Zoosyst. Evol. 94 (1) 2018, 195-209 | DOI 10.3897/zse.94.22289

> PENSUFT.

museum fur naturkunde

Ontogeny of Hemidactylus (Gekkota, Squamata)
with emphasis on the limbs

Wessel van der Vos!, Koen Stein?*, Nicolas Di-Poi*, Constanze Bickelmann!

BR WN

Museum fiir Naturkunde Berlin, Leibniz Institute for Evolution and Biodiversity Science, Invalidenstrasse 43, 10115 Berlin, Germany
Earth System Science — AMGC, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium

Royal Belgian Institute of Natural Sciences, Earth and History of Life, Rue Vautier 29, 1000 Brussels, Belgium

Institute of Biotechnology, Research Program in Developmental Biology, University of Helsinki, Finland

http://zoobank.org/122E692A-9E01-47F B-A386-D4E707620460

Corresponding author: Constanze Bickelmann (constanze.bickelmann@mfn.berlin)

Received 15 November 2017
Accepted 12 February 2018
Published 9 March 2018

Academic editor:
Johannes Penner

Key Words

Gecko

in-ovo development

staging table

limb development

bone histology

UCT

immunohistochemistry
ossification

ecomorphological specialization

Introduction

Abstract

Squamate reptiles constitute a major component of the world’s terrestrial vertebrate diver-
sity, encompassing many morphotypes related to ecological specialization. Specifically,
Gekkota, the sister clade to most other squamates, have highly specialized autopodia,
which have been linked to their ecological plasticity. In this study, a developmental stag-
ing table of the gecko Hemidactylus, housed at the Museum fiir Naturkunde, is estab-
lished. Twelve post-ovipositional stages are erected, monitoring morphological embryo-
logical transitions in eye, ear, nose, heart, limbs, pharyngeal arches, and skin structures.
Ecomorphological specializations in the limbs include multiple paraphalanges, hypothe-
sized to aid in supporting the strong muscles, that are situated adjacent to metacarpal and
phalangeal heads. Furthermore, some phalanges are highly reduced in manual digits II
and IV and pedal digits II, IV, and V. Development, composition, and growth of limb
elements is characterized in detail via wCT, histochemistry, and bone histological anal-
ysis. Using known life history data from two individuals, we found an average lamellar
bone accretion rate in the humeral diaphysis comparable to that of varanids. Various adult
individuals also showed moderate to extensive remodeling features in their long bone
cortices, indicating that these animals experience a highly dynamic bone homeostasis
during their growth, similar to some other medium-sized to large squamates. This study of
in-ovo development of the gecko Hemidactylus and its ecomorphological specializations
in the adult autopodia, enlarges our knowledge of morphological trait evolution and of
limb diversity within the vertebrate phylum.

Although squamate reptiles contribute greatly to verte-
brate diversity in modern ecosystems, and exhibit a wide

Gekkota (Squamata; lizards, snakes and amphisbaenians
sensu Wiens et al. 2012) is a speciose and ecologically
diverse clade with a cosmopolitan distribution (Carranza
and Arnold 2006). Yet, molecular and morphological data
for squamate interrelationships disagree substantially
(Losos et al. 2012); however, according to the molecular
data, Gekkota are, together with Dibamidae, sister to all
other squamates (Wiens et al. 2012, Pyron et al. 2013).

range of morphological variation in their limbs related to
ecological specialization, underlying developmental and
molecular mechanisms remain yet poorly studied (Sanger
2012, Pyron et al. 2013). However, importantly, the in-
corporation of taxa from all higher clades in the study of
development and molecules is essential for understanding
evolutionary patterns of development (Millinkovitch and
Tzika 2007, Sanger 2012).

Copyright Wessel van der Vos etal. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

196

Some gekkotan species have the extraordinary ability
of being able to cling to smooth surfaces and of inverted
locomotion because of the properties of their specialized
autopodia (Autumn et al. 2002, Russell 2002, Pianka and
Sweet 2005). Such ability 1s associated with adhesive
toepads which are located ventrally on the distal part of
the digits (Ruibal and Ernst 1965, Russell 2002, Gamble
et al. 2012). In Gekkota, adhesive toepads have first been
reported in fossils from Lower Cretaceous amber depo-
sits of Myanmar (Arnold and Poinar 2008). They evolved
and were lost independently several times within this li-
neage, and allowed them to enter novel ecological habi-
tats (Pianka and Sweet 2005, Gamble et al. 2012). Further
eco-specializations in Gekkota include the presence of
paraphalanges, unique structures only found in the gek-
kotan clade and hypothesized to aid both in the attach-
ment of strong muscles in the digits and in the grasping
power of the limb by applying pressure to the adhesive
toepads (Russell and Bauer 1988). These apomorphic el-
ements are located laterally to the interphalangeal joints
and occasionally on the dorsal and ventral sides, and vary
in number, shape and size (Wellborn 1933, Russell and
Bauer 1988, Gamble et al. 2012). Paraphalanges are either
cartilaginous or bony structures (Wellborn 1933, Russell
and Bauer 1988, Gamble et al. 2012). Paraphalanges have
been correlated with functional demands, such as grasp-
ing and/or climbing, rather than phylogeny (Russell and
Bauer 1988, Vickaryous and Olson 2007, Gamble et al.
2012). They have evolved and were lost multiple times
within Gekkota (Gamble et al. 2012).

Besides these general gekkotan traits, one gekkotan
genus shows additional ecomorphological specializations
in both fore- and hind limbs: Hemidactylus (Fig. 1). It is
placed within Gekkonidae, as sister to Cyrtodactylus (Py-

van der Vos, W. et al..: Ontogeny of Hemidactylus

ron et al. 2013). It has highly reduced phalanges (Russell
1975, 1977, Gamble et al. 2012). More specifically, the an-
tepenultimate phalanx is highly reduced in digits III and IV
of the manus, with a phalangeal count of 2-3-4-5-3 (Rus-
sell 1975, 1977). The pes shows a phalangeal count of 2-3-
4-5-4, with an additional reduced phalanx in digit V (Rus-
sell 1975, 1977). Although phalangeal elements are also
reduced in other geckos, the degree of reduction is never
as pronounced as in Hemidactylus (Gamble et al. 2012).
Here, we present a morphological staging table for
Hemidactylus based on external embryonic develop-
mental traits and compare these to other squamate taxa.
We make further references to characterize limbs which
display ecomorphological specializations, and which we
study using different approaches such as bone histology,
LCT, and immunohistochemistry. Enlarging the database
of well documented developmental character traits of or-
ganisms allows for a more detailed and comprehensive
knowledge of vertebrate morphological diversity. The de-
scribed ecomorphological specializations in the autopodia
of the adult phenotype make Hemidactylus an ideal can-
didate for eco-evo-devo studies (Gilbert and Epel 2009).

Material and methods

Institutional abbreviations

AC — Comparative Anatomy, Musée d‘Histoire Naturelle,
Paris, France; CA— Cold Archive, Museum fiir Naturkun-
de Berlin, Leibniz Institute for Evolution and Biodiver-
sity Science, Berlin, Germany; MK — Museum Koenig,
Bonn, Germany; ZMB — Museum fiir Naturkunde Berlin,
Leibniz Institute for Evolution and Biodiversity Science
(former Zoologisches Museum Berlin), Berlin, Germany.

Figure 1. An adult Hemidactylus female (three years old).

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Zoosyst. Evol. 94 (1) 2018, 195-209

Animal care

The study organism Hemidactylus is a yet unidentified spe-
cies based on a pregnant female which was collected in Su-
dan in 2011 (Fig. 1; ZMB 87075 to 87077; Kirchhof et al.
work in progress). Live animals are housed in an animal-care
facility at the Museum fiir Naturkunde Berlin; dead animals
are preserved in alcohol in the herpetological collection and
the Cold Archive at the Museum ftir Naturkunde Berlin.
Temperature in the breeding room is set to 26-28 °C and pho-
toperiod is maintained at 12 hours daylight: 12 hours of dark-
ness. Seasonal cycles are not mimicked. Animals are fed with
house crickets (Acheta domesticus), fruits, vitamins and cal-
cium powder twice weekly. Females are allowed to oviposit
naturally. Eggs are laid in clutches of one to two. Duration
of post-ovipositional development is approximately 60 days,
comparable to that of the Madagascar Ground Gecko Paro-
edura pictus (Noro et al. 2009). Embryos were sacrificed in
accordance with German Law on Tierschutzgesetz, and Tier-
SchVersV. Embryos were fixed in 4 % pFA in 1X Phosphate
buffered saline (PBS) for 24 h to 48 h, depending on size,
dehydrated and stored in 100 % methanol at -20 °C in the
Cold Archive at the Museum ftir Naturkunde Berlin (Tab. 1).

UCT analysis

UCT scans were performed using a Phoenix nanotom
X-ray|s at the Museum fiir Naturkunde Berlin (Suppl.
material 1: SOM Tab. 1). Scans were reconstructed to a
3D volume using datos|x (Version 2.0, GE Sensing & In-
spection Technologies GmbH phoenix|x-ray). Data were
analyzed and visualized with VolumeGraphics Studio
Max (Version 2.2., Volume Graphics). Scanned hands
were from an adult specimen (Fig. 2; ZMB 87075, SVL
42 mm, over one year old), and a juvenile (Fig. 5C; ZMB
87077, SVL 17 mm, two days old). Datasets and models
are deposited here https://doi.org/10.7479/qs1j-mnjp.

Bone Histology

We used two histo-technological procedures: (1) azan
stained microtome paraffin sections and (i1) ground pe-
trographic sections.

197

For azan staining, limbs were embedded (Leica EG
1160 and Shandon Hypercenter XP) in paraffin, and sec-
tioned with a microtome (Leica 2000R). Sections were
treated following the standard protocol by Heidenhain. In
detail, sections were washed or immersed in xylene paraf-
fin (ten minutes), xylene (twice five minutes), aniline alco-
hol (five minutes), wash in aquadest, azocarmine (25 min-
utes), wash in aquadest, wash in acetic acid, and 5 %
phosphotungstic acid (two times 20 seconds), one to three
hours in 5 % phosphotungstic acid. Hereafter, the tissue
was placed in the aniline blue orange dye (4.5 minutes),
washed with aquadest, 96 % alcohol, absolute alcohol, and
finally four times xylene for five minutes each. Azan stains
both bone and cartilage: dark blue indicates bone and light
blue cartilage. Red indicates muscle fibers, and the nuclei
are stained dark red. A forelimb of a hatchling (Fig. 5A;
cross section no. AS _F_13a, specimen ZMB 87077) and a
juvenile (Fig. 5B; cross section no. A4_F 21a, specimen
ZMB 87076, SVL 33 mm, one year old), and an adult (Fig.
6B, D, E; ZMB 87078, SVL 47 mm, over one year old)
were embedded in paraffin and sectioned. Cross sections
are stored at the Museum fiir Naturkunde Berlin.

For ground sections (Fig. 6A, C), macerated bones
were dehydrated in 70 % alcohol for 48 hours and embed-
ded in epoxy resin (Araldite 2020), cut along the desired
surface, mounted on glass slides and ground to a thickness
of 75 to 50 wm. To enhance contrast, prevent detachment
due to rehydration and generally protect the section, a
coverslip was added. Femoral sections of Jguana iguana
(AC1896 288), Varanus timorensis (MK52920) and Tu-
pinambis teguixin (MK53531) were studied at the histoth-
eca facilities of the Museum d‘ Histoire Naturell in Paris.

Morphometry

Photographs of embryos were taken with a Leica M205C
camera. Photomicrographs for histomorphometric analy-
sis of lamellar bone apposition rates were taken on a Zeiss
Axioskop (HBO 50) with Leica firecam (DFC 420) and
processed in a Leica Application Suite. All measurements
were taken using ImageJ (Rasband 1997) (Tabs 1, 2).

Table 1. Developmental age range and external length measurements of Hemidactylus stages A to L. All embryos were studied to

identify key features characteristic for each stage.

Stage Developmental days Snout vent length | Forelimb measurement Hind limb measurement Number of studied
post oviposition (range in mm) (mean in mm) (mean in mm) specimens
A 4-5 9.3-11.5 n/a n/a 3
B 6 17.4-20.1 Or 0:3 Z
C 9-14 14.1-17.6 0.7 0.6 4
D 11-30 18.9-24.7 135 1.4 11
E 8-30 21.0-24.0 1.8 1.8 Z
is 42-56 23.8-24.0 2.8 2.8 2
G 15-36 23.8-27.2 2.8 2.8 6
H 38-50 26.4-31.1 hl, ofa) 6
| 35-56 29.0-34.2 4.9 4.8 10
J 40 32.8-34.2 53 625 2
K 40-58 36.0-39.4 6.2 6.7 S
L 50-60 37.5-41.9 6.8 6.9 2

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Table 2. Histomorphometric data obtained from humeri of an
adult (ZMB 87078) and a ‘stage I’ (CA2017_ 014). These data
were used to calculate average apposition and lamellar bone ac-

cretion rates.
Humerus Humerus
ZMB 87078 CA2017 014
Adult,
Born 10.12.15,
deceased 22.03.17
Total days lived 468

Days since onset
ossification

Stage |

Life history data development

25 (unborn)

Humerus shaft width 987.6 161.0

posterior | anterior
Nomremogred 139.4 | 912 0.1
No. of lamellae in cortex 40 29 1

Cortical thickness (um)

Immunohistochemistry

Immunohistochemistry was performed on sections, with
a rabbit polyclonal anti-Sox9 antibody (Merck Millipore).
SOX9 is an early marker of cartilage, tendons and ligaments
(Sugimoto et al. 2013). Samples were deparaffinized in xy-
lene and rehydrated through ethanol steps into 1X PBS.
Antigen retrieval was accomplished by citrate buffer treat-
ment (pH = 6.0). Protease XXV treatment (Thermo Scien-
tific) for 15 min was followed by non-specific binding with
5 % goat serum (Sigma). Primary antibody (polyclonal,
rabbit, Millipore) was incubated overnight. Staining was
achieved by DAPI and Fluoroshield (Sigma).

Results

Morphological staging table

A morphological staging table of Hemidactylus estab-
lishing twelve post-ovipositional developmental stages
is presented here (Figs 2, 3; Tab. 1). For this purpose,
differences in pharyngeal arches, otic pit, olfactory pit,
optic cup, and other characters are studied in detail. In
the following, each stage is presented listing key features
which have been established in other squamate staging
tables and which have been considered suitable for char-
acterizing the ontogenetic development (Tab. 3; e.g. Noro
et al. 2009, Wise et al. 2009, Khannoon 2015).

Stage A

Ear: The otic pit is present and located dorsally to the hy-
oid arch (Fig. 2A, B). The cartilage capsule lies dorsally
to the otic pit (Fig. 2B).

Eye: The eye is composed of an optic cup that has not
yet fully enclosed the lens (Fig. 2A).

Heart: The heart shows the distinct s-shape resulting
from the curvature of the developing ventricle. It pro-
trudes from the thoracic cavity.

Nose: The snout starts to form at the rostral end of
the head. The olfactory pit is visible as a sickle shaped

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van der Vos, W. et al..: Ontogeny of Hemidactylus

rim with its curvature pointing to the cranial side and the
two points of the rim facing ventrally, towards the heart
(Fig. 2B). Between the anterior ends of the rim towards
the middle of the dorsal end of the rim, there is a small
membrane covering the pit.

Pharyngeal arches: The mandibular pharyngeal arch is
prominent, with a small bud from the maxillary that has
just started to bud out (Fig. 2A). Posterior to the mandib-
ular pharyngeal arch, the hyoid arch is present. It slightly
overlaps the mandibular arch ventrally at the proximal part.
Ventral to the hyoid arch is a small slit, marking the future
distinction between the hyoid arch and the third arch.

Stage B

Ear: The cartilage capsule dorsal to the otic pit 1s enlarged
(Fig. 2C).

Eye: The optic cup has almost enclosed the lens, ex-
cept for a small gap rostro-ventrally. Note that this is not
the characteristic choroid fissure. The optic cup shows
faint pigmentation at the caudal-dorsal margin (Fig. 2D).

Head: The mesencephalon protrudes prominently in
the cranial direction.

Heart: The heart and its placement in the thoracic cav-
ity have not changed in comparison to the previous stage
(Fig. 2D):

Limbs: Both fore- and hind limbs appear as small buds
(Fig. 2C).

Nose: The rim of the olfactory pit 1s still partially cov-
ered by a membrane.

Pharyngeal arches: The maxillary process has not pro-
ceeded its development compared to the previous stage. The
mandibular and hyoid arches, including the gap between
them (Fig. 2D; indicated by the plus), have increased in size
and show a sharper medial turn at their distal ends, and the
segregation of the ventral part of the hyoid arch is more
distinct than in the previous stage (Fig. 2D). The fourth arch
and the associated pharyngeal slit are present posterior to
the hyoid arch (Fig. 2C; indicated by the asterisk).

Stage C

Ear: The otic pit is enlarged and extends antero-posterior-
ly beyond the mandibular and hyoid arches. There is less
overlap with the cartilage capsule.

Eye: The eye is enlarged and pigmented; it is referred
to as the retina pigmented epithelium. The choroid fissure
is present, and an unpigmented caudal-rostral line divides
the optic cup into a distal and a proximal part (Fig. 2E, F;
indicated by the cross).

Limbs: Both limb buds are proximo-distally longer
than wide. The apical ectodermal ridge (AER) 1s distinct
(Fig. 2G).

Nose: The external nares have developed, replacing
the olfactory pit (Fig. 2E, F). This process of the fron-
to-nasal process replaces the olfactory rim. The dorsal
side of the covering membrane is now connected to the
maxillary process.

Pharyngeal arches: The maxillary process is linked to
the dorsal side of the fronto-nasal process. The mandibu-

Zoosyst. Evol. 94 (1) 2018, 195-209 199

Figure 2. Overview of embryonic developmental stages A to F, erected in this study. Stage A is depicted in (A, B) and represented by
CA2017_007. (C, D) show the representative CA2017_ 003 for stage B. Stage C (E-G) is represented by CA2017_006. CA2017_005 rep-
resents stage D in (H—J). Note the tmmunohistochemistry for SOX9 expression in a limb cross section in (J). Stage E is shown in (K-L),
represented by CA2017_010. CA2017_ 012 represents stage F in (M-O). Except for (J), all pictures in lateral view. Scale bars equal 1mm.
Abbreviations: aer — apical ectodermal ridge; au — autopod; ec — cartilage capsule; ef— choroid fissure; en — external nares; ep — eye pig-
mentation; fp — frontal nasal process; ha — hyoid arch; Ib — limb bud; Il — lateral lines; mda — mandibular arch; mxa — maxillary process;
oc — optic cup; of — olfactory pit; op — otic pit; pa — pharyngeal arches; st — stylopod; te — thorac cavity; zu — zeugopod.

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200

lar arch has budded out. The slit between the mandibular
and the hyoid arch is reduced. The fourth and the fifth
arches are present as small buds (Fig. 2E; indicated by
the double-cross).

Stage D

Ear: The otic vesicle is less prominent in comparison to
former stages.

Eye: The eye displays darker pigmentation, including
its extension rostrally and caudally to the lens (Fig. 2H;
indicated by the upside caret), which is here interpreted as
the precursor of the iris.

Heart: The heart is fully enclosed inside the thoracic
cavity.

Limbs: Fore- and hind limbs show the paddle shape
typically seen in other amniote embryos at corresponding
stages (Fig. 2H, I). An elbow angle between the stylopod
and zeugopod starts developing. SOX9, the precursor of
cartilage, is expressed in the presumptive stylopod, zeu-
gopod, and autopod regions (Fig. 2J). Expression 1s also
distinct in the presumptive regions of the strong limb
muscles (Fig. 2J).

Nose: The rim of the olfactory pit is completely closed.
The maxillary and the medial nasal prominence overlay
the stomodeum.

Pharyngeal arches: The mandibular arch and the hy-
oid arch are now indistinguishable from one another
(Fig. 2H; indicated by the downside caret), and the fourth
arch has begun to grow, becoming larger than the initial
bud. The mandibular arch reaches to the midline of the
eye, and the maxillary prominence has taken up a posi-
tion anterior to the eye.

Stage E

Ear: The otic vesicle further extends over the length of the
mandibular and hyoid arch.

Eye: Lines that are visible lateral to the eye, or primor-
dial iris, flanking the lens, have differentiated further.

Limbs: The most pronounced differences between
stages D and E are identifiable in the limbs. In both
fore- and hind limb, the interdigital tissue is retreating
(Fig. 2K, L; indicated by the circle). A blood vessel is
present at the distal margin of the forming autopod, which
is antero-posteriorly wider than it is proximo-distally
long (Fig. 2K).

Nose: Deep furrows between the nasal and maxillary
prominences are developing further and show invagina-
tions in this region.

Pharyngeal arches: The prominence of the mandibular
and hyoid arches is now located anterior to the lens.

Stage F

Ear: The otic capsule has now become a small pit on the
lateral side of the head.

Eye: The optic cup is darkly pigmented. The lateral
pigmented lines now enclose the lens (Fig. 2M).

Limbs: Recession of the interdigital tissue 1s extensive
(Fig. 3M-O; indicated by the rhomb). The autopodia have

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increased in size, most notably across the antero-posterior
axis (Fig. 2N).

Nose: The olfactory pit has disappeared. The fron-
to-nasal process 1s fully fused (Fig. 3M).

Pharyngeal arches: The pharyngeal arches do not show
the distinction between the different arches anymore
(Fig. 2M).

Stage G

Ear: No further development in comparison to the former
stage (Fig. 3A).

Eye: The optic cup has completely encircled the lens.
The lateral pigmented lines of the lens start to encircle the
lens and intensify in pigmentation.

Head: Cephalic projections, except for the mesencepha-
lon, have disappeared. The head resembles that of a hatch-
ling. Internally, only the calcified endolymphatic sacs and
statolithic masses are visible in the wCT scans (Fig. 3B).

Limbs: The autopodia show further recession of the in-
terdigital tissue, prominently segregating digits I to V. The
elbow angle is at 90°. First signs of slightly swollen patches
on the ventral side of the digits are visible (Fig. 3C).

Nose: The nose has become a pit laterally at the rostral
end of the snout (Fig. 3A).

Jaw: Upper and lower jaws are fully formed.

Urogenital bud: The urogenital bud is located medially
between the hind limbs (Fig. 3A).

Stage H

Eye: There are four distinct changes that are present in the
eye at this stage: (1) The lens has changed from a matte
white color to a more shining white color. (i1) The iris
starts to cover the margins of the lens. (111) The black pig-
mented tissue, starting out as the optic cup, has a glassy
appearance. (iv) The hollow pan surrounding the lens is
lighter than the rest of the eyeball.

Head: The head surface is smooth with few irregulari-
ties. The mesencephalon is still identifiable. Skull bones,
such as parietal and surangular, start ossifying (Fig. 3E).

Limbs: The digits are almost free of interdigital tissue,
and the distal phalanges start showing claw formation (Fig.
3D, F). Limb elements are still cartilaginous (Fig. 3E). On
the ventral side of the digits, swollen pads are distinct, repre-
senting the precursors of the adhesive toepads (Fig. 3D, F).

Stage I

Eye: The iris surrounding the lens starts to display a wavy
pattern but is still confined along the margin (Fig. 3G).

Head: More or less all skull bones are ossifyed
(Fig. 3H).

Limbs: Claws have developed on all digits. Adhesive
toepads are more prominent at this stage: one can dis-
criminate the wavy skin and swellings on the toepads
(Fig. 31). All long bones have started ossifying, as well
as the metacarpals in the forelimbs (Fig. 3H). In the hind
limbs, metatarsals have ossified further compared to the
forelimbs, and ossification of the proximal phalanges has
just set in (Fig. 3H).

Zoosyst. Evol. 94 (1) 2018, 195-209 201

Figure 3. Overview of embryonic developmental stages G to L, erected in this study, including wCT data. Stage G is shown in (A-C),
its representative is CA2017_ 004. CA2017_011 represents stage H in (D—F). (G—I) show stage I represented by CA2017_ 014. Stage
J is depicted in (J—M), represented by CA2017_ 008. Stage K is CA2017_013 and shown in (N, O). (P-R) show CA2017_002 for
stage L. (B) and (E) are in cranial view, (R) in dorsal view, and all others in lateral view. Scale bars equal Imm. Abbreviations: at — adhe-
sive toepads; ca — cornea; cw — claw formation; I— lens; np — nose pit; ot — otic capsule; s — scansors; sp — swollen pads; ub — urogenital
bud; ws — wrinkly skin.

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202

Skin: The skin starts to get wrinkly, a precursor stage
prior to scale formation of the skin (Fig. 3G). Scale for-
mation starts on the tail and proceeds cranially.

Stage J

Eye: Within the eye, the cornea is developing, and the
eye socket begins to enclose the eye (Fig. 3J). The iris has
a distinct shape, with projections pointing to the medial
part of the lens.

Head: The mesencephalon has started to retract, ex-
posing a smooth skull.

Limbs: The limbs are fully developed. Even the scales,
although not yet fully pigmented, are present on the limbs.
The scansors on the ventral adhesive toepads of the digits
now show a rough wavy texture (Fig. 3L). The most distal
part of the adhesive toepads no longer connects dorsally
to the ventral part of each digit. All phalangeal elements
have started ossifying (Fig. 3K, M).

Nose: The nose has moved to the most rostral side of
the snout; it will not change position hereafter.

Skin: Scales now cover the entire body. The skin is dark.

Urogenital bud: The urogenital bud has started retreat-
ing into the body (Fig. 3J).

Stage K

Eye: The eye is located inside the socket, and the scale
ridge surrounding it starts to develop (Fig. 3N). The scler-
al plates of the sclerotic ring start ossifying (Fig. 30).

Head: Fully scaled.

Limbs: Ossification of the long bones continues
(Fig. 30).

Skin: All scales are pigmented. Scales are present on
the upper and lower jaw.

Stage L

Eye: There 1s a rim on the cranial side of the eye, starting
dorsally and wrapping around the upper part of the socket
all the way to the midline (Fig. 3Q). The sclerotic ring is
fully ossified (Fig. 3P).

Limbs: Phalanges and metacarpals and -tarsals con-
tinue ossifying, with the epiphyses not yet being ossified
(Fig. 3R). Yet, neither paraphalanges nor carpals or tar-
sals have started ossifying (Fig. 3R).

Skin: The scales, present on tail, back, limbs, and head
show differences in pigmentation and color (Fig. 3Q).

Limb ossification

The phenomenon of limb reduction is of great interest in
evolutionary biology (e.g. Shapiro 2002), and especial-
ly among squamates, it is frequently found and has been
linked to functional demands. Among the many mecha-
nisms and patterns observed that go hand in hand with
limb reduction, skeletal heterochrony can be one of them
(Shapiro 2002, Hugi et al. 2012). Hemidactylus has re-
duced phalangeal elements, which could represent an
evolutionary step along the path of digit loss (Hopson
1995). In that regard, we analysed limb ossification in
Hemidactylus limbs.

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van der Vos, W. et al..: Ontogeny of Hemidactylus

Between fore- and hind limbs, no heterochronic differ-
ences in size and shape were monitored at any develop-
mental stage (Figs 2, 3; Tab. 1). Ossification sets in slightly
earlier in hind autopodial elements compared to the fore-
limb ones (Fig. 3H). Reduced phalanges ossify at the same
time and in the same order as other phalanges (Fig. 3).

Histochemical staining and wCT scanning show that the
paraphalangeal elements of Hemidactylus are present in
three different shapes (Figs 4, 5): (1) nubbin-like ones later-
ally and dorsally at the distal end of all metacarpals, as well
as at the dorsal side at the distal end of each last phalanx
(Fig. 4; labeled in orange); (11) rhomb-shaped ones distal-lat-
eral to phalanx 1 in digits II, III, IV, and V, as well as phalanx
2 in digit IV (Fig. 4; labeled in red); and (111) oval parapha-
langes located ventrally at metacarpal heads of digits II to
V (Fig. 4; labeled in yellow). All paraphalanges are bony in
one-year-old adults (ZMB 87075, ZMB 87076) (Fig. 4, 5B).
Yet, ossification starts only after hatching; in the two-day-
old specimen (ZMB 87077) it has not yet started.

Long bone histology and histomorphometry

The long bones of one-year-old adult individuals show
a lamellar bone matrix with no vascular spaces (Fig. 6).
Growth lines may sometimes be present in the primary
cortex of individuals over one year old, but in some in-
dividuals they are absent or have been resorbed by med-
ullary expansion. These individuals also show intense
endosteal remodelling features in the diaphyseal cortex,
as seen in longitudinal section (Fig. 6A, B). Different
generations of resorption and redeposition of endoste-
al bone can be seen along the margin of the innermost
cortex, expressed by the cross-cutting relations of the re-
sorption lines. Such remodeling features in the endosteal
region can also be observed in other lizard species, such
as varanid, tegu and iguanid lizards (Fig. 7), but only 7u-
pinambis shows true secondary osteons in the innermost
cortex among sampled squamates.

From an adult individual of known age (ZMB 87078,
468 days old, 493 days since onset of ossification, Fig. 6B,
D, E; Tab. 2), we calculated the average daily lamellar
bone apposition rate since the onset of ossification until
time of death at 0.1 lamellae per day, equivalent to an
average periosteal accretion rate of 0.4 wm per day. We
used the following formulae to calculate accretion rates:

Waauit.hum int ieee) x( gl )
2

O°
N days.onset.oss

Average|apposition.rate] = (

Average|lamellar. accretion. rate]
( Masatieis = Weissiot) x( 1

N days onset oss

N° i
) x ( inesiuce icone)

Worimcortex

Where apposition.rate is the daily accretion rate (in
um/day), lamellar.accretion.rate is the number of lamel-
lae deposited per day, W, 4.4; 18 the width of the adult
humerus (in wm), W....,,., iS the humeral width at the
onset of ossification (in wm), N° baer is the age of
the adult individuals, in days, since the onset of ossifi-
cation, Wa is the thickness of the primary cortex in
the adult individual, and ING etc Sy is the number of

lamellae counted in the primary cortex of the adult indi-
vidual (Measurements in Tab. 2).

Zoosyst. Evol. 94 (1) 2018, 195-209 203

Figure 4. wCT images of the manus of an adult Hemidactylus (ZMB 87075). Left (A) and right (B, C) manus in dorsal (A, B)
and ventral (C) view. Large lateral paraphalanges are shown in red, small nubbin-like ones laterally and dorsally in orange, and
ventral ones in yellow. Note the reduced antepenultimate in pink in (B, C). Scale bar is 500 wm. Abbreviations: de — distal carpals;
m — metacarpals; p —pisiform; pp — paraphalanges; R — radius; r—radiale; rp — reduced phalanges; U — ulna; u — ulnare; 1-5 — pha-
langes 1-5; I-V — digits I-V.

ZMB 87077 (cross section no. in A is AS_F_ 13a), and (B) of the juvenile ZMB 87076 (cross section no. A4_F 21a). Note the yet
unossified paraphalanges in both. In contrast, reduced phalangeal elements have started ossifying. Radius and ulna are not yet fully
ossified, and carpal elements are as yet unossified. Numericals indicate: 1 — trapezium; 2 — metacarpal; 3 — phalanx; 4— paraphalanx;
5 — metacarpal; 6 — phalanx; 7 — paraphalanx. Scale bars in (A) and (B) equal 200 wm, and scale bar in (C) is 550 wm.

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204 van der Vos, W. et al..: Ontogeny of Hemidactylus

Figure 6. Bone histology of Hemidactylus long bones. Adult specimen CA2017_001 (cross section no. H63_FR_B5; A, C), ZMB
87078 (cross section no. A12_FL_6b, one year old, SVL 47 mm; B, D, E), and developmental stage I specimen CA2017_015 (F) and
CA2017_014 (G). (A-E) Longitudinal sections (HE staining) of the humeral shaft showing a clear pattern of alternating bone lamellae
in the periosteal cortex and remodeling in the endosteal region. (C) Close-up of boxed area in (A). (D, E) Magnification of cortical bone
of similar areas in (B). (F, G) Onset of ossification in individuals, 25 days before hatching, as seen in histological sections (F) and wCT
scans (G). Abbreviations: eb — endosteal bone; f — femur; h — humerus; hl — Howship lacunae; me — medullary cavity; pb — periosteal
bone; r— radius; u— ulna. Scale bars in (A, B) equal 200 wm, in (C—E) 100 um, in (F) 500 wm, and 1 mm in (G).

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Zoosyst. Evol. 94 (1) 2018, 195-209 205

Figure 7. Lamellar bone histology and endosteal remodeling in other squamate lizards. (A) Cross section of an /guana iguana femur
(AC1896 288). (B) Cross section of a Varanus timorensis femur (MK52920). (C) Cross section of Tupinambis teguixin femur (MK53531).
All images were taken under cross polarized light. Note the cross cutting relations in the endosteal bone of V. timorensis and Tupinambis.
Also note the more extensive layer of remodeling with secondary osteons in the innermost cortex of Tupinambis. Abbreviations:
eb — endosteal bone; pb — periosteal bone; so — secondary osteon. Scale bar in (A) equals 1 mm, in (B) 250 wm, and in (C) 500 wm.

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206

Discussion and conlusions

Comparative embryology

The in-ovo development of the gecko Hemidactylus
was defined into twelve stages post-oviposition based
on morphological embryonic characters (Figs 2, 3). In
contrast to other staging tables, somite formation is not
taken into account as they are not clearly identifiable
(Hamburger and Hamilton 1951, Muthukkaruppan et al.
1970, Theiler 1989, Sanger et al. 2008). The comparisons
of days post-oviposition proved to be a poor indicator
of actual development of the embryo (Tab. 1), which
we attribute to variations in oviposition timing between
females and/or to fluctuation in temperature (26-
28°C) in the breeding room. Such discrepancy is not
unusual considering the poikilothermic physiology of
Hemidactylus, shifts in temperature are known to have
effects on the developmental age of individuals (Atkinson
1994, Andrews 2004, Goodman 2008, Dayananda
et al. 2017). A similar range was also monitored in

van der Vos, W. et al..: Ontogeny of Hemidactylus

Eublepharis macularius (Wise et al. 2009), Nothobachia
ablephara and Calyptommatus sinebrachiatus (Roscito
and Rodrigues 2012). Instead, our study groups erected
stages based on morphological events regarding the ear,
eye, head, heart, limbs, fronto-nasal process, pharyngeal
arches, skin, and thorax development (Figs 2, 3; Tabs
1, 3). Since limb morphogenesis is an often used and a
very informative criterion in vertebrate developmental
biology, it is also a focus in this study. We not only
generated a framework in which to communicate the
development of Hemidactylus, but we also made direct
comparisons with other staging tables available for a
diverse range of squamate species (Tab. 3), thus allowing
comparative heterochronic studies. In fact, in squamates,
the differential timing of fore- and hind limb is rather
synchronous (Bininda-Emonds et al. 2007). Interestingly,
this is also the case in Hemidactylus, which shows no
heterochrony in limb development (Tab. 1), 1n contrast
to other gekkotan taxa in which forelimb development is
slightly advanced (Noro et al. 2009, Wise et al. 2009).
Furthermore, in Paroedura, which has a comparable

Table 3. Comparison of squamate staging tables. Differing stages were horizontally aligned based on key characters. Comparing
different staging tables allows for the study of heterochrony within squamates, here including geckos, lacertids and iguanians. Fo-
cused on limb development, darkening shades of gray indicate (1) both limb buds present after oviposition, (11) interdigital tissue
starts retreating, (ii1) develops claws on all 5 digits, (iv) first sign of scales on limbs, and (v) limbs fully developed apart from size.
Abbreviations: dpo — days post-oviposition; S, St — embryonic stage.

E Noro et al. | Khannoon | Wise et al. ROSEINO ang Boseie Je. Py-Daniel et al.| Sanger et al. |Muthukkarruppan
This.study,!' 009) || “¢2015)° | ‘(@o09). || ROdHeues; | “Rodrigues (2017) (2008) et al. (1970)
(2012) (2012)
Hemidacty- | Paroedura | Tarentola | Eublepharis | Nothobachia | Calyptommatus Tropidurus :
[US Sp. pictus annularis | macularius ablephara_ | sinebrachiatus torquatus PHIDUS SAS IEE |aP atCies IS ICalor
OQ dpo Stage 27
1 dpo Stage 28
Stage A 2 dpo
3-4-5 dpo Stage 29
Stage B Sa seine
8-9-10 dpo Stage 30
Stage C
12 dpo Stage 31
14 dpo
Sool St
ee St 6 Stage 32
i a ee
StageE | 18 dpo S32 St 33 St 6 St 7 Stage 33
Stage F 20-22 dpo 9 Stage 34
24 dpo SiG Cle: 10 Stage 35
Stage Gea 11
28 dpo | size fT | Stage 36
30 dpo 3 a ee eee)
Stage | —E—— ll Stage 37
—— J Stage 38
ie Stage 39

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Zoosyst. Evol. 94 (1) 2018, 195-209

developmental period, limb budding starts at the third day
of oviposition (Noro et al. 2009). In contrast, we observe
that in Hemidactylus, limb budding does not start as early
as stage B (Fig. 2, Tab. 1). Later in development, Noro et
al. (2009) identify a pattern of heterochrony in the limbs
of Paroedura, which they attribute to the development of
the digits in the gekkotan clade. However, findings of this
study do not support this assumption (Figs 2, 3). Instead,
we found interspecific variation during limb development
within eight squamate species including geckos, lacertids,
and iguanians: (1) concerning limb bud formation after
Oviposition, (11) retraction of the interdigital tissue, (111)
claw development on the ultimate phalanges, (iv) scale
formation on the limbs, and (v) termination of limb
development (Tab. 3). Among geckos, for example, scale
formation on the limbs starts at stage J and 35 dpo in
Hemidactylus and Paroedura, respectively, whereas in
Tarentola and Eublepharis, it is developed at an earlier
stage (S35 and St 37, respectively).

Long bone histomorphometrics

Bone histology shows rapid accretion and endosteal re-
modeling of lamellar bone in the long bone cortices at
later phases in ontogeny. The lamellar bone formation
rate is similar to rates seen in the nearly avascular fib-
ulae of wild Varanus niloticus (de Buffrénil and Casta-
net 2000), suggesting that absolute matrix production
rate could be similar in different squamates. The intense
remodeling in adult Hemidactylus indicates a dynamic
balance of cortical bone thickness during growth. Inter-
estingly, in contrast to Hemidactylus, Iguana, and Vara-
nus timorensis, the Tupinambis (Fig. 7) in our study also
produces secondary osteons, and generally experiences
more advanced remodeling. This difference may be the
result of the combination of the relatively large size and
foraging behavioral ecology of Tupinambis. The studied
Hemicactylus and V. timorensis also typically forage for
food, but they retain relatively small body sizes, and their
lacuno-canalicular networks may provide enough flux to
maintain a healthy bone homeostasis without the need for
additional blood vessels inside the bony cortex.

Eco-specializations of the limbs of Hemidactylus

Our detailed analysis of ecomorphological specializa-
tions in the limbs of Hemidactylus also reveal that, in
contrast to earlier observations (Russell and Bauer 1988),
paraphalanges are bony in the adult, as revealed by wCT
(Figs 3, 4). However, these ossify only after hatching, as
shown by wCT scanning and azan staining (Figs 4, 5).
Furthermore, data from the wCT indicate that more para-
phalanges in various different shapes are present in Hemi-
dactylus (Fig. 4), when compared to other gekkotans
(Gamble et al. 2012). These structures have been sug-
gested to aid in the support of the scansors (Russell and
Bauer 1988, Gamble et al. 2012). In fact, they evolved
also multiple times independently in geckos, alongside
adhesive toepads (Gamble et al. 2012). Paraphalanges
have been suggested to represent sesamoid bones (Rus-

207

sell and Bauer 1988, Gamble et al. 2012). Sesamoids are
neomorphic ossifications present in tendons and/or liga-
ments, located near joints. They are often mistaken for
accessory ossicles because both share various imaging
characteristics (Nwawka et al. 2013). However, we agree
with their previous identification as paraphalanges, be-
cause their existence correlates with the modified tendons
and muscles controlling the scansors (Russell and Bauer
1988, Gamble et al. 2012). Among Squamata, a variety of
different sesamoid bones are present in the hands and feet
(Fontanarrosa and Abdala 2016, Regnault et al. 2016).
In fact, phalangeal sesamoids, located dorsally on the
penultimate phalanges, are ancestral to all Lepidosauria
(squamates and rhynchocephalians), whereas metacarpal
sesamoids likely evolved only later within squamates
(Regnault et al. 2016). Penultimate phalangeal sesamoids
are, in fact, present in most squamates (Regnault et al.
2016). Metacarpal and metatarsal sesamoids are of vari-
able occurrence in Gekkota (Regnault et al. 2016). Al-
though some sesamoids have been linked to lifestyle,
functional interpretations are to be inferred with caution
(Fontanarrosa and Abdala 2016, Regnault et al. 2016). In
humans, e.g., sesamoids likely result from an interplay
of intrinsic genetic factors and phylogeny, and extrinsic
mechanobiological factors (Sarin et al. 1999).

Reduced phalanges ossify at the same time sched-
ule as the other phalangeal elements in Hemidactylus
(Figs 3, 4), but their ecological relevance is yet to be de-
termined in this species. This trait is also found in non-re-
lated and ecologically different fossil synapsids, such as
Biarmosuchus, Titanophoneus, Lycaenops, Thrinaxodon,
and even the mammaliaform Docofossor (Hopson 1995,
Luo et al. 2015). However, phalanx number varies wide-
ly in non-mammalian tetrapods (Richardson and Chip-
man 2003). Hopson (1995) suggested that these reduced
disc-like structures were eventually lost. Gamble et al.
(2012), on the other hand, linked phalanx reduction with
the evolution of adhesive toepads. Furthermore, Russell
(1977) suggested that the reduced phalanx is used to
raise the penultimate phalanx, thereby altering the angle
of the claw relative to the substrate for a more beneficial
grip. Phalanx variation has been developmentally asso-
ciated with either heterochronic shifts (Richardson and
Oelschlager 2002), or the involvement of mutations in
expression patterns and molecular pathways of BMPs and
other transcription factors (Cooper et al. 2014, Luo et al.
2015). Studying the latter in Hemidactylus remains an av-
enue for future research. This study provides the morpho-
logical framework essential for the study of such kinds
of ecological traits from a molecular perspective, that is,
by means of gene expression and transcriptome analyses.

Author contributions
CB designed the study. CB and WvdV collected gecko

embryos. WdV carried out experiments in the morphology
& histology lab. CB compiled wCT datasets. KS carried

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208

out bone histology experiments. CB, KS, and WvdV
analyzed data. NDP provided reagents and lab facilities
for immunohistochemical analyses, and commented on an
earlier version of the manuscript. CB, KS, and WvdV wrote
the manuscript. All authors approved the final version.

Acknowledgements

Johannes Muller (Berlin) provided the parental Hemidacty-
Jus generation and helped with animal husbandry; we thank
him also for discussion. Annett Billepp and Petra Grimm
(Berlin) are thanked for animal care-taking. We thank Ju-
lia Eymann (Helsinki) for help with immunohistochemical
analyses. We thank Jutta Zeller (Berlin) for help with and
preparation of histological sections and staining. Kristin
Mahlow (Berlin) is thanked for help with wCT scanning.
Frank Tillack (Berlin) kindly helped with taking photos
of live animals. We furthermore thank Vivian de Buffrénil
(Paris) for access to comparative material of Varanus timo-
rensis, Tupinambis teguixin, and Iguana iguana at the his-
tology library of the Musée d’ Histoire Naturelle. We also
thank Brandon Kilbourne (Berlin) for language editing of
an earlier version of the manuscript. We acknowledge criti-
cal and helpful comments by Torsten Scheyer (Zurich) and
Johannes Penner (Freiburg). CB and WvdV were funded
by the German Research Foundation (DFG, BI 1750/3-1
to CB); KS was funded by a postdoctoral mandate of the
Research Foundation Flanders (FWO).

References

Andrews RM (2004) Patterns of embryonic development. In: Deeming
DC (Ed.) Reptilian Incubation: Environment, Evolution And Be-
haviour. Nottingham University Press, Nottingham. 75—102.

Arnold EN, Poinar G (2008) A 100 million year old gecko with sophisti-
cated adhesive toe pads preserved in amber from Myanmar. Natural
History 1847: 6-68.

Atkinson D (1994) Temperature and organism size - a biological
law for ectotherms? Advances in Ecological Research 25: 1-58.
https://doi.org/10.1016/S0065-2504(08)602 12-3

Autumn K, Sitti M, Liang YA Peattie AM, Hansen WR, Sponberg S, Ken-
ny TW, Fearing R, Israelachvili JN, Full RJ (2002) Evidence for van
der Waals adhesion in gecko setae. Proceedings of the National Acad-
emy of Sciences of the United States of America 99: 12252-12256.
https://doi.org/10.1073/pnas. 192252799

Bininda-Emonds O, Cardillo M, Jones KE, MacPhee RDE, Beck
RMD, Grenyer R, Price SA, Vos RA, Gittleman JL, Purvis A (2007)
The delayed rise of present-day mammals. Nature 446: 507-512.
https://doi.org/10.1038/nature05634

de Buffrenil V, Castanet J (2000) Age estimation by skeletochronology
in the Nile monitor (Varanus niloticus), a highly exploited species.
Journal of Herpetology 34: 414-424. https://doi.org/10.2307/1565365

Carranza S, Arnold EN (2006) Systematics, biogeography, and evolu-
tion of Hemidactylus geckos (Reptilia: Gekkonidae) elucidated using
mitochondrial DNA sequences. Molecular Phylogenetics and Evo-
lution 38: 531-545. https://doi.org/10.1016/j.ympev.2005.07.012

zse.pensoft.net

van der Vos, W. et al..: Ontogeny of Hemidactylus

Cooper KL, Sears KE, Uygur A, Maier J, Baczkowski KS, Brosnah-
an M, Antczak D, Skidmore JA, Tabin CJ (2014) Patterning and
post-patterning modes of evolutionary digit loss in mammals. Nature
511: 41-45. https://doi.org/10.1038/nature13496

Dayananda B, Penfold S, Webb JK (2017) The effects of incubation tem-
perature on locomotor performance, growth and survival in hatchling
velvet geckos. Journal of Zoology. https://doi.org/10.1111/)zo.12460

Fontanarrosa G, Abdala V (2016) Bone indicators of grasping hands in
lizards. PeerJ 4: e1978. https://doi.org/10.7717/peerj.1978

Gamble T, Greenbaum E, Jackman TR, Russell AP, Bauer AM (2012)
Repeated origin and loss of adhesive toepads in geckos. PLoS ONE
7: e39429. https://doi.org/10.1371/journal.pone.0039429

Gilbert F, Epel D (2009) Ecological developmental biology: integrating
epigenetics, medicine and evolution. Sinauer Associates Inc, Sun-
derland, Massachusetts, 480 pp.

Goodman RM (2008) Latent effects of egg incubation temperature on
growth in the lizard Anolis carolinensis. Journal of Experimental
Zoology Part A: Ecological Genetics and Physiology 309: 525-533.
https://do1.org/10.1002/jez.483

Hamburger V, Hamilton H (1951) A series of normal stages in the devel-
opment of the chick embryo. Journal of Morphology 88: 231-272.
https://do1.org/10.1002/jmor. 1050880104

Hopson JA (1995) Patterns of evolution in the manus and pes of non-mam-
malian therapsids. Journal of Vertebrate Paleontology 15: 615-639.
https://doi.org/10.1080/02724634.1995.10011252

Hugi J, Hutchinson MN, Koyabu D, Sanchez- Villagra MR (2012) Hete-
rochronic shifts in the ossification sequences of surface-and subsur-
face-dwelling skinks are correlated with the degree of limb reduction.
Zoology 115: 188-198. https://doi.org/10.1016/j.zo0l.2011.10.003

Khannoon ER (2015) Developmental stages of the climbing gecko
Tarentola annularis with special reference to the claws, pad
lamellae, and subdigital setae. Journal of Experimental Zoology
Part B Molecular and Developmental Evolution 324: 450-464.
https://doi.org/10.1002/jez.b.22630

Losos JB, Hillis DM, Greene HW (2012) Who speaks with a forked tongue?
Science 338: 1428-1429. https://doi.org/10.1126/science. 1232455

Luo Z-X, Meng Q, Ji Q, Zhang Y-G, Neander AI (2015) Evolutionary
development in basal mammaliaforms as revealed by a docodontan.
Science 347: 760-764. https://do1.org/10.1126/science. 1260880

Millinkovitch MC, Tzika A (2007) Escaping the mouse trap: The selection
of new evo-devo model species. Journal of Experimental Zoology
308B: 337-346. https://doi.org/10.1002/jez.b.21180

Muthukkaruppan V, Kanakambika P, Manickavel V, Veeraraghaven K
(1970) Analysis of the development of the lizard, Calotes versicol-
or. Journal of Morphology 130: 479-490. https://doi.org/10.1002/
jmor. 1051300407

Noro M, Uejima A, Abe G, Manabe M, Tamura K (2009) Normal devel-
opmental stages of the Madagascar Ground Gecko Paroedura pictus
with special reference to limb morphogenesis. Developmental Dy-
namics 238: 100-109. https://doi.org/10.1002/dvdy.21828

Nwawka OK, Hayashi D, Diaz LE, Goud AR, Arndt III] WF, Roemer FW,
Malguria N, Guermazi A (2013) Sesamoids and accessory ossicles of
the foot: anatomical variability and related pathology. Insights Imaging
4: 581-593. https://doi.org/10.1007/s13244-013-0277-1

Pianka ER, Sweet SS (2005) Integrative biology of sticky feet in geckos.
BioEssays 27: 647-652. https://do1.org/10.1002/bies.20237

Py-Daniel R, Kennedy Soares De-Lima A, Campos Lima F, Pic-Taylor
A, Rodrigues Pires Junior O, Sebben A (2017) A staging table of

Zoosyst. Evol. 94 (1) 2018, 195-209

post-ovipositional development for the South American collared 1iz-
ard Tropidurus torquatus (Squamata: Tropiduridae). The Anatomical
Record 300: 277-290. https://doi.org/10.1002/ar.23500

Pyron RA, Burbrink FT, Wiens JJ (2013) A phylogeny and revised classifi-
cation of Squamata, including 4161 species of lizards and snakes. BMC
evolutionary biology 13: 93. https://doi.org/10.1186/1471-2148-13-93

Rasband WS (1997) ImageJ. US National Institutes of Health, Bethes-
da, MD.

Regnault S, Jones MEH, Pitsillides AA, Hutchinson JR (2016) Anato-
my, morphology and evolution of the patella in squamate lizards and
tuatara (Sphenodon punctatus). Journal of Anatomy 228: 864-876.
https://doi.org/10.1111/joa.12435

Richardson MK, Oelschlager HHA (2002) Time, pattern, and heter-
ochrony: a study on hyperphalangy in the dolphin embryo flipper.
Evolution and Development 4: 435-444. https://doi.org/10.1046/
j.1525-142X.2002.02032.x

Richardson MK, Chipman AD (2003) Developmental constraints in
a comparative framework: a test case using variations in phalanx
number during amniote evolution. Journal of Experimental Zool-
ogy Part B Molecular and Developmental Evolution 296: 8-22.
https://doi.org/10.1002/jez.b.13

Roscito JG, Rodrigues MT (2012) Embryonic development of the fossorial
gymnophthalmid lizards Nothobachia ablephara and Calyptommatus
sinebrachiatus. Zoology 115: 302-318. https://doi.org/10.1016/j.
zool.2012.03.003

Ruibal R, Ernst V (1965) The structure of the digital setae of lizards.
Journal of Morphology 117: 271-293. https://doi.org/10.1002/
jmor. 1051170302

Russell AP (1975) A contribution to the functional analysis of the foot
of the Tokay gecko, Gekko gecko. Journal of Zoology 176: 437-476.
https://doi.org/10.1111/j.1469-7998.1975.tb03215.x

Russell AP (1977) The phalangeal formula of Hemidactylus Oken: A cor-
rection and a functional explanation. Anatomia, Histologia, Embryo-
logia 6: 332-338. https://doi.org/10.1111/j.1439-0264.1977 tb00443.x

Russell AP (2002) Integrative functional morphology of the gekkotan
adhesive system (Reptilia: Gekkota). Integrative and Comparative
Biology 42: 1154-1163. https://doi.org/10.1093/icb/42.6.1154

Russell AP, Bauer AM (1988) Paraphalangeal elements of gekkonid liz-
ards: a comparative survey. Journal of Morphology 197: 221-240.
https://doi.org/10.1002/jmor. 1051970208

Sanger TJ, Losos JB, Gibson-Brown JJ (2008) A developmental staging
series for the lizard genus Anolis: a new system for the integration
of evolution development, and ecology. Journal of Morphology
296: 129-137. https://doi.org/10.1002/jmor. 10563

Sanger TJ (2012) The emergence of squamates as model systems for
integrative biology. Evolution and Development 14: 231-233.
https://doi.org/10.1111/.1525-142X%.2012.00541.x

209

Sarin VK, Erickson GM, Giori NJ, Bergmann AG, Carter DR (1999)
Coincident development of sesamoid bones and clues to their evolu-
tion. The Anatomical Record 257: 174-180. https://doi.org/10.1002/
(SICI)1097-0185(19991015)257:5<174::AID-AR6>3.0.CO;2-O

Shapiro MD (2002) Developmental morphology of limb reduction in
Hemiergis (Squamata: Scincidae): chondrogenesis, osteogenesis,
and heterochrony. Journal of Morphology 254: 211-231. https://do1.
org/10.1002/jmor. 10027

Theiler K (1989) The House Mouse: Atlas Of Embryonic Development.
Springer-Verlag, New York Inc, New York.

Vickaryous MK, Olson MW (2007) Sesamoids and ossicles in the ap-
pendicular skeleton. In: Hall BK (Ed.) Fins into Limbs: Evolution,
development, and transformation. The University of Chicago Press,
Chicago, 323-341.

Wellborn V (1933) Vergleichende osteologische Untersuchungen an
Geckoniden, Eublephariden und Uroplatiden. Sitzung der Gesell-
schaft Naturfreunde, Berlin.

Wiens JJ, Hutter CR., Mulcahy, D. G., Noonan BP, Townsend TM,
Sites Jr JW, Reeder TW (2012) Resolving the phylogeny of liz-
ards and snakes (Squamata) with extensive sampling of genes and
species. Biology Letters 8: 1043-1046. https://doi.org/10.1098/
rsbl.2012.0703

Wise PAD, Vickaryous MK, Russell AP (2009) An embryonic staging table
for in ovo development of Eublepharis macularius, the leopard gecko.
The Anatomical Record: Advances in Integrative Anatomy and Evo-
lutionary Biology 292: 1198-1212. https://do1.org/10.1002/ar.20945

Supplementary material |

SOM Table

Authors: Wessel van der Vos, Koen Stein, Nicolas Di-Poi,
Constanze Bickelmann

Data type: Adobe PDF file

Explanation note: wCT scanning details for specimens used
in this study and depicted in Figures 2, 4-6.

Copyright notice: This dataset is made available under
the Open Database License (http://opendatacommons.
org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow us-
ers to freely share, modify, and use this Dataset while
maintaining this same freedom for others, provided
that the original source and author(s) are credited.

Link: https://do1.org/10.3897/zse.94.22289 suppl 1

zse.pensoft.net