and ungulates. The replacement 133 Leu -+ Val is a synapomorphy for potto and galago, although it occurs independently in the horse. Aotus is unambiguously joined to the anthropoid lineage by two unique replacements, 148 Ser -+ Thr and 155 Ser -+ Thr, while residue 13 Thr, which occurs in

MOLECULAR

disparate

mammalian

PHYLOGENY

OF

taxa, also supports the anthropoid

Glu -+ Asp and a rare deletion,

at position

579

TARSIUS

monophyly.

153, are synapomorphies

The replacement

91

for human and rhesus

monkey LYAchains. Two replacements of threonine for serine, at positions 132 and 134 (or 142), are unique characters for Aotus CYA,while 55 Thr -+ Ser also occurs independently in some other mammals. unambiguously

The branching

deduced from sequence

arrangement

of the primate

lineages

comparisons

of aA-crystallin

chains is depicted in

that can be

owl MONKEY

HUMAN

Figure 3.

WA

LEMUR

POTTO

GALAGO

TARSIER

0

RHESUS MONKEY

10

20

30

40

50

60

Figure 3. Phylogenetic tree based on primate uA-crystallin sequences. replacements in the different lineages are deduced from the alignment 153 is present in the hominoid arA sequences. The asignment of 146 tarsier and anthropoid CYA is discussed in the text. Divergence approximations, and are largely based on palaeontological evidence 1987; Romero-Herrera et al. 1978; Andrew, 1985; Simons, 1976).

Although

the sequence

information

Positions and types ofthe inferred in Figure 2. A deletion at position Val + Ile as a synapomorphy of times should be considered as (Gingerich, 1981, 1986; Martin,

from the CYAchains of Strepsirhini

(Lemuriformes

and Lorisiformes) and Anthropoidea smoothly fits and confirms their well-established pattern of relationships, the a:A sequence of Tarsius occupies an intermediate and more ambiguous sequence

position

(as befits this taxonomically

most closely resembles

problematic

that of guinea-pig

case). In fact, the Tarsius CXA

and springhare,

differing only by the

replacement of isoleucine for valine at position 146 (Figure 2). This does not reflect a specific phylogenetic relationship, but is only due to the low number of amino acid replacement’s since the divergence of the lineages leading to these taxa. This same lack of replacements makes it impossible to recognize tarsier CYAas resembling other primate aA sequences. However, if we accept the undisputed Figure 2 that, within the primate (YA chains,

primate affiliation of Tarsius, we see from residue 146 Ile is the only informative

580

W. W.

character.

DE JONG

AND M. GOODMAN

Tarsius with the Anthropoidea.

It groups

146 in all orders, except certain ungulates, the closest relatives of primates),

Because

edentates

it seems most probable

primate residue at this position, making character of the Haplorhini, as depicted

valine is present

and elephant

at position

(which are unlikely to be

that valine is indeed the ancestral 146 Val +

the replacement in Figure 3.

Ile a shared derived

However, because 146 Ile occurs in several placental orders (Figure 2) as well as in marsupials (de Jong et al. 1984) we cannot discount the fact that isoleucine may have been at position 146 in the ancestral primate aA sequence. To solve this problem we must employ strict parsimony trees obtained

analyses

for 59 vertebrate

of all available LXA sequences

cllA sequences.

The most parsimonious

and two aB sequences

(which

are 58%

related to LXA) (de Jong et al. 1984; Stapel et al. 1984), starting from initial cladograms as described in de Jong & Goodman (1982), re q uire 377 nucleotide substitutions (NS). In these trees the tarsier is eitherjoined to the anthropoids or to a guinea pig-springhare-pika branch.

In the latter

taxonomically replacements

the rabbit

alternative

groups

well-established

to become

parsimonious

biologically

the constrained

in certain

than

tarsier.

This

the lack of amino acid

and primates.

The tree construction

by forcing the species into their

groups, according

et al., 1979; de Jong

trees, varying

tarsier and anthropoids.

other

considering

more realistic

orders and certain subordinal

earlier (Goodman

with primates

is not surprising,

between rodents, lagomorphs

can be constrained described

case,

unrealistic

& Goodman,

non-primate

procedure

to criteria and algorithms

1982). In that case, the most

branches,

require

It adds 1 NS, both to the most parsimonious

382 NS trees, to join tarsier to the lemur-lorises

382 NS, and join 377 NS trees and to

branch

or to place Tarsius

as sister group of all other primates. The grouping

of Tar&us with the Primates

based on tandem and p hemoglobin trees, joined

tarsier

alignments

and olA-crystallin

is constrained

to the Anthropoidea.

strepsirhines

or exchanges

due to arA-crystallin). To provide insight residue at position

(Czelusniak

by the hemoglobins positions

primate

into the question

et al., 1988). to remain

of whether

ancestral

for the topologies

residue

constructions by a

In the most parsimonious

within

the Primates,

with them (four NS due to hemoglobins

at position

and is

reconstructions

the

and one NS

was the original (Goodman

et al.,

to the lowest (377) NS tree (tarsier

382 NS tree, and a 386 NS tree following the & Goodman (1986). In all cases, valine is the

146, and hence the mutation

Tarsius with the Anthropoidea,

as depicted

concluded

sequence

that the aA-crystallin

valine or isoleucine

sequence

corresponding

grouped with primates), the constrained tandem alignment results of Miyamoto ancestral

in parsimony

At least five NS are added to this tree when tarsier joins

146 in primates,

1979) were performed

is also obtained

for seven protein chains, in which Tarsius is represented

in Figure

146 Val -+ Ile joins

3. In summary

data contribute

then,

it can be

to the growing evidence,

both

morphological and molecular, for a haplorhine clade. The reconstructed mutational events in the primate CLA sequences (Figure 3) reveal differences in rates of change that seem to go beyond the normally expected fluctuations. The absence of any change in the tarsier aA chain since its divergence from the other primates contrasts sharply with the condition in anthropoid lineages, where the rate of change has increased from the average of 3% replacements per 100 million years in 5% replacement in 100 mammalian cllA chains (de Jong et al., 1984), to approximately million years. There is evidence that in the blind mole rat, Spalax ehrenbergi, decreased functional constraints have led to an increased rate of change in aA-crystallin (Hendriks

MOLECULAR

1987).

et al.,

There

of

is little reason

PHYLOGENY

to propose

581

OF TARSIUS

that this may also apply to

in the

In contrast

to the

in Spalax, in the

to the

of the

et al.,

a remarkable

the ten replacements an increase

in

aA chains

of five

It

directionality;

in a net loss of six

of interest

to note

of

change of (YA in the

a decelerated

rate of

in higher Li & Tanimura, of change

at the 1987).

to assume

in the

is caused by positive a very long history of

to consider primates.

We are

a causal relation

with the different evolutionary

it should be realized of a-crystallin in the

to daylight it patterns

of

in

of knowledge it

to warrant

to of Willeke is gratefully

by the

Aiello, L. C. (1986). The relationships ofthe Tarsiiformes: a review ofthe case for the Haplorhini. In (B. Wood, L. Martin & P. Andrews, Eds) Major Topics in Primate and Human Evolution, pp. 47-65. Cambridge: Cambridge University Press. Andrews, P. (1985). Improved timing of hominoid evolution with a DNA clock. Nature 314, 498499. Baba, M. L., Weiss, M. L., Goodman, M. & Czelusniak, J. (1982). The case of tarsier hemoglobin, S)&.2001.31, 156165. Bloemendal, H. (Ed.) (1981). Molecular and Cellular Biology of the Eye Lens. New York: Wiley. Britten, R. (1986). Rates of DNA sequence evolution differ between taxonomic groups. Science 231, 1393-1398. Cartmill, M., MacPhee, R. D. E. & Simons, E. L. (1981). Anatomy of the temporal bone in early anthropoids, with remarks on the problem of anthropoid origins. Am. J. phys. Anthrop. 56, 3-21. Cronin, J. E. & Sarich, V. M. (1980). Tupaiid and archonta phylogeny: the macromolecular evidence. In (W. Luckett, Ed.) Comparative Biology and EuolutionaT Relationships of Tree Shrews, pp. 293-312. New York: Plenum Press. Czelusniak, J., Koop, B., Tagle, D., Shoshani, H., Goodman, M., Braunitzer, G., Kleinschmidt, T., de Jong, W. W. & Matsuda, G. (1988). Perspectives from amino acid and nucleotide sequences on cladistic relationships among higher taxa of Eutheria. Current Mammalogy (in press). de Jong, W. W. (1986). Protein sequence evidence for monophyly of the carnivore families Procyonidae and Mustelidae. Mol. Biol. Euol. 3, 276281. de Jong, W. W. & Goodman, M. (1982). Mammalian phylogeny studied by sequence analysis of the eye lens protein cu-crystallin. 2. Siiugetiarkunde 47, 257-276. de Jong, W. W., Zweers, A., Versteeg, M. & Nuy-Terwindt, E. C. (1984). Primary structures ofthe cu-crystallin A chains of twenty-eight mammalian species, chicken and frog. Eur. J. Biochem. 141, 131-140. Gebo, D. L. (1986). Anthropoid origins: the foot evidence. J. hum. Evol. 15, 421-430. Gingerich, P. D. (1981). Early Cenozoic Omomyidae and the evolutionary history of tarsiiform primates. J. hum. Evol. 10, 345-374.

582

W. W.

DE JONG

AND

M.

GOODMAN

Gingerich, P. D. (1986). Early Eocene Cantius torresi-oldest primate of modern aspect from North America. Nature 319, 319-321. Goodman, M. (1976) Toward a genealogical description ofthe primates. In (M. Goodman, R. E. Tashian &J. H. Tashian, Eds) Molecular Anthropology, pp. 321-353. New York: Plenum Press. Goodman, M., Hewett-Emmett, D. & Beard, J. M. (1978). Molecular evidence on the phylogenetic relationships of Tarsius. In D. J. Chivers & K. A. Joysey, Eds) Recent Advances in Primatology (Vol. 3, Evolution) pp. 215-226. New York: Academic Press. Goodman, M., Czelusniak, J., Moore, G. W., Romero-Herrera, A. E. & Matsuda, G. (1979). Fitting the gene lineage into its species lineage, a parsimony strategy illustrated by cladograms constructed from globin sequences. SyJt. Zool. 28, 132-163. Hendriks, W., Leunissen, J. A. M., Nevo, E., Bloemendal, H. & de Jong, W. W. (1987). The lens protein olA crystallin in the blind mole rat, @alax ehrenbergi: evolutionary change and functional constraints. Proc. Natl. Acad. Sci. USA 84, 5320-5324. Le Gros Clark, W. E. (1959). The Antecedents ofMun. Edinburgh: Edinburgh University Press. Li, W.-H. & Tanimura, M. (1987). The molecular clock runs more slowly in man than in apes and monkeys. Nature 326, 93-9.5. Luckett, W. P. & Szalay, F. S. (1978). Glades versus grades in primate phylogeny. In (D. J. Chivers & K. A. Joysey, Eds) Recent Advances in Primatology (Vol. 3, Evolution), pp. 227-235. New York: Academic Press. Mai, L. L. (1985). Chromosomes and the taxonomic status of the genus Tarsius: preliminary results.]. hum. Euol. 14,229-240. Martin, R. D. (1985). First fossil tarsier from Africa. Nature 313, 430-43 1. Martin, R. D. (1987). Long night for owl monkeys. Nature 326, 639-640. McKenna, M. C. (1987). Molecular and morphological analysis of high-level mammalian interrelationships. In (C. Patterson, Ed.) Molecules versus Morphology in Phylogeny: Con&t or Compromise?, pp. 55-93. Cambridge: Cambridge University Press. Miyamoto, M. M. & Goodman, M. (1986). B iomolecular systematics of eutherian mammals: phylogenetic patterns and classification. Syst.2001. 35, 230-240. Novacek, M. J. (1982). Information for molecular studies from anatomical and fossil evidence on higher eutherian phylogeny. In (M. Goodman, Ed.) Macromolecular Sequences in Systematic and Euolutiona7y Biology, pp. 3-41. New York: Plenum Press. Pocock, R. I. (1918). On the external characters of lemurs and of Tarsius. Proc. 2001. Sot. Land., 19-53. Pollock, J. I. & Mullin, R. J. (1987). Vitamin C biosynthesis in prosimians: evidence for the anthropoid affinity of Tarsius. Am. J. phys. Anthrop. 73, 65-70. Romer, A. S. (1966). Vertebrate Paleontology. Chicago: University of Chicago Press. Romero-Herrera, A. E., Lehmann, H., Joysey, K. A. & Friday, A. E. (1978) On the evolution ofmyoglobin. Phil. Trans. R. SOL. B 282, 61-163. Rosenberger, A. L. & Szalay, F. S. (1979). On the tarsiform origins of Anthropoidea. In (R. L. Ciochon & A. B. Chiarelli, Eds) Evolutionary Biolqgy ofNew World Monkeys and Continental Drift, pp. 243-274. New York: Plenum Press. Schwartz, J. H. (1984). What is a tarsier? In (N. Eldredge & S. M. Stanley, Eds) Living Fossils, pp. 38-49. New York: Springer Verlag. hum. Evol. 16,23-40. Schwartz, J. H. & Tattersall, I. (1987). T arsiers, adapids and the integrity 0fStrepsirhini.J. Simons, E. L. (1976). The fossil record of primate phylogeny. In (M. Goodman, R. E. Tashian &J. H. Tashian, Eds) Molecular Anthropolou, pp. 35-62. New York: Plenum Press. Simpson, G. G. (1945). The principles of classification and a classification of mammals. Bull. Amer. Mus. nut. Hist. 85, l-350. Stapel, S. O., Leunissen, J. A. M., Versteeg, M., Wattel, J. & de Jong, W. W. (1984). Ratites as oldest offshoot of avian stem: evidence from or-crystallin A sequences. Nature 311, 257-259. van den Heuvel, R., Hendriks, W., Quax, W. & Bloemendal, H. (1985). Complete structure of the hamster @A crystallin gene. J. mol. Biol. 185, 273-284. van Druten, J. A. M., Peer, P. G. M., Bos, A. B. H. & de Jong, W. W. (1978). Reciprocal amino acid substitutions in the evolution of homologous peptides. J. theor. Biol. 73, 54%561.