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Munrmul Rev. 1989, Volume 19, No.1, 1-26.

Printed in Great Britain

The origins of lactation and the evolution of milk:


a review with new hypotheses
DANIEL G. BLACKBURN*, VIRGINIA HAY SSENt and CHRISTOPHER

J. MURPHY$
"Department of Biology, Trinity College, Hartford CT 061 06, U.S.A.; +Department
of Biology, Smith College, Northampton, Massachusetts 01063 U.S.A.; $Department of
Anatomy, N e w York State College of Veterinary Medicine, Cornell University, Ithuca,
New York 14853 U.S.A.

CONTENTS
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Previous scemrios . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Origins of lactation . . . . . . . . . .
Fossil evidencefor lactation . . . . . . .
Characteristicsof the ancestral lactstors . . .
Mammary gland origins . . . . . . . .
A missing link? . . . . . . . . . . .
Developmentofahypothesis . . . . . .
Evolutionof milk . . . . . . . . . . .
The trend towards nutritional lacteal secretions
Carbohydrates . . . . . . . . . . .
Proteins . . . . . . . . . . . . .
Lipids . . . . . . . . . . . . . .
A revised scenario . . . . . . . . . . .
Conclusion . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . .
References . . . . . . . . . . . . .

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ABSTRACT
Lactation is central to mammalian reproduction, and an understanding of the origins of
lactation is necessary to comprehend the early evolution of the Class Mammalia. In
modern mammals, lactation is a staggeringly complex phenomenon involving morphological, physiological, biochemical, ecological, and behavioural adaptations. Because
such a complex web of adaptations could not have arisen de nmo, lactation must have
begun as a much simpler process. Hypotheses regarding the original adaptive value of
proto-lacteal secretions invoke thermoregulatory, antibiotic, behavioural or nutritive
functions. In this review, we evaluate each of &ese hypotheses and attempt to reconstruct the origins of lactation in the light of current concepts of the biology of early
mammals and their ancestors, the origin of mammary glands, the evolution of extravitelline nutrient provision, and the physiology and biochemistry of milk synthesis and
secretion. Drawing upon empirical evidence and theoretical considerations, we present
a gradualistic scenario involving the following steps. First, incubation of eggs was aided
by development of a vascularized abdominal incubation patch. Secondly, anti-microbial
secretions from cutaneous glands of the incubation patch enhanced survival of eggs or
offspring. At this stage, these secretions could have supplemented vitelline nutrients
$Current address: Department of Ophthalmology, Veterinary Medical Teaching Hospital, University of
California, Davis, -95616 U.S.A.

D. G . Blackburn, V . Hayssen and C .J. Murphy

(in much the same way as oviductal secretions do in monotremes), perhaps facultatively.
Subsequently, hypertrophy and specialization of cutaneous glands accompanied the
controlled production of a copious, nutritious secretion, which eventually evolved into a
primary source of energy for development and growth of the offspring. We outline
important events in the evolution of milk, and suggest studies likely to provide indirect
tests of the evolutionary hypotheses discussed.

INTRODUCTION
Since the time of Darwin, the origin of lactation has remained a major unresolved issue
in evolutionary biology. The highly integrated nature of the morphological, physiological and behavioural features of milk production, ingestion and digestion renders
lactation a complex adaptation (sensu Frazzetta, 1975) that challenges gradualistic
explanations for its evolution. Particularly difficult to explain are the incipient stages
in the evolution of milk production, and the adaptive value of early proto-lacteal
secretions. Other unresolved issues include the origins of the mammary gland and the
evolutionary transformation from production of proto-lacteal fluids to the complexities
of milk synthesis.
Several conflicting scenarios have been advanced as possible explanations for the
evolution of lactation. However, in the absence of living species representi- transitional stages, it has not been easy to evaluate the validity of these scenarios or to resolve
the discrepancies between them. Little attempt has been made to test predictions
based on these evolutionary hypotheses, and the major criterion for acceptance of each
scenario remains that of plausibility.
The importance of the phenomenon of lactation to mammalian evolution and ecology
has been discussed recently by Pond (1977, 1983, 1984), Parker (1977), Oftedal
(1980), Renfree (1983), and Hayssen, Lacy, & Parker (1985). Other recent works have
briefly considered the origins of lactation (Graves & Duvall, 1983) (Griffiths, 1983)
(Hayssen & Blackburn, 1985) and evolution of the mammary gland (Blackburn,
1985a). However, there exists no thorough, modem review of lactation from the standpoint of its origin and subsequent evolutionary development. Hayssen & Blackburn
(1985) suggested that an understanding of the evolutionary history of lactation would
require a complex synthesis of a wealth of molecular, developmental, physiological,
morphological, and ethological information. They proposed that the validity of the
various hypotheses on the origin of lactation could best be assessed through careful
reconsideration of such issues as mammary gland origins, the nature of proto-lacteal
secretions, the transition to milk production and the reproductive biology of
mammalian ancestors.
This paper reviews and evaluates traditional and contemporary explanations for the
origin and subsequent evolution of lactation in mammals, as an initial step towards a
synthetic concept of the evolution of lactation. A new hypothesis on the origin of
proto-lacteal secretion is developed in the context of therapsid reproductive biology
and mammary-gland origins. Drawing upon diverse bodies of evidence, we present a
scenario for the provision of nutritional lacteal secretions, and outline significant events
in the evolution of milk. Finally we discuss lines of investigation that are likely to
produce evidence bearing on the evolutionary hypotheses discussed herein. Because
this paper focuses on the origins of lactation, the significant implications of lactation for
neonatal and maternal physiology lie outside of the scope of our discussion. Other
peripherally relevant issues dealt with elsewhere include the absence of lactation in
male mammals (Daly, 1982) and the evolutionary origins of the major mammalian

D. G . Blackburn, V . Hayssen and C.J.Murphy

Long (1972) cited numerous objections to Haldanes (1965) hypothesis, including


the following:
1 simultaneous embryological development of mammary glands and hair indicate
that they probably evolved concomitantly;
2 homeothermy preceded development of a hairy coat;
3 evidence is lacking that ancestral mammals arose under hot and arid conditions;
4 parental secretion of a salty sweat would be of little value to the offspring, whereas
secretion of a watery sweat would tend to dehydrate the parent;
5 small mammals (as were the Mesozoic forms) generally lack sweat glands,
because the relatively large surface area of their bodies provides effective cooling;
6 Haldanes theory fails to account for the origin of the marsupium.
Long (1972) called for a return to what he called Darwins theory, although the
scenario he described was more a product of contributions by Bresslau (1907,1920) and
Gregory (1910) than of Darwin himself.
The issue of the origin of lactation was re-opened by Graves & Duvall(l983; also see
Duvall, 1986),who hypothesized that ancestral maternal secretions contained an aggregation pheromone that behaviourally bonded the offspring to the mother. They
suggested that these secretions were accidentally imbibed by young therapsids during
introduction of the pheromone into the vomeronasal organs. Continued ingestion of
these secretions eventually selected for females that secreted an increasingly nutritious
product.
Current scenarios for the evolution of lactation (e.g. Hopson, 1973; Long, 1969,
1972; Graves & Duvall, 1983; Guillette & Hotton, 1986) can be summarized by the
following sequence of events:
1 maternal development of a vascularized abdominal incubation patch;
2 enhancement of egg survival by secretions of maternal cutaneous glands;
3 utilization of maternal secretions as food or drink through absorption by the yolk
sac or ingestion by the hatchlings;
4 concomitant evolution of suckling behaviour, increase of the nutritional value of
the maternal secretions, and the development of localized areolar regions.
The most controversial elements of the accepted sequence are related to steps 2 and 4
above-the possible value of the proto-lacteal secretion to the eggs or young, and the
transition from non-nutritional secretions to milk. Although the various hypotheses
summarized above differ both with regard to the selective advantages of proto-lacteal
secretion and with respect to their plausibility, they have several features in common:
they provide imaginative explanations that are relatively compatible with gradualistic
models of evolutionary change; they have little predictive value and are difficult to
falsify; they require that the original functions of proto-lacteal secretions have been lost;
they do not explain the selective pressures leading to post-paritive nutrient provision
and the production of altricial young, and they fail to account for intermediate stages in
the evolutionary transition to milk secretion.
Of the availablehypotheses on the origins of proto-lacteal secretion, perhaps the least
credible is that survival of the eggs was enhanced by their being glued to the abdomen of
the mother (Gregory, 1910). Also difficultto accept is the premise (Bresslau, 1907) that
cutaneous secretions provided a means of warming the eggs. Unless the eggs were
drowned in a continuous and copious flow, maternal secretions would decrease egg
temperatures through evaporative cooling. In addition, gas exchange across the eggshell
would be difficult under such conditions. Moreover, such an elaborate mechanism

Origins of lactation and evolution of milk

reproductive patterns (e.g. Lillegraven, et al. 1987; Tyndale-Biscoe & Renfree, 1987;
Blackburn et al., 1988).

PREVIOUS SCENARIOS
Is it conceivable that the young of any animal was ever saved from destruction by
accidentally suckling a drop of scarcely nutritious fluid from an accidentally hypertrophied cutaneous gland of its mother? And if one was so, what chance was there for the
perpetuation of such a variation? (Mivart, 1871)
If it could be demonstrated that any complex organ existed, which could not
possibly have been formed by numerous, successive slight modifications, my theory
would absolutely break down. (Darwin, 1872).
In response to Mivarts (1871) eloquent challenge to his theory, Darwin (1872)
formulated what apparently was the first formal hypothesis as to how lactation
evolved.* Unaware of the egg-laying habits of monotremes, Darwin (1872) believed
that mammals descended from a marsupial-like ancestor. He envisaged a scenario in
which mammary glands arose through hypertrophy and concentration of cutaneous
glands located in the marsupium. He assumed the growing young learned to lick up the
secretory products of these glands, leading to selection of females that could produce the
most nutritious glandular secretions. Support for Darwins hypothesis derived from
belief that the incubatory pouch antedated the mammary apparatus and that the absence
of a pouch in the platypus Ornithorhynchus represented secondary loss due to adoption
of aquatic habits (see Bresslau, 1920).
Two subsequent developments led to substantial modification of Darwins (1872)
scenario. First, the confirmation of inonotreme oviparity (Caldwell, 1887) suggested
that lactation had evolved prior to the adoption of viviparous habits. Secondly, evidence
that the pouches of monotremes and marsupials are not homologous (Bresslau, 1907,
1920) raised the likelihood that these structures evolved after lactation.
Bresslau (1907, 1920) suggested that the primordial mammary gland arose from a
highly-vascularized region of skin that served to warm the eggs, like the incubation
patch of certain birds. In his view, the rich blood supply of the region led to the
development of tubular glands, secretions of which helped to raise the temperature of
the egg during incubation. Gregory (1910) added considerable detail to Bresslaus
(1907) scenario. He suggested than an early function of the proto-lacteal fluid was to
adhere the eggs to the incubation patch. After hatching, these cutaneous secretions
would have served to keep the young warm. Gregory postulated that the pleasant
flavour of the maternal secretions would have induced the young to lick them up,
eventually leading to suckling of nutritious fluids.
In a major departure from previous hypotheses, Haldane (1965) postulated that
lactation arose as a means of providing water to the young in arid environments. He
hypothesized that tropical mammal-like reptiles interspersed episodes of bathing in
water with egg incubation, with the result that egg temperature (and parental body
temperature) remained cool. According to this view, hatchlings learned to lick water
from parental fur, much as the young of certain tropical birds (Pterocles, Vanellus) sip
water from the feathers of their parents. After establishment of this behaviour pattern,
hypertrophy of maternal sweat glands led to secretion of water for the young, followed
evolutionarily by secretion of more nutritious fluids.
*Some recent papers have attributed Darwins views on lactation to the 1859 edition of Ongin of the
Species, an error that obscures the interesting historical development of his ideas.

O r i g h of lactation and evolution of milk

would seem superfluous in a species in which the adults were most probably
endothermic (see below).
Somewhat more plausible is the hypothesis (Haldane, 1965) that the ancestral
mammary gland provided water for the growing hatchlings. Elements of this hypothesis
have also been supported in other papers (Hopson, 1973; Chadwick, 1977; Guillette &
Hotton, 1986). The fact that water is a major component of milk could be considered
as evidence for this hypothesis, although it should be noted that water is a major
component of most biological secretions and waste products.
Some of Longs (1972) objections to Haldanes (1965) scenario are of questionable
relevance, such as that the origin of the marsupium is not explained and that
homeothermy preceded the development of hair. In addition, recent evidence suggests
that mammalian ancestors may well have arisen under arid conditions (Kemp, 1982,
pp. 65,252), contrary to Longs (1972) inferences. Furthermore, the assumption that
the sequence of development of integumentary structures necessarily recapitulates
evolutionary history (Long, 1972) is also dubious (Hopson, 1973) in the light of current
concepts oc the relationships between ontogeny and phylogeny (e.g. Gould, 1977). On
the other hand, Haldanes assumption that the ancestral mammary gland necessarily
produced a copious watery secretion is not particularly supported by phylogenetic
considerations (Blackburn, 1985a).
The hypothesis that the proto-lacteal secretions contained pheromones (Graves &
Duvall, 1983) is certainly plausible in view of the pheromonal nature of certain
cutaneous gland secretions in living mammals, as is the corollary hypothesis that offspring could have imprinted on such phermones. Because very minute quantities of
maternal secretions would be needed for imprinting, this hypothesis does not specifically address the selective advantages of subsequent glandular hypertrophy and
production of copious secretions.
Taking note of the lack of consensus, the paucity of testable predictions, and the
absence of explanations for the evolution of nutritional from non-nutritional secretions,
we begin our evolutionary reconstruction by considering the early ancestors in which
proto-lactation originated.

ORIGINS OF LACTATION
Fossil evidence for lactation
The numerous, detailed similarities in mammary gland structure and function evident
among living mammals (e.g. Griffiths et al., 1973) provide overwhelming evidence that
a complex form of lactation was established prior to divergence of the extant lineages.
From such evidence, both Lillegraven (1979) and Pond (1983) have inferred that
mammary glands were present in mammals of the Late Triassic, inferences that are
compatible with recent suggestions of lactation in multituberculates (Griffiths, 1983)
and morganucodontids (Kemp, 1982). On the other hand, recent evidence and analyses
suggest that monotremes are closely related to therians (Kemp, 1983; Archer et al.,
1985; Kielan-Jaworowska et al., 1987), raising the possibility that lactation might not
have been universal among the numerous groups of extinct mammals.
Because fossils have yielded little information on the nature or even the existence of
lactation in extinct forms, the origins of lactation cannot as yet be pin-pointed with
precision. The range of opinions expressed recently by paleontologists and other
writers reflects the inadequacy of the fossil evidence available. Some have suggested that
lactation evolved in cynodont reptiles (Brink, 1955a, 1956; Guillette & Hotton, 1986)

D . G . Blackburn, V . Hayssen and C .J.Murphy

or (undefined) therapsids (Van Valen, 1960), whereas others have concluded that no
evidence for lactation exists for any forms predating the earliest mammals (Crompton,
1980; Hopson, 1971,1973; Kemp, 1982).
The feature most commonly invoked as an indicator of lactation in extinct forms is a
mammalian pattern of tooth replacement. Living mammals exhibit a delay in initial
tooth eruption-viewed with good reason as an adaptation associated with suckling
(Ewer, 1963)-as
well as diphyodonty (two generations of teeth). Pond (1977) indicated that diphyodonty was made possible by the evolution of lactation. In marked
contrast to mammals, cynodonts and other reptiles exhibit continuous tooth replacement (polyphyodonty) throughout life (Hopson, 1964, 1971; Osborn & Crompton,
1973). Consequently, many investigators have regarded the diphyodonty of early
mammals as strong evidence that the young of these creatures fed by sucking.
However, Wright (1983) recently demonstrated that the assumption that
diphyodonty must be associated with delayed tooth eruption and lactation may not be
valid. Wright indicated that delayed tooth eruption is one of four potential evolutionary
pathways by which diphyodonty can be achieved, and was probably not the means by
which mammalian diphyodonty originated. Not only did she find no evidence in the
literature for postponed tooth acquisition in early mammals, but she cited data on the
morganucodontid Morganucodon ( =Euzuszrodon) suggestive of a functional dentition
very early in life (Wright, 1983). Consequently, the inference that diphyodonty of early
mammals was directly associated with suckling should be reconsidered.
Lactation has also been inferred from dental characteristics of certain cynodonts of
the Early Triassic. Evidence cited for this inference includes a delicate dentition and
nearly mammalian tooth replacement pattern in Diademodon (Brink, 1955a, 1956).
However, more extensive information has shown that cynodonts exhibited continuous
tooth replacement, and more significantly, that even the smallest juveniles apparently
had a fully functional dentition (Hopson, 1971, 1973).
On occasion, osteological structures purportedly associated with muscular lips and a
movable snount have been cited as indirect evidence for suckling (and lactation) in fossil
forms (see Van Valen, 1960). For example, in various advanced therapsids, foramina in
bones of the snout hypothetically carried blood vessels and nerves to muscles of a
movable lip that might have functioned in suckling. However, as noted by Van Valen
(1960; also see Bennett i?i Ruben, 1986),similar foramina are present in living reptiles,
where they supply no such structure. Some have argued that the absence of grooved
bone surfaces adjacent to the foramina suggests the presence of fleshy labial tissue (see
Kemp, 1982, p. 248). However, even the presence of lip musculature would not
necessarily indicate that the young therapsids fed by sucking, given the potential
alternative roles for such musculature (Van Valen, 1960).
In summary, fossils have thus far provided little substantial evidence for lactation in
specific extinct reptiles and early mammals. Even fewer data are available to indicate the
identity of the extinct group in which lactation first originated. Thus, although lactation
is not precluded for animals on either side of the reptile-mammal interface, compelling
evidence for lactation in the form of such features as delayed tooth eruption or fossilized
mammary integument has yet to be discovered.

Characteristics of the ancestral lactators


Despite the paucity of definitive fossil evidence for lactation, certain characteristics of
the animals in which proto-lactation originated may be inferred from information on
extinct and extant forms. The fundamental similarities of living mammals with respect

Origins of lactation and evolution of milk

to the nature of suckling, as well as structure, function, development, and neuroendocrine control of the mammary gland, indicate the virtual certainty that all extant
mammals stemmed from a common ancestor that exhibited lactation. In fact, the complete absence of both primitivemammary glands (Griffiths, 1978;Griffiths e t al., 1973)
and a simple level of lactation (Oftedal, 1980) among existing species indicates that a
complex form of lactation was well-established before the ancestors of monotremes and
therians diverged phylogenetically .
One can deduce from extant mammals that lactation probably arose in endothermic,
oviparous ancestors that exhibited some degree of maternal care. Assuming that the
transition from oviparity to viviparity is irreversible in amniotes (Fitch, 1970;
Blackburn, 1985a, 1985b), the presence of oviparity in monotremes clearly indicates
that lactation had to precede the evolution of mammalian viviparity. The evolutionary
development of the mammary gland from glands associated with abdominal hair
follicles (Bresslau, 1920;Jollie, 1962; Blackburn, 1985a)suggests that hair evolved prior
to lactation; in fact, abdominal hair (as distinct from sensory vibrissae of the rostrum)
suggests a pelage. The presence of hair and abdominal cutaneous glands in turn implies
that lactation arose in animals that had achieved a degree of endothermy.
Did this endothermic ancestor incubate its eggs, as is often presumed (e.g., Bresslau,
1907; Hopson, 1973)?Although direct evidence is unavailable, egg-incubation seems
reasonable on several grounds. First, a continued, close association between females
and oviposited eggs is a virtual pre-requisite for maternal production of proto-lacteal
secretions that benefit the offspring. Secondly, because the rate of embryonic development is temperature-dependent in amniotes, incubation by endothermic females would
speed development, therby decreasing the time between clutches and minimizing
exposure of the female and egg to mortality (Blackburn & Evans, 1986).Therefore, eggincubation could have been selected in the ancestors of mammals due at least in part to
thermal benefits, as appears to have occurred in birds and, independently, in certain
reptiles (e.g., Python).
The animals in which lactation originated may have produced fully precocial young,
as do living reptiles, or somewhat altricial offspring, as do many birds. Clearly, the
degree of altriciality exhibited by living monotremes, marsupials, and many eutherians
could not have preceded the evolution of milk. However, given the likelihood of maternal care, the hatchlings may not have been fully capable of nutritional or physiological
autonomy.
Whether the ancestral lactators accomplished incubation via a pouch wiil probably
never be known. Developmental and morphological evidence indicates that the
incubatoriun of the monotreme echidna is not homologous with the marsupia of
extant metatherians (Bresslau, 1907, 1920; Grifliths, 1968). Moreover, marsupial
pouches exhibit considerable structural diversity; Woolley (1974) recognized four distinct types of pouches among the Dasyuridae, and two further types have been distinguished among non-dasyurids (Russell, 1982). This morphological diversity
strongly suggests that pouches evolved repeatedly within the infraclass Marsupialia
(Shaman, 1976; Kirsch, 1977). Given that pouches are lacking in the platypus
Ornirhorhynchus and in some species of the marsupial families Dasyuridae and
Didelphidae (Walker, 1975), the only alternative to multiple origins is multiple losses,
with pouches having been lost independently among monotremes, some marsupials,
and, by implication, ancestral eutherians. Whereas production of altricial offspring
plausibly could have led to convergent evolution of pouches, we have difficulty identifying a selective advantage for pouch loss in these various lineages, many representatives

D. G . Blackburn, V . Hayssen and C.J. Murphy

of which produce highly altricial offspringfor which a pouch would be beneficial. In any
case, given that all mammalian pouches are evidently not all homologous, pouches
cannot be assumed to have been present in the earliestmammals (Hopson, 1973; Kirsch,
1977), much less in their cynodont ancestors.
Were the animals in which lactation originated mammals or reptiles? A tautological
response to this question could be to define mammals by the presence of mammae,
which by default would place proto-lactation on the reptilian side of the reptilemammal boundary. But such an answer obviously is of little value from a paleontological standpoint and is useless in identifying the particular taxa involved.
With the possible exception of hair (see Bennett & Ruben, 1986), the various characteristics inferred for the inventors of lactation were probably shared by early mammals
and their cynodont ancestors. For example, a glandular integument was present
early in therapsid history, as indicated by excellent skin fossils of the dinocephalian
Estemmenosuchus (Chudinov, 1968, 1970). That cynodonts and early mammals were
relatively endothermic is suggested by several lines of evidence (e.g., see Hopson, 1973;
McNab, 1978; Van Valen, 1960; but see Crompton, Taylor & Jagger, 1978). In their
critical review, Bennett & Ruben (1986) cited the presence of a nasal turbinal complex
and vascularized fibro-lamellar bone as being particularly suggestive of therapsid
endothermy. Early mammals and their reptilian ancestors were oviparous, as is indicated by the retention of egg-laying habits in monotremes. Oviparity is plesiomorphic
for the class Reptilia, and clear evidence is lacking for viviparity in reptiles other than
ichthyosaurs and squamates (Blackburn, 1982,1985b). Assuming that extant mammals
are a monophyletic group, as the consensus now indicates (e.g., Kemp, 1982, 1983;
Archer et al., 1985; Kielan-Jaworowska e t al., 1987), if any therapsids were viviparous
(Brink, 1956) they could hardly have been ancestral to living mammals. In addition to
oviparity, endothermy and skin glands, some cynodonts might even have had maternal
care. Tenuous evidence for a maternal-offspring relationship in Thrinaxodon liorhinus
is provided by the oft-cited fossil associationof a tiny, immature specimen with an adult
female of that species (Brink, 1955b).
In summary, the phylogenetic origins of lactation cannot be pin-pointed at present.
However, because cynodonts and early mammals share many of the characteristics
inferred for the ancestral lactators, the evidence is consistent with an origin of lactation
having occurred in either of these two groups.

M a m m a r y gland origins
The evolutionary origin of the mammary gland was reviewed recently by Blackbum
(1985a). As indicated above, the detailed similarities of the mammary glands of extant
monotremes, marsupials and eutherians are strong evidence for a monophyletic origin
of the mammary gland (Bresslau, 1920; Jenness, 1974a; Raynaud, 1961). Conventional
wisdom holds that mammary glands originated from eccrine-like sweat glands.
However, developmental, physiological and phylogenetic evidence suggests that the
mammary gland is actually more closely related to the epitrichial (sebaceous and
apocrine) glands than to the eccrine glands. Nevertheless, the two types of epitrichial
glands have specializations that seem to preclude a direct ancestry to the mammary
gland. On this basis, Blackbum (1985a) suggested that the mammary gland probably
either arose from some evolutionary precursor of extant epitrichial glands, or represents
a neomorphic hybrid assemblage of characteristics of these two gland populations.
The inferred relationship between the mammary gland and epitrichial glands may
have implicationsfor the nature of proto-lacteal secretions. Specifically,the evolutionary

Origins of lactation and evolution of milk

precursor to the mammary gland could have already possessed the capacity to synthesize
carbohydrates, proteins, and lipids, as do modern epitrichial glands. Thus, whatever
their function, proto-lacteal secretions may have had some weak nutritive potential at
their inception, rather than simply being composed of water and electrolytes, as are
eccrine gland secretions (Blackburn, 1985a).

A missing link?
Scenarios for the evolution of lactation generally hypothesize functions for proto-lacteal
secretions that are unlike those of milk, secretions of integumentary glands, or both.
According to such scenarios, the evolutionary history of lactation is not reflected in the
composition of the mammary secretions of living mammals. In contrast, Hayssen &
Blackburn (1985) noted structural and functional links between the components of milk
and the antimicrobial secretions of other integument-derived glands. The most notable
of these relationships is that between the milk protein a-lactalbumin and the enzyme
lysozyme.
Lysozyme is a bacteriocidal enzyme that destroys Gram-negative bacteria by lysing
their mucopolysaccharide cell walls (Tizard, 1981). It is also known to be effective
against fungi (Marquis et al., 1982). Lysozyme is found in the small intestine, most body
secretions, and the egg whites of most birds (Dickerson & Geis, 1969; Tizard, 1981).
This substance has a bacteriostatic effect on the intestinal flora (Tizard, 1981) and
probably helps to protect the mammary gland from bacterial infection (Smith, 1959).
Milk itself has bacteriocidal properties that have been attributed in part to lysozyme
(Smith, 1959; Vakil e t al., 1969).
a-Lactalbumin is a crucial component of the lactose-synthetase system, the other
component being a galactosyltransferase.Lactose and its derivatives comprise the great
majority of milk carbohydrates in most mammals (Jones, 1977). Features of the lactosesynthetase system were summarized by Brew (1969) and Jones (1977). Galactosyltransferase is an enzyme present in the liver and a variety of other organs including the
mammary gland. This enzyme catalyzes the transfer of galactose from UDP-galactose
to a variety of acceptors, including N-acetylglucosamine (NAG):
galactosyltransferase
UDP-galactose +NAG A N-acetyllactosamine + UDP.
a-Lactalbumin forms a bimolecular complex with galactosyltransferase, altering its
substrate complex to include glucose and thereby favouring the synthesis of lactose:
galactosy ltransferase
UDP-galactose + glucose A lactose+ UDP.
a-lactalbumin
During gestation, the concentration of galactosyltransferase in mammary tissue
steadily increases, without stimulating milk production. Only when a-lactalbumin synthesis is triggered near the end of pregnancy does lactose synthesis and milk production
begin (Jones, 1977). Unlike galactosyltransferase, a-lactalbumin is almost confined to
the mammary gland (Dickerson & Geis, 1969; Hamilton, 1981). As noted by Jones
(1977, p. 78), it is the occurrence of a-lactalbumin which confers upon the mammary
gland its unique ability to synthesize lactose and its derivatives.
Biochemists have been aware of the structural, functional and evolutionary relationships between lysozyme and a-lactalbumin for nearly 2 decades (Brew, Vanaman
& Hill, 1967, 1970; Gordon, 1971). Both substances exhibit an &ity
for a B 1-4

10

D.G . Blackburn, V.Hnyssen and C.3. Murphy

glucopyranosyl structure (Smith, 1970), and the reactions in which they participate are
clearly related. Lysozyme catalyzes hydrolysis of a 1,4 glycosidic bond, whereas
a-lactalbumin is involved in the synthesis of a p1,4 glycosidic bond (Ebner &
Schanbacher, 1974). The amino acid sequences and three-dimensional configurations
of a-lactalbumin and lysozyme from various species reveal that the two proteins share a
common ancestry, suggesting that evolution of a-lactal bumin involved modification of a
duplicated lysozyme gene (Brew er al., 1967). Could & Vrba (1982) used the alactalbumin/lysozyme relationship as an example of a structure co-opted for a new
function. As Dickerson & Geis (1969:78) stated:
It appears that when a milk-producing system was being developed during the
evolution of mammals, and when the need for a polysaccharide-synthesizingenzyme
arose, a suitable one was found in part by modifying a pre-existing polysaccharidecutting enzyme. This adoption of pre-existing structures to new uses has been seen
many times in macroscopic anatomy . . . but this is one of the first documented examples
of the same process at the molecular level.
The link between lysozyme and a-lactalbumin may provide the key to understanding
the function of the proto-lacteal secretions and the origin of lactation itself. The fact that
a-lactalbumin evolved from lysozyme implies that lysozyme was secreted by the
mammary-gland precursor, as it is currently secreted by certain other glands of ectodermal origin (see Miyauchi, 1984; Sapse et nl., 1968; Tizard, 1981). If the lysozyme of
this ancestral secretion served an important function, selection could have led to
enlarged cutaneous glands and production of a more copious secretion with a higher
lysozyme content. Moreover, if the original function of these hypertrophied glands was
associated with reproduction, then glandular enlargement and secretion would have
been likely to come under the control of the reproductive hormones. And finally, if the
function of this secretion was associated with the eggs or young, these hypertrophied
glands would be likely to be located on the ventral abdomen, as mammary glands almost
invariably are.
Development of a hypothesis
Lysozyme is but one of several microbial inhibitors common to milk and the secretions
of mucosal membranes and glands derived from the integument (Table 1). Among these
inhibitors are lactoferrin, immunoglobulins, peroxidase, xanthine oxidase, transferrin
and fatty acids. These substances act against a wide range of bacteria, fungi, protozoa
and viruses by means of a variety of mechanisms (e.g., see Aisen & Listowsky, 1980;
Arnold, Cole & McGhee, 1977; Bortner, Miller & Arnold, 1986; Broekhuyse, 1974;
Masson, Heremans &Dive, 1966; Newbold, 1974; Reiter & Oram, 1967; Tizard, 1981;
Weinberg, 1977). Milk also contains anti-parasite factors unreported in other mucosal
secretions (Gillin, Reiner & Wang, 1983). Although the importance of coIostrum in the
transfer of immuno-competence is widely appreciated, other anti-microbial functions
of milk are often overlooked (McClelland, 1982; Butler, 1979). Thus, the roles of many
of these microbial inhibitors in protection of the milk, mammary gland, and offspring in
extant mammals are not well understood.
Nevertheless, at least some of the microbial inhibitors of milk are similar or identical
to substances that protect eggs and other structures from microbial infection. For
example, a lysozyme similar to that in milk protects avian eggs from microbial and
fungal attack (Board & Fuller, 1974; Marquis et al., 1982). Likewise, milk lactoferrin
chelates iron vital for bacterial proliferation (Bortner et al., 1986; Masson, Heremans &
Dive, 1966), as does the lactoferrin of other exocrine secretions (Arnold, Cole &

Origins of lactation and evolution of milk

11

Table 1
Microbial inhibitors of milk
Substances

Targets

Lysozyme
Peroxidase

bacteria, viruses, fungi


bacteria, viruses, protozoa

Xanthine oxidase

bacteria, viruses, protozoa

Lactoferrin
Transferrin
Immunoglobulins

bacteria
bacteria
bacteria

Interferon

infectious agents?

Fatty acids

bacteria, fungi

Mechanisms
hydrolyzes cell walls
generates inhibitory
metabolites
generates inhibitory
metabolites
chelates iron
chelates iron
transfer passive immunity;
protect gut and pharynx
may provide non-specific
defence
increase acidity

Most of these inhibitors are also present in the secretions of other integument-derivedglands. Documentation
can be found in Aisen & Listowsky (1980); Arnold cz al. (1977); Butler (1979); Groves (1970); McClelland,
McGrath & Samson (1978); Morrison & Allen (1966); Reiter & Oram (1967); Selsted & Martinez (1982);
Tizard (1981), and other sources listed in the text. Mechanisms are not exclusive.

McGhee, 1977; Broekhuyse, 1974; Masson, Heremans & Dive, 1966; Reiter & Oram,
1967) and the related protein ovorransferrin, of bird albumen (Frelinger, 1972;
Board & Fuller, 1974). Milks of some species contain transferrin, a protein which has
bacteriostatic properties in blood serum (Masson & Heremans, 1971; Aisen &
Listowsky, 1980).
The fact that milk shares anti-microbial substances and properties with the
secretions of other glands may be evidence of functions that predate the nutritional role
of milk. Hayssen & Blackburn (1985) postulated that the secretory products of the
mammary-gland precursor enhanced survival of the eggs or young by virtue of their
anti-microbial properties, although they did not detail ways in which this enhanced
survival might have been accomplished. Below are three possibilities, which are not
mutually exclusive.
1 . Proto-lacteal secretions controlled or destroyed bacteria, fungi, or other potential
pathogens on or near the surface of the eggshell. Accordingly, anti-microbial substances
that originally functioned in protection of the integument incidentally increased survival of incubated eggs, leading to selection for females with more potent or more
copious secretions.
A major rationale behind this hypothesis is that microbes can be significant pathogens of eggs (see Packard & Packard, 1980). In oviparous species, an eggshell or egg
membrane constitutes the first line of defence of the egg against microbial attack. Thus,
egg survival can depend on selection of a commensal microfloral population on the egg
surface and maintenance of that flora at appropriate levels. For example, in fish, the egg
capsule is colonized by a characteristic flora, and embryo viability depends upon establishment of a dynamic ecosystem that precludes colonization by pathogenic bacteria,
(Bell, Hoskins & Hodgkiss, 1971). Similarly, the nest of birds provides a characteristic

12

D. G . Blackburn, V . Hayssen and C.J.Murphy

microfloral environment to which the egg is well adapted (Board & Fuller, 1974).
Whereas avian eggs possess a highly calcified shell which acts as a very effective barrier
to microbial attack (Board & Fuller, 1974), amniote eggs with less-calcified shells are
relatively susceptible to such attack (Packard & Packard, 1980).
Although secretions with microbicidal properties might well have controlled microflora populating the eggshell and nest micro-environment, anti-microbial actions need
not have been limited to this role. A further, if more distant, possibility is that lysozyme
of these secretions complexed with egg albumen to form a physical barrier to microbial
attack. In birds, oviductal lysozyme forms a network of fibers with the ovomucin of the
albumenous layer, probably forming a physical barrier to any micro-organisms that
have managed to penetrate the eggshell and egg membranes (Board & Fuller, 1974).
Lysozyme of avian albumen apparently does not chemically attack bacteria, presumably
because bacteriolysis would release substances toxic to the cells of the embryo (Board &
Fuller, 1974). A corollary of this hypothesis is that the therapsid egg was capable of
absorbing glandular secretions; given the range of eggshell permeabilities exhibited by
extant amniotes (Packard, Tracy & Rothj 1977; Packard & Packard, 1980; Packard,
Packard & Boardman, 1982), this may well have been the case. In living oviparous
mammals, the eggshell of monotremes is known to be permeable to liquids, for considerable quantities of oviductal secretions are absorbed by the monotreme egg during
the period of shell deposition (Hughes & Carrick, 1978).
2. Proto-lacteal secretions, ingested in small quantities by the hatchlings, enhanced offspring survival by controlling microflora of the pharynx anddigestive tract. These secretions
may also have provided immunity to the o8spring in the form of maternal immunoglobulins
or cellular elements. This hypothesis is based strongly on evidence from living mammals.
In extant species, milk plays a central role in protecting the mucosal surfaces and
establishing the gastrointestinal flora of the offspring, in part by virtue of its bacteriostatic and microbicidal properties. For example, human milk provides continuous local
control of bacterial and viral levels in the mouth, nose and gut, a function that can be
critical for offspring survival (Gunther, 1977). Thus, human milk kills pathogenic gut
protozoa such as Giardia, Trichomonas ar,d the dysentery amoeba Entamoeba (Gillin et
al., 1983); controls levels of commensal bacteria such as Escherichia coli(Burdon, 1973),
and inhibits growth of yeast and Shigella bacteria (Butler, 1979). In humans and other
mammals, lacteal IgA agglutinates bacteria of the gut and oral cavity, presumably
preventing colonization (Butler, 1974; Lascelles, 1977; Williams & Gibbons, 1972).
Milk also functions in the transmission of passive immunity to the offspring. This
transmission involves lacteal transfer of IgA, and in many species, IgG, both of which
provide the offspring with humoral immunity to invading agents (Butler, 1979;
Lascelles, 1977). Most milks also contain immunocompetent phagocytic cells that are
thought to protect the gut of the suckling young from bacteria and fungi (Head & Beer,
1978; Richards, 1979). In addition, milk contains T-lymphocytes concerned with
cell-mediated immunity (Richards, 1979).
The fact that milks universally protect suckled offspring from microbial attack is
evidence of a function that predates the common ancestor of living mammals. Given the
general association of mucosal surfaces with IgA (Gregory & Allansmith, 1986) and
anti-microbial factors, such substances could have been incidentally produced by
glands of an incubation patch in a mammalian or reptilian ancestor. In fact, if ingested
proto-lacteal secretions did offer hatchlings anti-microbial protection, the dilute and

Origins of lactation and evolution of milk

13

immunoglobulin-rich colostrum of living mammals may be more similar to ancestral


milk than is the nutrient-rich milk produced later in lactation.

3. The proto-lacteal secretions regulated pouch fIora through their anti-microbial


properties. Because this hypothesis rests on the questionable assumption that pouches
were present in the ancestors of mammals, we regard it as the least likely alternative. As a
warm, moist and dark environment, a pouch provides ideal conditions for bacterial
proliferation and development of cutaneous mycoses. Fecal contamination from the
pouch young can also contribute significantly to heightened bacterial levels (Charlick
et al., 1981). For eggs or hatchlings to be incubated successfully in a pouch could require
that an appropriately non-pathogenic microflora be physiologically selected and maintained at levels compatible with offspring viability. The study by Charlick et al. (1981)
on the Quokka Setonix brachyurus is particularly interesting in this regard. In this
species, the epithelium of the pouch and the pouch microflora undergo marked changes
during the course of gestation and pouch incubation of the neonate (A. Cockson, personal communication). Although Gram-negative bacilli constitute 99y0 of the pouch
bacteria during the course of oestrus, by the end of pregnancy these bacteria are
virtually non-existent. The mammary gland or pouch epithelium possibly produce
unidentified anti-bacterial substances responsible for this dramatic decrease (Yadav,
Stanley & Waring, 1972; Charlick et al., 1981). Given that the mother licks the pouch
clean prior to birth, lysozyme of salivary origin may contribute to the qualitative change
in the flora of the pouch (Charlick et al., 1981).
The postulate that anti-microbial properties of proto-lacteal secretions enhanced egg
or offspring survival rests in part on the inference that microbial attack on eggs constitutes an important selective pressure. Strong, if indirect, evidence for this inference is
presented by the broad spectrum of adaptations that have evolved in vertebrates for the
protection of eggs from microbial attack. Some of these adaptations were mentioned
previously, such as the lysozyme, ovotransferrin and shell of avian eggs. In amphibians
and osteichthyans, the jelly coat of the egg capsule protects the eggs from bacterial
invasion (Gabaeva, 1962; Movchan & Gabaeva, 1967; Uhlenbruch et al., 1972). The
albumen of chelonian eggs provides similar protection (Movchan & Gabaeva, 1967), as
may the phenols of the chondrichthyan egg case (Wourms, 1977). In certain frogs and
salamanders, parental care enhances survival of the eggs by reducing pathogenic infection of the clutch (Salthe & Mecham, 1974; Forester, 1979). Removal of spoiled eggs by
nest-guarding lizards has also been reported (Tinkle & Gibbons, 1977). In reptiles, a
selective advantage of maternal egg retention and viviparity over simple oviparity may
be the reduced exposure of the eggs to bacterial and fungal infection (Fitch, 1970).
Intense mortality via soil microbes and invertebrates may even have been the selective
pressure leading to evolution of the specialized calcareous eggshell of birds (Packard
& Packard, 1980). Additional examples of selective pressures exerted by microbial
infection are provided by Janzen (1977).
Our hypothesis also suggests that the original protective functions of the protolacteal secretions have been partially obscured by the nutritional importance of milk.
However, certain peculiarities of the milks of some species may reflect the original antimicrobial functions of the proto-lacteal secretions. For example, milk of the monotreme
Tachyglossus aculeatus contains at least two potentially microbicidal substances in very
high concentrations, lysozyme and iron-binding protein. Hopper & McKenzie (1974)
reported lysozyme concentrations in samples of Tachyglossus milk ranging from 6 to
450 mg/dl, in contrast to concentrations of < 1 to 24 mg/dl in other mammals. Echidna

14

D . G . Blackburn, V . Hayssen and C.J. Murphy

lysozyme is reportedly unusual in that it exhibits enzymatic and iso-electric properties


typical of lysozyme, but also has a weak ability to stimulate lactose synthesis (Hopper &
McKenzie, 1974). The precise role of Echidna lysozyrne is not known, and is one of
several aspects of this molecule currently under detailed investigation (Teahan,
McKenzie & Shaw, 1986; McKenzie, personal communication). However, no direct
evidence of an a-lactalbumin has been found in Echidna milk and efforts to isolate such a
molecule have not succeeded (McKenzie, personal communication, 1986). Information
currently available raises the possibility that Echidna lysozyme has structural and
functional attributes of both lysozyme and a-lactalbumin, although further research
is necessary to confirm whether the protein actually performs the functions of both
substances.
Another potentially bactericidal substance found in high concentrations in
Tachyglossus milk is an iron-binding protein identified as transferrin (Jordan & Morgan,
1969). Although largely synthesized by the mammary gland (Jordan & Morgan, 1970),
milk transferrin is similar or identical to blood serum transferrin (e.g., Dayal et al.,
1982), which has well known bacteriostatic properties (Reiter & Oram, 1967). Transferrin is thought to be important in providing iron to the young of Tachyglossus and certain
marsupials, in which both iron and transferrin concentrations of the milk are high
(Griffiths, 1978; Griffiths, McIntosh & Coles, 1969). However, this functional relationship is not invariant, for the concentrations of iron-binding protein in milks of other
species do not closely correlate with iron content (Masson & Heremans, 1971). For
example, in rabbit milk, which also exhibits high transferrin concentrations, this protein apparently does not facilitate iron transfer from the mother to the suckling and the
actual iron content of the milk is low (Tarvydas, Jones & Morgan, 1968; Loh & Calder,
1973).

Most likely, both transferrin and lysozyme originally had bacteriostatic functions in
proto-lacteal secretions, and were co-opted for nutritional functions in some lineages
during milk evolution. In contrast to these substances, the several other anti-microbial
factors of milk serve functions similar to those hypothesized for ancestral forms by
protecting the offspring and mammae from infection. In other words, rather than
being lost in antiquity, the original functions of proto-lacteal secretions may have been
supplemented (but not supplanted) by the evolution of a nutrient-rich secretion.

EVOLUTION OF MILK
The trend towards nutritious lacteal secretions
In considering the selective pressures that led to the production of nutrient-rich
secretions, one must distinguish between advantages associated with the early stages in
the transition towards milk and those that accrued only after the capacity for production
of highly nutritious secretions had been achieved. The situation is comparable to the
evolution of vertebrate matrotrophy (extra-vitelline nutrient provision to the offspring, see Wourms, 1981) in general. Many of the hypothetical advantages of vertebrate matrotrophy were probably not responsible for the evolutionary origins of that
nutritional pattern, but could have operated only after a substantial degree of
matrotrophy had already evolved (Blackburn, 1985a).
The earliest stages in milk evolution did not necessarily involve a fundamental qualitative change from production of non-nutritive secretions. The inferred evolutionary
relationship between epitrichial glands and the mammary gland suggests that protolacteal glands may have had the ability to secrete complex organic molecules already

Origins of lactation and evolution of milk

15

(supra vide). Substances produced by these ancestral glands could have been ingested by
the hatchlings (Graves & Duvall, 1983) and/or secreted onto the eggs and taken up by
the yolk sac (see Hogarth, 1976) or the allantoic vasculature. (Absorption of glandular
fluids by shelled eggs has some precedent; prior to oviposition, monotreme eggs swell
through absorption of oviductal secretions to 4-5 times their original diameter; Hughes
& Carrick, 1978). In the incipient evolutionary stages, lacteal provision of extravitelline nutrients to the offspring was conceivably an incidental consequence of a
secretory process that satisfied an alternative function.
Immediate advantages of the ingestion or absorption of weakly-nutritious lacteal
secretions would accrue to the offspring, which could increase their body size or energy
stores with minimal energetic cost to themselves and the mother. As a consequence, a
selective regime would be provided for more efficient absorption or ingestion of the
maternal secretions. The indirect benefits to the female of enhanced offspring survival
would be increased in turn through evolutionary development of physiological control
over the secretory process. This control could have been manifested through facultative
addition of nutrients to the lacteal secretions and/or termination of the secretory process, according to availability of food or maternal energy reserves. Such facultative
provision of nutrients for offspring growth could have provided the female with a means
of tailoring reproductive investment to environmental conditions.
Once proto-lacteal secretions began to account for a significant share of nutrient
provision to the offspring, further advantages could begin to accrue to the mother. At
this stage, any of a variety of selective pressures could have produced an evolutionary
trend towards obligatory production of nutritious lacteal secretions, as a replacement
for vitelline (yolk) nutrient provisions. Several possible selectiveadvantages of lactation
have been discussed by Pond (1977,1983, 1984). By providing energy to the offspring
in the form of milk, lactation allows adults to exploit environments lacking in food
resources suitable for the young (Pond, 1983). Furthermore, because the lactating
female can draw upon nutrient reserves in synthesizing milk, lactation offers the female
and her dependent offspringsome independence from spatial and temporal fluctuations
in food resources (Pond, 1977, 1984). In addition, lactation appears to be related to
rapid offspring growth and early attainment of reproductive maturity (Pond, 1977).
Lactation also tends to decrease the physical burden imposed on the mother by the
yolky oocyte or egg prior to oviposition.
Other potential advantages of lactation reflect a change in the timing of maternal
energy investment into reproduction. Whereas females of typical oviparous vertebrates
make the bulk of their reproductive investment at ovulation, highly matrotrophic
females spread their investment over a longer time-period. This postponement of
nutrient investment may:
1 allow the female to minimize the energy drain associated with vitellogenesis
(Packard, Tracy & Roth, 1977);
2 diminish the potential costs of loss of the developing reproductive product
(Wourms & Cohen, 1975), and
3 offer the female an extended time period in which to terminate her reproductive
investment with minimal loss of energy, should this be warranted by deteriorating
environmental conditions (Parker, 1977; Low,1968; Vitt & Blackburn, 1983; Hayssen,
Lacy &Parker, 1985).
Although these last three selective advantages have been proposed in reference to
pre-pamun matrotrophy, they also appear applicable to lactation, which, in a broad
sense, can be viewed as a type of post-pamun matrotrophy (Blackburn, 1985a).

16

D.G . Blackburn, V.Hayssen and C.3. Murphy

The general evolutionary trend we postulate can be summarized as three successive


stages:
1 provision of small quantities of nutrients to the offspring as an incidental
consequence of a process satisfying alternative functions;
2 yolk supplementarion, possibly of a facultative nature;
3 obligatory yolk replacement.
This scenario is derived from a more general scenario for the evolution of vertebrate
matrotrophy (Blackburn, 1985a)and is similar to a scenario for the evolution of placental nutrient transfer in reptiles (Stewart, 1989).Only achievement of the last of the three
stages (obligatory matrotrophy) would have made possible the oviposition of altricial
offspring that require mammary secretions for survival. Hence, if production of highly
altricial offspring characterized the common ancestor of prototherian and therian
stocks, as is often suggested (e.g., Lillegraven, 1975, 1979), then evolution of a highly
nutritious milk certainly must have preceded divergence of these two stocks.
The milk of all known mammals, including monotremes, is rich in carbohydrates,
proteins and lipids (e.g., Oftedal, 1980; Griffiths, 1983). Thus, the sequence in which
various nutritional components of milk were added evolutionarily will probably never
be known. Nonetheless, reconstruction of important events in the evolution of milk may
be possible through reference to living species. We shall attempt to discern some of
these events, concentrating chiefly on those that preceded radiation of the three major
extant groups of mammals. In so doing, we must focus on features that milks have in
common, rather than on the extraordinary interspecific and ontogenetic diversity
exhibited among mammals. Such diversity, and its functional significance, have been
reviewed several times in considerable detail (e.g., Jenness, 197O,1974ayb; Jones, 1977;
Oftedal, 1981).

Carbohydrates
Lactose and its derivatives constitute the great majority of milk carbohydrates in most
mammals. Carbohydrates make up between 1 and 181.1 of the calorific value of milks
studied. However, in many species, milk carbohydrates may be more important as a
source of raw materials for synthesis thon as a source of energy (Jones, 1977). Milk
carbohydrate also plays a crucial role in drawing water osmotically into milk; consequently, when lactose secretion is inhibited, water secretion is also arrested (Peaker,
1977). Lactose also promotes calcium absorption from the gut of the suckling mammal
(Wasserman & Lengeman, 1960; Wasserman, 1964) and other evidence suggests that
these carbohydrates have the capacity to determine the flora of the digestive tract of the
suckling (Jenness, 1974a).
Lactose and lactose derivatives are ideal carbohydrates for lumenal storage.
Compartmentalization of this carbohydrate is feasible because the epithelium lining
the alveoli and ducts is impermeable to lactose (Peaker, 1977). In contrast, monosaccharides such as glucose are inappropriate for bulk storage; the permeability of the
cell membranes to glucose would require energy espenditure to maintain the concentration difference of the monosaccharide across the apical surface of the alveolar cells
(Jones, 1977). In fact, even if cell membranes could be made impermeable to monosaccharides, replacement of the lactose of carbohydrate-rich milks by monosaccharide
would make the milk hypertonic to intracellular fluid, requiring energy expenditure to
maintain the same concentration (Jones, 1977). The mammary gland is not the only case
in which carbohydrate compartmentalization has involved differential synthesis of a

Origins of lactation and evolution of milk

17

particular polysaccharide; for example, fructose secreted by the seminal vesicles


constitutes the primary energy source for spermatozoa (Bloom & Fawcett, 1975).
Given the nutritional and non-nutritional importance of milk carbohydrates, the
evolution of a-lactalbumin from lysozyme would have been one of the most significant
events in the development of milk. This event had at least four important consequences:
1 it made possible the synthesisofmilk carbohydrates from ubiquitous precursors;
2 it provided a means of precisely controlling the extent and timing of lactose
synthesis (see Brew, 1969);
3 it provided carbohydrates in a form suitable for lumenal storage;
4 it provided an osmotically active substance that permitted storage of a secretion
with a high water content.
Additional consequences may have included promotion of gut absorption of calcium
and establishment of the intestinal flora of the hatchling.
The directionality of evolutionary trends in milk carbohydrate synthesis is unclear.
Although lactose is the principle milk sugar of eutherian mammals, oligosaccharide
lactose/derivativesand various monosaccharides predominate in monotremes (Messer
& Kerry, 1973; Messer et al., 1983) and marsupials (Jenness, Regehr & Sloan, 1964;
Bergman & Housley, 1968). One proposed explanation is that non-lactose carbohydrates are primitive (plesiomorphic) for mammals and that subsequent evolution in
eutherians led to modifications that prevented acceptance of additional sugar residues
by lactose (Jones, 1977). However, oligosaccharide production does not imply an
inability to synthesize lactose, for studies on the Tammar Wallaby Macropus eugenii
reveal an ontogenetic shift early in lactation from lactose synthesis to production of
larger oligosaccharides (Messer & Green, 1979; Messer, Griffiths, & Green, 1984; also
see Findlay, Ward & Renfree, 1983). Thus, lactose was quite possibly present in
ancestral milks as it is in eutherian milks, with living monotremes and marsupials being
derived with respect to milk-carbohydrate content. Production of non-lactose carbohydrates may have its advantages; oligosaccharides in eutherians have been shown to
select intestinal flora of the newborn (Gyorgi & Rose, 1955; Kuhn, 1958) and monotreme oligosaccharides are thought by some to have an important nutritional role as well
(Messer & Kerry, 1973).

Proteins
The milks of all mammalian species studied contain considerable quantities of protein.
Protein serves as the source of amino acids for the suckling young, and generally provides between 15 and 30% of the energy of milk (Jenness, 1974a; Jenness & Sloan,
1970). There are two major groups of milk proteins, the caseins and the whey proteins.
Most of these proteins are specific to milk and are synthesized from amino-acid precursors by the same processes typical of other eukaryotic cells. Some whey proteins,
however, are derived from proteins of the blood (Larson & Jorgensen, 1974; Mepham
1977).
Although it is not known whether casein was a constituent of the earliest milks, we
can infer from its functional importance that casein secretion must have preceded any
substantial trends towards production of highly altricial young. Milk caseins serve two
very important nutritional functions; they are the major source of essential amino acids
to the suckling young, and they can bind the phosphorus and calcium that are necessary
for bone growth (Mepham, 1977). The ability of caseins to aggregate in stable micelles
greatly increases the carrying capacity of milk for those minerals (Jenness, 1974a).

18

D. G . Blackburn, V . Hayssen and C.3. Murphy

Larson & Jorgensen (1974) speculated that caseins originally performed some cellular
structural role that has been supplanted by their nutritional function.
For reasons discussed previously, the evolution of a-lactalbumin, a major milkspecific whey protein, was an important step in the transition to nutritional secretions.
However, in addition to its role in lactose synthesis, a-lactalbumin itself is of nutritive
value (Jenness, 1970, 1974a). Whether this nutritional role preceded or accompanied
the synthetic function during the transition from lysozyme to a-lactalbumin is unclear,
although further study of monotremes can be expected to clarify the issue.
The immunoglobulins of lacteal secretions have been implicated in the transfer of
passive immunity from the mother to the offspring. In addition, lacteal IgA coats the
oral cavity and the gut of the neonate and agglutinates resident bacteria, thereby protecting the mucosal surfaces from microbial invasion (Butler, 1974; Lascelles, 1977).
IgA universally is transferred in mammalian colostrum and milk, whereas IgG may be
transferred through mammary secretions, placental means, or both, depending on the
species (Butler, 1974).
Certain evolutionary inferences can be drawn from the taxonomic distribution of
immunoglobulin provision. IgG is supplied via the yolk in reptiles, birds and probably
monotremes, presumably representing the primitive condition for mammals
(Pumphrey, 1977). According to a scenario by Pumphrey (1977), the evolution of
microlecithal eggs eliminated the source ofmaternal IgG, presenting a problem that was
solved differently and independently by various therian groups. Some eutherians
transfer IgG via a specialized yolk sac placenta, others via a haemochorial placenta and
still others (such as ungulates, carnivores, and perhaps marsupials) via the milk. As
indicated above, the widespread provision of IgA via the milk is probably primitive for
mammals.
Among the other whey proteins are the iron-binding proteins and other microbial
inhibitors that were discussed previously, and proteins (i.e. 8-lactoglobulin) which are
taxonomically restricted and almost certainly not primitive for mammals. The remaining whey proteins are found in minute quantities, and their taxonomic distribution and
functional significance are poorly known (Groves, 1970; Jenness, 1970,1974a).
Lipids
The lipid compositionof milk in numerous species was summarized by Jenness (1974a).
Lipid concentration varies from trace quantities to 500 g/l and can furnish over 750," of
the total milk-calories in some species. Milk lipid consists mainly of triglycerides synthesized from fatty acids in the mammary gland. These fatty acids are taken up from the
blood or synthesized de n m o (Bauman & Davis, 1974). 'The long-chain fatty acids that
predominate in numerous species are probably derived mainly from the diet. This
pattern is found in such diverse groups as monotremes, marsupials, rodents, carnivores,
cetaceans and artiodactyls (Jessess, 1974a; Griffiths, 1978),and presumably is primitive
for mammals.
There are at least two potential advantages to producing a milk rich in short- or
medium-chain fatty acids. First, such fatty acids are more easily absorbed into the
blood stream of the suckling young (Dils, Clark & Knudsen, 1977). Secondly, a way of
ensuring that milk fat remains liquid at body temperature is by lowering its melting
point through increasing the proportion of short- and medium-chain fatty acids
(Jenness, 1974b). As these fatty acids are absent from the diet, they must be synthesized
by mammary tissue. In many non-ruminants, such fatty acids are synthesized from

Origins of lactation and evolution of milk

19

glucose via the citric acid cycle (Bauman & Davis, 1974). In at least some such species,
an acylthioester hydrolase terminates fatty acid elongation at short- and medium-chain
lengths (Dils et al., 1977).
Reconstruction of the evolutionary history of the production of short- and mediumchain fatty acids awaits further information on the taxonomic distribution of these
synthetic mechanisms. Another mechanism is exhibited by ruminants, which synthesize short- and medium-chain fatty acids from acetate and 8-hydroxybutyrate derived
from rumenal fermentation (Bauman & Davis, 1974). This mechanism clearly is linked
to ruminant specializations for herbivory, and thus cannot be primitive for mammals.
T o sum up, the evolutionary development of nutrient-rich milks appears to have
involved all of the following: use of pre-existing synthetic machinery; modification of
pre-existing synthetic pathways and enzymes; development of new synthetic pathways,
and mammary uptake of certain blood constituents of hepatic or dietary origin. Close
attention to the taxonomic distribution of particular synthetic mechanisms, products
and enzymes are likely to reveal more details of the evolutionary history of milk among
extant orders.

A REVISED SCENARIO
Based on the evolutionary reconstruction presented above, we suggest the following
scenario (as modified from those of Gregory, 1910; Long, 1969; Graves & Duvall, 1983)
to be most consistent with available evidence and theoretical considerations:
1 Evolution of egg-incubation behaviour in a mammal-like reptile,
following development of hair, endothermy, and cutaneous glands.
2 Evolution of a well-vascularized incubation patch on the ventral
abdomen.
3 Enhancement of egg survival through the anti-microbial properties of
secretions of cutaneous glands of the incubation patch. Offspring survival also
may have been enhanced at or following this stage through ingestion or egg absorption
of the weakly-nutritious secretions, which constituted a supplement to vitelline (yolk)
nutrients.
4 Hypertrophy of cutaneous glands of the incubation patch, with production of a somewhat more copious secretion. Hormonal control of glandular
hypertrophy and secretion may have been developed by this stage.
5 Gradual evolutionary shqt towards maternal secretion of a more
nutritious product, associatedfirst with a supplementation and then with a
replacement of yolk nutrient provision. The beginning of this evolutionary
change may have been concomitant with the trend towards cyclically hypertrophied
lacteal glands. The mammary-gland prototype evolved synthesis capabilities in part
through co-optation and modification of existing synthetic pathways, enzymes and
end-products, The trend towards production of nutritious secretions may have been
associated with a general shift from facultative to obligatory provision of extra-vitelline
nutrients, as evidence suggests has occurred in non-mammalian matrotrophy.
6 Concomitant increases in milk-nutrient content, ingestion emciency
(suckling.), and delivery eficiency (areola formation, hormonal control of
milk expulsion). The similarities of monotreme and therian lactation, as demonstrated in publications by Griffiths and his colleagues (Griffiths, 1965, 1968, 1978;
Griffiths et al., 1969, 1973) indicate that these features evolved prior to divergence of
monotremes from therians.

20

D.G . Blackburn, V . Hayssen and C .J . Murphy

7 Following divergence of the ancestors of extant mammals, the evolution


of specializations such as teats (then'ans), and a degree ofphysiological control that allows concurrent production of v e v darerent milksfrom adjacent
glands. Simultaneous asynchronous lactation has been demonstrated among
macropodid marsupials (Griffiths, McIntosh & Leckie, 1972; Lincoln & Renfree,
1981), but not in eutherians. As a group, marsupials rely more heavily than eutherians
on lactation for offspring development (Pond, 1977; Hayssen et al., 1985; Renfree,
1983) and some marsupials exhibit the most specialized and complex form of lactation
known for mammals (Griffiths et al., 1972).
This scenario withholds judgement on the timing of pouch evolution. Unlike Long
(1972), we do not believe that a valid lactation scenario must account for the evolution of
the pouch as well. By making extreme altriciality possible, lactation made pouches
adaptive. If pouches are neither homologous among marsupials (Woolley, 1974;Russell,
1982) nor between marsupials and monotremes (Bresslau, 1920), pouches probably
evolved independently in multiple lineages after the divergence of prototherian and
therian stocks, and long after the development of lactation.
Reconstruction of the role of hormones in the evolution of lactation is beyond the
scope of this review. However, we note that for mammary gland hypertrophy and
regression to be effectively synchronized with the reproductive cycle would have
required hormonal control early in the evolution of lactation. In fact, a degree of hormonally controlled gland development and secretion may have predated substantial
glandular hypertrophy, particularly if mammary glands are more closely related to
sebaceous glands than to sweat glands. Prolactin is known to affect the growth and
activity of sebaceous glands and mammary glands (Ebling, 1967; Nicoll & Bern, 1972),
as well as a-lactalbumin and casein synthesis (Rosen, 1981; Ray et al., 1986).An investigation of the effects of prolactin on lysozyme synthesis may prove enlightening in this
regard.
Our scenario makes little attempt to explain the transition to ingestion of maternal
secretions by the hatchlings. Given the evolutionary plasticity of behavioural change
(Bartholomew, 1964), we tend to view the evolution of ingestion as involving microevolutionary modifications of existing behaviour patterns. Accordingly, ingestion of
maternal secretions could have been accomplished through minor changes in the context and timing of expression of the reflexes associated with typical ingestion behaviour.
An alternativt-but not necessarily contradictory-aplanation for this evolutionary
transition is presented by Graves & Duvall(l983) and Duvall(l986).

CONCLUSION
Ideally, evolutionary scenarios are an early stage, rather than a final step, in reconstructions of evolutionary history. Such scenarios are not directly testable because they are
complex explanations based on inferences about historical events long past. Rather,
estimates of the plausibility of a given evolutionary hypothesis largely depend on the
predictive value of that hypothesis, and its congruence with observable facts and
accepted paradigms. Unfortunately, the paucity of facts bearing on the origins of
lactation and milk renders any evolutionary explanation speculative, the present ones
included.
However, an attractive feature of the scenario developed herein is that several lines of
investigation may produce evidence on its validity. Studies addressing the following
questions seem particularly likely to provide information relevant to the hypotheses
presented.

Origins of lactation and evolution of milk

21

1 . Microbial predation.
If microbial attack was a major cause of egg or hatchling mortality during the origins of
lactation, evidence may still exist among the living reptiles and monotremes. What types
of micro-organisms populate the eggshells and hatchlings of (non-avian) amniotes, and
what is their degree of pathogenicity?How significantis microbial infection of eggs and
hatchlings in living monotremes and reptiles? Do monotreme eggs exhibit any defences
against microbial attack? Do prototherians provide any indirect evidence that microbial
infection of eggs or hatchlings occurred in ancestral forms?

2. Evolution of microbial inhibitors.


In addition to lysozyme and lactoferrin, are other microbial inhibitors found in milk
also produced by mammalian (or even reptilian) integumentary glands? What role do
lactenins play in the protection of the neonate or hatchling in conservative mammals?
How is the microflora of the pouches and nests of mammals regulated? Are any of the
microbial inhibitors in milk and cutaneous gland secretions effective against microorganisms h a t populate the eggshells of monotremes and reptiles? Is there any possibility that egg survival is enhanced by cutaneous gland secretions during incubation in
extant monotremes? What is the function of lysozyme in monotreme milk and transferrin in lagomorph milk? Do taxonomic comparisons or cladistic analyses suggest high
concentrations of lysozyme, transferrin, or other lactenins to be plesiomorphic for
mammals? What do such comparisons indicate about the evolution of mechanisms of
immunoglobulin transfer?

3. Matrotrophy.
Given that lactation is a special type of matrotrophy, information on the evolution of
matrotrophy may be relevant to the evolution of lactation. Matrotrophy is known to
have arisen in numerous animal groups (see Hogarth, 1976; Mossman, 1987); a recent
analysis indicates that substantial matrotrophy has evolved independently in at least 27
vertebrate lineages (Blackburn, 1985a, unpublished data; Blackburn, Evans & Vitt,
1985). What selective pressures have led to matrotrophy in these lineages? What
historical sequences and evolutionary transformations are involved?Does phylogenetic
analysis support the hypothesized transformation from incipient matrotrophy to facultative matrotrophy to obligatory matrotrophy? What is the adaptive significance of
matrotrophy? How broadly applicable is evidence on pre-partum matrotrophy to the
post-partum phenomenon of lactation?
4. Milk evolution.
The evolution of a-lactalbumin from lysozyme conferred upon the mammary gland the
ability to synthesize and store carbohydrates. What can be deduced of the evolutionary
histories of other enzymes involved in milk synthesis?Can any other enzymes trace their
origins to those of other integument-derived glands?What other synthetic pathways are
shared by such glands? Both prolactin and progesterone have been shown to affect alactalbumin synthesis and secretion (Quirk et d.,1985; also see Ray et d.,1986), as well
as the development and function of sebaceous glands and mammary glands. Do these
hormones affect lysozyme synthesis?Are there any other similarities in hormonal effects
upon mammary glands and cutaneous glands?
We expect that answers to these and numerous other questions that we undoubtedly
have not anticipated have the potential to produce a sound, empirically based, synthetic
concept of the evolution of lactation and its role in the early history of mammals.

22

D . G . Blackburn, V . Hayssen and C. 3. Murphy

ACKNOWLEDGMENTS
We wish to thank Drs Howard E. Evans, Jason A. Lillegraven, James R. Stewart and
D. W. Yalden for reading and commenting on previous drafts of the manuscript. We are
also grateful to D r Hugh A. McKenzie and Dr A. Cockson for generously sharing
information derived from unpublished studies.
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