The Dermal Chromatophores of Anolis carolinensis

(Reptilia, Iguanidae)
N A N C Y J. ALEXANDER

AND

WOLF H. FAHRENBACH

Departments of Cii tuneous Biology and Electron Microscopy, Oregon Regzonnl

Primate Research Center. Beaverton, Oregon 97005

ABSTRACT
Skin of the back, belly, dewlap and parietal eye were studied as illustrations of
the principal variations of chromatophore arrangement and color range present in Anolis
carolinensis. The fine structure of the chromatophores, disposed in sharply stratified layers,
was investigated. The most superficial chromatophore layer consists of xanthophores, which
impart a greenish-yellow color to the skin. These cells are filled with two types of membranebounded inclusions containing pteridines and carotenoids, respectively. Basal to the xanthophores is a 2-4 cell-deep layer of iridophores. These cells are filled with undulating layers of
birefringent rodlets, presumably guanine, arranged parallel to the skin surface. The most basal
chromatophores are large melanophores, positioned above a thick collagenous basement lamella.
Migration of melanin granules into the dendritic processes, which terminate at the dermoepidermal junction, cause the lizard to change from green to brown. The back skin, which
ranges in color from bright green to brown, contains the full complement of dermal chromatophores. The xanthophore region is much reduced in the cream-colored belly. The dewlap, which
is bright red when extended, bears a thick layer of iridophores through which are scattered
melanophores, and erythrophores containing red pigment. The dermis over the parietal eye
contains only xanthophores and a basement lamella.

Anolis carolinensis, an iguanid lizard,

varies in color from bright green to deep
brown depending upon the background
color of its environment, the illumination,
and temperature. It is usually green at
night and brown in the daytime. The
green-to-brown transformation is under
humoral control (Kleinholz, '38a, b) and
results primarily from migration of pigment granules within the extensions of
melanophores. Historically, the color
changes were considered to result from
an expansion of the melanophores themselves. Dispersal of melanosomes i n
dermal melanophores is controlled by
melanocyte-stimulating hormone (MSH).
When the effect of adrenalin is superimposed on that of MSH, a brownish mottling
of the green skin is produced (Kleinholz,
'38a). Migration of melanosomes toward
the middle of the malanophore is an active
process that requires the expenditure of
energy (Horowitz, '58) and takes about 20
to 30 minutes, whereas the dispersal of
the melanosomes radially (mediated by
MSH) is completed in 4 to 15 minutes
(Carlton, '04; Kleinholz, '35). Waring ('63)
and Bagnara ('66) have published general
reviews of the mechanism of color change
and the cytophysiology of nonmelanophores, respectively.
AM. J. ANAT.,126.41-56

The complex epidermal changes associated with the molting cycle have been
investigated in Anolis carolinensis by
Maderson and Licht ('67) and in various
snakes by Maderson ('65). Ultrastructural
studies of reptile skin have been restricted
to the epidermis of the boa constrictor
(Roth and Jones, '67) and Lacerta vivipara
(Bryant et al., '67). The present study deals
with the morphological corollaries of color
and color change in Anolis carolinensis;
hence, it is limited to the dermis.
METHODS AND MATERIALS

Samples of skin from the back, belly,

dewlap and parietal eye were fixed in
osmium tetroxide or 6.5% glutaraldehyde
in s-collidine buffer; in a solution containing 1% glutaraldehyde, 4% formalin, 3.5%
sucrose in phosphate buffer; or in osmium
tetroxide in Veronal. Thin sections of
Araldite-embedded blocks were stained
with uranyl acetate, potassium permanganate, and lead citrate and examined with
a Philips EM200 or Philips EM300 electron
microscope. Thick sections (l/2or 1 p)
were stained with toluidine blue in 1%
borax for light microscopy.
I Publication nu. 363 from thc Oregon Regional Primatc Research Center, supported in part by postdoctoral training fellowship 1-TIAM-5521-02 and grant no FRO0163 from the National
Institutes of Health.

41

42

NANCY J. ALEXANDER AND WOLF H. FAHRENBACH

bounded and 0.5 p or less in diameter. The

KESULTS
The skin of Anolis carolinensis is modi- more superficial cell type primarily confied into scales that vary in shape and size; tains granules with a n internal array of
each scale possesses both epidermal and concentric membranes typical of pteridinedermal components. The hexagonal scales containing inclusions, called pterinosomes
of the back measure 250 to 400 p i n extent. (Matsumoto, '65). These cells, therefore,
The slightly larger elongate ventral scales correspond more strictly to the allophores
(300 by 500 p) partly overlap each other of Schmidt ('17), i.e., nonmelanin chroand have a keel on the posterior third. The matophores with nonlipid pigment. The
cream-colored scales of the dewlap, a n predominant inclusion of the more basal
extensible reddish throat flap in males, cell type consists of empty-appearing vesialso overlap considerably and measure up cles, their contents having presumably
to 400 by 700 p. The transparent spot over- been extracted. This alcohol-soluble piglying the parietal eye is situated in the ment gives a blue reaction with concencenter of a single scale, whose remaining trated sulfuric acid and represents the
area has the same structure as back skin. carotenoid component of the xanthophores.
The four areas of the skin chosen for The cells contain a few mitochondria and
study illustrate the principal variations of some glycogen, and, like all the other
chromatophore arrangement and thus dermal pigment cells, each is surrounded
cover the color range of this species. The by a thin external lamina, i.e., a n envelopdermis of the back represents the most ing basal lamina (Coggeshall and Fawcett,
complex disposition of chromatophores in '64). A layer of collagen separates the
their usual layers (fig. 1 ) and will be used xanthophores from the epidermis, and
as a basis of comparison. Two subtly differ- thick bundles of collagen pass between
entiated layers of lipophores -more spe- xanthophores t o the deeper regions of the
cifically, xanthophores on the basis of their dermis.
At the periphery of each scale, the
yellow pigment -lie underneath the stratum germinativum of the epidermis. These melanophores and xanthophores termiare followed by a thick stratum of reflect- nate in the dermis and both the epidering iridophores, basal to which are large mis and dermis become thinner. Flattened
melanophores. A thick collagenous base- dendritic cells that contain both pterinoment lamella defines the base of the somes and melanosomes occur amid the
dermis. I n the keeled scales of the belly, iridophores of this area. The pterinothe xanthophore layer is greatly reduced melanophores (fig. 4) have larger pigment
in thickness and the malanophores are droplets than other pigment cells and the
reduced in number (only 5 to 12 as com- pterinosomes are twice the size of those
pared to about 50 per scale in the back found in the xanthophores (0.4 to 1.O p).
The belly xanthophores (fig. 3 ) differ
skin). The dermis over the parietal eye
lacks melanophores and iridophores. The radically from back xanthophores in two
disordered layer of xanthophores over the respects. First, they contain few pigment
parietal e y e is interspersed with much droplets; pigment present is of the pterinocollagen. The dewlap is the only region some type. The cells contain sparse quantithat lacks xanthophores. Instead, a layer ties of glycogen, fibrils, and organelles.
of iridophores directly underneath the Secondly, the regularly-shaped nuclei do
epidermis is followed by erythrophores, not appear in the narrow (0.6 to 3.5 p ) layer
which, like the scattered basal melano- subjacent to the dermo-epidermal juncphores, are particularly prevalent between tion; instead the main portion of the cell is
the scales.
interspersed within the iridophore layer.
Xu~ztkophores. The xanthophores (fig.
I n the parietal eye, again the xantho2) form the most superficial layer (10 p ) phores are of the pterinophore type. The
of the dermis and are indistinctly divided 2 p layer of xanthophores is laced with
into two subsidiary cell types. The cells collagen fibrils and the organization of
contain elongated or deeply lobed nuclei the layer is less uniform.
and massive quantities of two types of
Iridopkores. Below the xanthophores
inclusions, both of which are membrane- is a layer of iridophores (fig. 5), 1 0 to 20 p

DERMAL CHROMATOPHORES OF A N O L I S C A R O L I N E N S I S

deep and 2 to 4 cells thick. The nucleus,

generally basal, lies in a region of cytoplasm containing mitochondria, Golgi
vesicles, a n d s o m e incipient g u a n i n e
platelets in membrane-bounded, electrondense vesicles having granular contents
and varying in size from 120 to 3,500 A
The rest of the cell, as much as 20 p in
horizontal extent, is filled with orderly,
undulating layers of guanine platelets,
each in a membrane-bounded vesicle. The
presumptive identification of guanine a s
the main component of the platelets rests
on the analogous disposition of similar
birefringent inclusions seen in the integuments of fish and frogs (Barraud et al., '59;
Taylor, '66), plus their solubility characteristics (Setoguti, '67). The platelets aTe
short, square rods (1000 x 1000 x 2000 A)
packed in layers predominantly parallel to
the surface of the skin; successive layers
are spaced about 1000 A apart. The arrangement of the platelets in a given cell
is sufficiently uniform that cells appear
variously red or blue when a cross-section
of skin is viewed under polarized light with
a quarter-wave plate in the beam. This
effect is probably due to the rodlets being
viewed on end or from the side, their internal molecular structure being responsible for the sign of the birefringence. In
thin sections the platelets almost invariably fall out of the section, leaving regular
arrays of holes.
Melanophores. The melanophores (fig.
5), the largest chromatophores (14 to 25 p),
usually form a single layer. Their dendritic
processes project a n additional 20 to 40 p
through the iridophore and xanthophore
layers. These extensions taper upward and
terminate in bulbous ends immediately
above the xanthophores. The usually multilobate nucleus lies basally, whereas a
separate, centrally located region of cytoplasm contains most of the mitochondria
and endoplasmic reticulum. The melanosomes are dense, ovoid, or rod-shaped
bodies 0.25 to 0.6 p long and 0.15 to 0.25 p
wide. Many of the granules show traces of
a limiting membrane. Premelanosomes are
common regardless of the molting stage of
the epidermis.
Microtubules, averaging 180 A in diameter, lie in bundles in the processes of the
melanophores. They are, however, unusu-

43

ally difficult to preserve and have the appearance of spindle tubules rather than the
more robust cytoplasmic tubules of 250 A
diameter usually associated with migrating pigments.
Erythrophores. The erythrophores (fig.
S), inserted within the basal zone of the
iridophore layer, are found only in the
dewlap. These cells are flattened and irregular (up to 20 p in maximal extent).
Their cytoplasm is filled with a variety of
granules and droplets of widely differing
opacity, glycogen, and a large, often multilobate nucleus. Various pigments appear
to contribute to their red pigmentation. A
blue color that develops on contact with
concentrated sulfuric acid indicates the
presence of a carotenoid. Pterinosomes in
the same cell and the solubility of the red
pigment in water suggest the presence of
drosopterins. Similar pigments have been
reported for several species of Anolis (Ortiz
and Williams-Ashman, '63; Ortiz et al., '64)
and for the swordtail, Xiphophorus helleri
(Matsumoto, '65).
The red color of the erythrophores is
concealed when the dewlap is in its normal
resting position, but when a cartilage rod
from the hyoid is lowered, the skin between
the scales becomes visible and a bright
orange-red color is seen.
Collagen. The lowest level of the
dermis consists of the basem.ent lamella
(fig. 6), a n orderly array of about 20 layers
approximately 50 p thick in the back skin.
Each layer is about 2 to 3 p thick, its component fibrils running parallel and almost
at right angles to the direction of those in
adjacent layers. The diameter of the nontapering fibrils varies from 100 A to
2400 A.
Pacinian corpuscles occupy the upper
regions of the basement lamella at the
anterior end of the scale. Many unmyelinated nerves course through the dermis
and terminate in the epidermis. These
nerves, particularly prevalent in the dewlap, contain large quantities of glycogen
near their ends. Below the collagen layer,
bundles of myelinated nerves parallel the
blood vessels.
Collagen in the dewlap and parietal eye
is not so well organized into orthogonal
layers. In the dewlap, thick bundles of
collagen are found between chromato-

44

N A N C Y J. A L E X A N D E R A N D WOLF H. F A H R E N B A C H

phores (fig. 7). The diameter of the fibrils

in the dewlap, parietal eye, and belly ranges
up to 1200 A.

DISCUSSION

Color and color change in the skin of

Anolis are effected by various devices. The
basic color of a given area is produced by
the relative thickness and position of the
various layers of chromatophores, color
modulation being produced by the melanophores alone. In the back, deep-lying melanophores, together with the scattering
effect of the semi-transparent iridophores,
provide reflected blue light modified to
green by passing through the yellow lipophores. No green pigment exists. Pigment
dispersed into the melanophore processes
masks the chromatic effect of the deeper
layers. It is unlikely that the scattered
epidermal melanocytes contribute significantly to the tint of the skin.
The cream color of the belly skin depends
primarily on reflection from the iridophores combined with the light passing
through scanty lipophores. This countershading, a common camouflage device, is
maintained in appropriate contrast to the
varying darkness of the back by a thinner
distribution of melanophores, about onefifth of that found in the back skin.
The varying color of the dewlap results
from the mechanical rearrangement of the
light scales and intervening red skin, i.e.,
by the extension or deep wrinkling of this
skin fold. This extensibility is paralleled
by a much less rigid stratification of the
chromatophores than in the back-skin and
is probably aided by the wider septa between them that allows for some slippage.
Similarly, the less developed basement
lamella is presumably related to the mechanical property of this skin being more
flexible than that of the back. It is not clear
whether the extraordinarily large diameter
of collagen fibrils in the back and their
lesser diameter in the dewlapare correlated
with d i f f e r e n t ph>ysical properties or
whether the 2400 A diameter, twice that
observed in any mammalian tissue, is
simply a peculiarity of lower vertebrates.
Kawaguti et0 al. ('65) observed collagen
with a 2000 A diameter in a tree frog.

The dermis of the transparent "pupil"

over the parietal eye is distinguished principally by the absence of shielding pigment, i.e., melanin and guanine, and by the
quite disorderly disposition of the remaining xanthophores and basement lamella.
Of all the chromatophores described,
only the melanophores appear to provide
any degree of pigment movement. Microtubules are found only in the processes of
melanophores and appear to be a corollary
of pigment mobility in such cells, a s described in the melanophores of F u n d u l u s
(Bikle et al., "66; Green, '68). By contrast,
the iridophores provide no morphological
suggestion of cytoplasmic mobility even
though in some fishes and amphibians
these cells respond to MSH (Bagnara, '58,
'66). The regularity of the packing of platelets in the iridophores suggests the degree
of order encountered in (reflecting) tapetal
cells and would be incompatible with pigment migration. The melanophores are
exclusively u n d e r hormonal control
(Kleinholz, '38a, b). Blind animals are
capable of color change in response to
light or darkness, but this phenomenon is
probably mediated by the parietal eye
rather than by a direct response of the
melanophores. The parietal eye, which has
been inspected with light and electron
microscopy in our laboratory, has the same
morphology as that described for other
lizards (Eakin and Westfall, '60; Eakin et
al., '61). Direct innervation of chromatophores, which is known to exist in true
chameleons (Zoond and Eyre, '34) is absent
in Anolis.
The preceding discussion shows that
color production in the dermis of Anolis
depends on the extensive, horizontal layering of chromatophores. Anolis skin basically differs in this particular characteristic
from that of amphibians. In frogs a
grouped disposition of chromatophores has
been described by Schmidt ('20) in which
one cup-shaped melanophore envelopes
several iridophores and xanthophores. This
a r r a n g e m e n t h a s been restudied by
Bagnara etal. ('68)and has been termed the
"chromatophore unit." No such unitary
disposition of chromatophores is seen in
the diverse skin areas of Anolis; hence the
chromatophore unit appears to be a n amphibian (or anuran) characteristic.

D E R M A L C H R O M A T O P H O R E S OF ANOLIS CAROLINENSIS

LITEKATURE CITED
Bagnara. J . T . 1966 Cytology and cytophysiology of
non-mclanophore pigment cells. Internl. Rev. Cytol.,
20: 173-205.
- 1968 Hypophyseal control of guanophores in
anuran larvae. J . Exp. Zool., 137: 265-283.
Barraud. J.. J-M. Bassot and P. Favard 1959 Identification r a d i o c r i s t a l l o g r a p h i q u e e t a s p e c t s cytologiques de la guanine dans le reflecteur d r s
photophores chez Miriirolicris p e r c i i t i i t t i Walbaum
(Teleosteen Mnurolicidac). C.R. Soc. Biol.. ,249:
2633-2635.
Bikle, D., D. G. l i l n e y and K. R . Porter 1966 Microtubules and pigment migration in the melanophores
of Firttdrilfis heteroclitfis L. Protoplasma. 62: 322345.
Bryant. S. V.. A. S. Breathnach and A. A. Bellairs 1967
Ultrastructure of the epidermis of the lizard (Lrrcrrto
uiuipcrrir) at the resting stage of the sloughing cycle.
J. Zool., 252: 209-219.
Carlton, F. C. 1904 T h e colour changes of the skin
of the so-called Florida chameleon. Proc. Amer.
Acad. Arts Sci., 39: 259-276.
Coggeshall. R. E.. a n d D. W. Fawcett 1964 The fine
structure of the central nervous system of the Icceh.
H i r u d o nit~diciirrrlis.J . Neurophysiol., 27: 229-289.
Eakin, K. M.. W. B. Quay and J . A. Westfall 1960
Cytochemical and cytological studies of the parietal
eye of t h e lizard, Scoloporifs occirlentctlis. Z.
Zellforscli.. 53: 449-470.
Eakin. R. M.. and .J. A. Westfall 1960 Further observations on the fine structurc of the parietal eye of
lizards. J . Biophys. Biochem. Cytol.. 8: 483-499.
Green. L. 1968 Mechanism of movements of granules in melnnocvtes of F I I ndir l i r s he t e roclit t i s . Proc.
Natl. Acad. Sci. U . S . A . .59: 1179-1186.
Horowitz. S . B. 1958 The energy requirements of
melanin granule aggregation and dispersion in the
melanophorcs of Aitolis rctro/iifc,iisis. J. Cell and
Comp. Physiol.. 5 1 . 341-347.
Kawaguti. S . . Y. Kamishima and K. Sato 196-5 Electron microscopic study on the green skin of the tree
frog. Biol. J . Okayama U n i v . . 1 1 : 97-109.

45

Kleinholz, L. H. 1938a Studies in reptilian colour

changes. 11. T h e pituitary and adrenal glands in the
r e g u l a t i o n of t h e m c l a n o p h o r e s of A i f o l t s c u r o / i n e ~ ? s i , sJ . Exptl. Biol., 1 5 : 474-491.
-~
1938b Studies in reptilian colour changes.
111. Control of the light phase and behaviour of isolated skin. J . Exptl. Biol., 1 5 . 492-499.
Maderson, P. F. A. 1965 Histological changes in the
epidermis of snakes during the sloughing cycle. J.
Zool., 146: 98-113.
Maderson, P. F. A., and P. I k h t 1967 Epidermal
morphology and sloughing frequency in normal and
prolactin treated Anolis curoliiierisis (lguanidae.
Laccrtilia). J . Morph., 123: 157-172.
Matsumoto. J . 1965 Studies on fine structure and
cytochemical properties of erythrophores in w o r d tail, Xiphoptiorus helleri, with special reference to
their pigment granules (pterinosomcs). J. Cell B i d .
27: 493-504.
Ortiz, E., E. Biiichli. D. Price and H. G. Williams-Ashman
1963 Red pteridine pigments in the dewlaps of some
anoles. Physiol. Zool., 36: 97-103.
Ortiz, E., and H. G. Williams-Ashman 1963 Idcntification of skin pteridines in the pasture lizard Anolis
pti k he l l t t s . Comp. Biochem. Physiol., 10: 181-190.
Roth. S . I . , and W. A. Jones 1967 T h e ultrastruclure
and enzymatic activity of the boa constrictor (Constrictor constrictor) skin during the resting phase.
J . Ultrastruct. Res., 18: 304-323.
Schmidt. W.J . 1917 Die Chromatophoren der Reptilienhaut. Arch. microskop. Anat., 90: 98-259.
1920 Ueber' das Verhalten der verschiedenartigen Chromatophoren beim Farbcnwechsel des
Laubfrosches. Arch. mikroskop. Anat., 93: 41 4-455.
Setoguti. T. 1967 Ultrastructure of guanophores. J .
Ultrastruct. Res., 18: 324-332.
Taylor. J. D. 1966 Electron microscopy of iridophores
in hypophysectomized Runci p i p i e i i s larvae. Am.
Zool.. 6 . 587.
\Vnring. H. 1963 Color change mechanisms of eoldblooded vertebrates. Academic Press, N.Y. 266 p.
Zoond. A,, and J . Eyre. 1934 Studies in reptilian
colours response. Phil. Trans. Roy. Soc. London B,
223: 27-35.
~

PLATE 1
EXPLANATION OF FIGURE

46

Low power electron micrograph of a vertically sectioned back scale. The resting
epidermis consists of the stratum corneum (Co) and the stratum germinativum
(G). The dermis contains three layers of chromatophores. The most superficial
layer contains xanthophores, differentiated into pteridine- (P) and carotinoidcontaining (C) cells. A layer of iridophores (I) is followed by melanophores (M)
with processes that end at the dermo-epidermaljunction. The base of the dermis
consists of an orthogonally layered, collagenous basement lamella (L). x 3,300.

DERMAL C H R O h I A I'OPlIORES OF ANOLIS CAROLINENSIS

Nancy J . Alexander a n d Wul! H.Fnhrenbach

PLATE 1

47

PLATE 2
EXPLANATION OF FIGURE

2 Xanthophore layer subjacent to the epidermis (E), subdivided into two cell
types. The more superficial pterinophores are filled with pigment granules,
called pterinosomes (P), which consist of a concentric lamellar array. Adjacent
to these cells are xanthophores filled with carotinoid pigment droplets (C).
Glycogen is interspersed between the pigment droplets. Subjacent to the
xanthophore is the iridophore layer (I). Occasional extensions of melanophores
containing melanin granules are seen. x 16,000.

48

DERMAL CHROMATOPHORES OF ANOLIS C A R O L l N E N S I S

Nancy J. Alexander and Wolf H. Fdirenbach

PLATE 2

49

PLATE 3
EXPLANATION OF FIGURES

50

Xanthophores of the belly skin. The layer is much reduced in size as compared
to the back skin. The cells contain glycogen and some mitochondria. but practically no pigment. (E). epidermis; (XI, xanthophore; (I), iridophore. x 15,000.

Xanthophores at the pheriphery of the back scales. These cells occur subjacent
to the epidermis and adjacent to iridophores and contain both pteridines and
melanin. x 16,000.

DEKMAL CHROMATOPHOKES 01.

ANOLIS CAROLINENSIS
Nancy J. Alewaiider arid \ W f H.Fahrenbach

Pl.ATk: 3

PLATE 4
EXPLANATION OF FIGURES

Boundary zone between iridophore (I) and melanophore (M) layers in the back
skin. Formation of guanine platelets (represented only as holes in the section)
occurs in the narrow region basal to the iridophore nucleus. x 4.500.

6 Vertical section of the basement lamella. Collagen fibrils, measuring 200 to

2400 A in diameter, are arranged in up to 20 layers of alternating orientation.
x 14,000.

DEHMAL C I I H O V A I OPHORES OF ANOLIS C A R O L I N E N S I S

N m r v J Ale\.indei dnd Wolf H F.ihrenhach

PLATE 4

53

PLATE 5
EXPLANATION OF FIGURES

7 Ii-idophore layer of the dewlap. The iridophores are separated by loose partitions
of connective tissue. X 7,000.
8 An erythrophore, located in the dewlap below the iridophore layer. The varied
pigment droplets are presumed to contain drosopterins. x 43,000.

54

DERMAL CHROMATOPIIOKES OF A N O L l S CAROLlNEiVSlS

Nancy J Alrxmdcv and Wolf H.Fnlircribacli

PLATE 5

55