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. 2015 Sep 18:15:184.
doi: 10.1186/s12862-015-0464-4.

Hand/foot splitting and the 're-evolution' of mesopodial skeletal elements during the evolution and radiation of chameleons

Raul E Diaz Jr  1   2 , Paul A Trainor  3   4
Affiliations

Hand/foot splitting and the 're-evolution' of mesopodial skeletal elements during the evolution and radiation of chameleons

Raul E Diaz Jr et al. BMC Evol Biol. .

Abstract

Background: One of the most distinctive traits found within Chamaeleonidae is their split/cleft autopodia and the simplified and divergent morphology of the mesopodial skeleton. These anatomical characteristics have facilitated the adaptive radiation of chameleons to arboreal niches. To better understand the homology of chameleon carpal and tarsal elements, the process of syndactyly, cleft formation, and how modification of the mesopodial skeleton has played a role in the evolution and diversification of chameleons, we have studied the Veiled Chameleon (Chamaeleo calyptratus). We analysed limb patterning and morphogenesis through in situ hybridization, in vitro whole embryo culture and pharmacological perturbation, scoring for apoptosis, clefting, and skeletogenesis. Furthermore, we framed our data within a phylogenetic context by performing comparative skeletal analyses in 8 of the 12 currently recognized genera of extant chameleons.

Results: Our study uncovered a previously underappreciated degree of mesopodial skeletal diversity in chameleons. Phylogenetically derived chameleons exhibit a 'typical' outgroup complement of mesopodial elements (with the exception of centralia), with twice the number of currently recognized carpal and tarsal elements considered for this clade. In contrast to avians and rodents, mesenchymal clefting in chameleons commences in spite of the maintenance of a robust apical ectodermal ridge (AER). Furthermore, Bmp signaling appears to be important for cleft initiation but not for maintenance of apoptosis. Interdigital cell death therefore may be an ancestral characteristic of the autopodium, however syndactyly is an evolutionary novelty. In addition, we find that the pisiform segments from the ulnare and that chameleons lack an astragalus-calcaneum complex typical of amniotes and have evolved an ankle architecture convergent with amphibians in phylogenetically higher chameleons.

Conclusion: Our data underscores the importance of comparative and phylogenetic approaches when studying development. Body size may have played a role in the characteristic mesopodial skeletal architecture of chameleons by constraining deployment of the skeletogenic program in the smaller and earliest diverged and basal taxa. Our study challenges the 're-evolution' of osteological features by showing that 're-evolving' a 'lost' feature de novo (contrary to Dollo's Law) may instead be due to so called 'missing structures' being present but underdeveloped and/or fused to other adjacent elements (cryptic features) whose independence may be re-established under changes in adaptive selective pressure.

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Figures

Fig. 1
Fig. 1
Chameleon autopodia. a The Veiled Chameleon (Chamaeleo calyptratus) has a laterally compressed body, prehensile tail, turreted and independent eyes, a projectile tongue and zygodactylous cleft autopdia. b Chameleon hand digits I, II, III are syndactylous medially with digits IV, V are bound laterally. Feet (c) have digits I, II syndactylous medially while digits III, IV, V are bound laterally. d The Desert Grassland Whiptail Lizard (Aspidoscelis uniparens) was used as for comparison due to its ‘typical’ terrestrial lizard body plan
Fig. 2
Fig. 2
Comparative morphogenesis of lizard autopodia. In Aspidoscelis uniparens, the digital plate progresses through a round shape with the pentadactyl digital rays developing within and visible through the thin tissue (a). Subsequent thickening of digital rays and thinning of the interdigital mesenchyme (b) ultimately leads to loss of the interdigital tissue through apoptosis (c). In the chameleon hand, a semi round digital plate develops (d) with subsequent distal flattening. Clefting initiates at this distally flattened location (e). Two syndactylous digit bundles are approximately 180° apart in the anterior-posterior plane. The A. uniparens foot also passes through a semi round digital plate stage (g) which subsequently presents digit differentiation with subsequent interdigital cell death (h-i). The chameleon foot undergoes the same stages as in the hand (d-f), but terminates with a different complement of digits in syndactyly (j-l). dpo = days post oviposition; A = Anterior; P = Posterior
Fig. 3
Fig. 3
SEM of Aspidoscelis uniparens hand and foot morphogenesis. Aspidoscelis uniparens hands and feet undergo the same stages of morphogenesis as presented in Fig. 2 in dorsal view (a, e, i; c, g, k), respectively. In distal view (b, f, j), the hand shows an Apical Ectodermal Ridge (AER) which is initially very robust as a distal ectodermal thickening and is present at the midline between the dorsal and ventral half of the developing limb. At later stages, the AER narrows significantly as it spans a greater anterior-posterior domain. In the foot (d, h, l), we see a similar situation with a very straight and stereotypical AER which is initially robust and later thins
Fig. 4
Fig. 4
SEM of Chamaeleo calyptratus hand and foot morphogenesis. Early stages of morphogenesis show that the chameleon hand develops as a round digital plate (a) which subsequently develops a distal flattening (d, g). In dorsal view, these stages present a very robust AER, relatively larger than that present in A. uniparens (Fig. 3) while in distal view the AER is seen as having a greater thickness along the dorsoventral midline (b, e, h). Significantly, during the stage at which the distal autopodium begins to flatten (g), the distal AER is no longer the stereotypical A-P flattened ectodermal thickening but is instead arched ventrally (h, i). Surprisingly, despite having a robust distal AER, proximal mesenchymal cleft formation has already begun (j, k). At later stages (l-o, p-s), both the forelimb and hindlimb expand the cleft while maintaining the AER quite robust until the thickness tapers and narrows at later stages (while the AER also returns to its expected conformation of a straight distal ridge)
Fig. 5
Fig. 5
C. calyptratus autopodial Sonic Hedgehog (Shh) expression. Wholemount in situ hybridization indicates that Shh is expressed in a similar pattern to other amniotes along the posterior margin of the developing limb within the Zone of Polarizing Activity (ZPA) in the hands (a, c) and feet (b, d). Significantly, the expression of Shh is lost upon distal flattening of the limb bud in both the hands (e, g) and feet (f, h)
Fig. 6
Fig. 6
C. calyptratus Fibroblast Growth Factor 8 (Fgf8) expression in the Apical Ectodermal Ridge. In wholemount in situ hybridization, the Apical Ectodermal Ridge expresses Fgf8 in the highly conserved pattern as seen in tetrapods (a-d) along the distal anteroposterior boundary of the limb. Fgf8 expression is maintained in the AER at stages where mesenchymal clefting of the autopodia has already commenced in both the hands and feet, though at a reduced amount prior to complete cleft formation (e-h)
Fig. 7
Fig. 7
Nile Blue staining for cell death during autopodial morphogenesis. During autopodial morphogenesis, Nile Blue granules are concentrated along the distal periphery of the developing limb within the mesenchyme as well as delimiting the Apical Ectodermal Ridge in both the hands and feet (a-j). The AER is also highlighted as ventrally arched in distal view (b, d) and is also clearly dorsoventrally thinner at stages when cleft formation is underway (h, j). During final stages of cleft morphogenesis, nile blue staining is concentrated within the interdigital mesenchyme (k-n), with a significantly greater concentration in the interdigital mesenchyme between digits III and IV in the hand (k, l) and II and III in the foot (m, n). Reduced Nile Blue is present in the tissue within syndactylous digits. Nile Blue is visible in the AER on the anterior and posterior regions of the limb while absent in the cleft domain in the hand (k, l)
Fig. 8
Fig. 8
C. calyptratus Gremlin 1 (Grem1) expression during autopodial morphogenesis. Grem1 is expressed in the mesenchyme of the autopodium anterior to the ZPA where a feedback loop is known to be present between Grem1 and SHH, Bmps, and FGF8 (a-f). Grem1 appears to decrease its anterior expression during digital plate formation in the forelimb (e) and hindlimb (f). During distal flattening and cleft formation, Grem1 does not appear ectopically between syndactylous digits (g-l)
Fig. 9
Fig. 9
Disruption of Bmp signaling during autopod development. Dorsomorphin (LDN193189) at 15 μM was used to disrupt Bmp signaling during whole embryo in vitro culture for 4 days. In the ‘younger’ group of embryos (a-b) collected prior to distal flattening of the digital plate, treatment led to impaired cleft formation. Asterisks highlight reductions in cleft formation relative to controls. b Older embryos collected during the onset of clefting (c-d) had no effect from the LDN193189 treatment over a 4 day growth period (d)
Fig. 10
Fig. 10
Tetrapod autopodia are dorsoventrally flattened, except in chameleons. Chameleon autopodia (C. calyptratus; a-d) while retaining the pentadactyl complement of digits, not only have a distal cleft separating the digits III and IV in the hand (a, b) and II and III in the foot (c, d) but also develop a rosette shaped cluster of metacarpals and metatarsals around a central enlarged distal carpal element. In species (humans) with congenital distal clefting (e, f), clefting does not alter the dorsoventrally flattened morphology
Fig. 11
Fig. 11
Chondrogenic differentiation in chameleon carpus. During distal flattening and expansion (a-c), the digital rays are already present and are lightly stained by Alcian Blue. The first elements of the mesopodium to form as cartilage are the Fi, dc4, and metacarpals 3–5. Distal carpal 3 and 5 form next (d, e) with later appearance of the intermedium, metacarpal 2 (f-h) and ultimately the radiale and pisiform (i-j). All embryos were at 107 dpo from clutchmates
Fig. 12
Fig. 12
Chondrogenic differentiation in chameleon tarsus. During distal flattening expansion, the first elements to appear are the ulnare and distal tarsal 4 (a) followed by distal tarsal 3 and metatarsals 3–5 and the intermedium (b-c). Metatarsal 2 is followed along with an appearance of the tibiale (e-g). All embryos were at 107 dpo from clutchmates
Fig. 13
Fig. 13
Outgroup lizard species autopodial chondrification. Desert grassland whiptail lizard (Aspidoscelis uniparens; Teiidae) and bearded dragon (Pogona vitticeps; Agamidae) chondrification patterns show post-axial dominance in elements appearance along the metapterygial axis. A. uniparens (a, b) hands show early formation of the ulnare, distal carpals 3 and 4, metacarpals 2–5 and a diffuse staining in the area of the radiale, lateral centrale and intermedium a. This empty domain later fills with a large medial and lateral centrale b. The foot develops from less elements and completes with a fused metatarsal 5 and distal tarsal 5 and a large distal tarsal 4 c-d. A large proximal tarsal element comprising the fibulare, intermedium and tibiale + lateral centrale develops in the ankle. In P. vitticeps e-j, a very similar sequence of cartilage elements in the hand (e-g) develop relative to A. uniparens with the added intermediate stage of seeing the medial carpale segment from the proximal end of distal carpal 1 (f) while the lateral centrale shifts distally adjacent to dc2 and dc3. The tarsus is more difficult to follow due to a small sample size and diffuse staining, but the proximal tarsal element is at least formed by the tibiale, intermedium and fibulare (h-j) with centralia not distinguishable
Fig. 14
Fig. 14
Contentious skeletal elements of the chameleon mesopodium. While broadly considered to be a sesamoid, we see the pisiform segmenting from the ventrolateral margin of the ulnare, underscoring its origin from the primary skeleton (a). In ‘true chameleons’ we see what appear to be ectopic and possibly de novo elements on the posterior tarsus. b The two distal elements are actually the tibiale and the intermedium which become shifted distally toward distal tarsals by the enlarged distal epiphysis of the tibia. c Despite the highly modified autopodium, chameleons retain distal tarsal 5
Fig. 15
Fig. 15
Histological examination of mesopodial mesenchymal and cartilaginous condensations. To complement the wholemount alcian cartilage stains, standard H&E paraffin sections were made to look for condensations which may not have shown due to a failure to reach the chondrification stage. In the carpals, we were able to find additional support for elements such as the intermedium, radiale and ulnare (a-c, g-j). In addition, we were able to identify a condensation which appears to be small and transient as distal carpal 1 (j) which does not chondrify and has a significant contribution of mesenchyme from the preaxial side of the limb. In the tarsals, we see a significant amount of shared mesenchymal cells between the distal tarsal 4, fibulare, intermedium and the tibiale (d-f), with the tibiale and intermedium always in closer association and smaller
Fig. 16
Fig. 16
Chamaeleo calyptratus atuopodial skeletogenic condensations. Despite a very specialized and element reduced adult chameleon autopodial skeleton, most ancestral skeletal elements remained present through ontogeny with the exception of the centralia. Between the manus and pes, the preaxial elements were always significantly delayed (ra, ti) in appearance. The intermedium appeared to have a ‘dual’ origin, with its condensation occurring closer to the radius in the forelimb and the fibulare in the hindlimb. We found that elements condensed and completed skeletogenesis (metacarpals, at least half of the distal carpals/tarsals, pisiform). There was a high tendency for fusion amongst proximal elements of the foot in C. calyptratus despite delayed appearance of the tibiale. Distal carpal 1 and distal tarsal 2 were seen to condense as mesenchyme but later fuse to adjacent elements while we were unable to find any condensation for distal tarsal 1. Distal carpal 3 remains cartilaginous. The pisiform was found to be derived through segmentation of the ulnare and the fibularis brevis tendon to enclose a sesamoid
Fig. 17
Fig. 17
Evolutionary trends in chameleon autopodial morphology. Outgroup comparison was conducted with the extant Rhynchocephalian Sphenodon punctatus (a) and 2 outgroups within Squamata (Teiidae [Aspidoscelis; (b)], Agamidae [Pogona; (c)]). Fusion of proximal tarsal elements arose during amniote evolution by formation of the Astragalus (tibiale + intermedium) and the Calcaneum (fibulare) (see [87] for review). Aspidoscelis (b) and Pogona (c) both show an increase in fusion of mesopodial elements relative to Sphenodon (a). The small and early diverged chameleons (Brookesia (d), Palleon) show the distal clefting of the autopodia between the forelimb digits III and IV and hindlimb II and III characteristic of chameleons. These two genera also present the greatest modified proximal autopodial skeleton within chameleons. The larger Rieppeleon and Rhampholeon (with shorter tails; (e, f)) presents a sesamoid in the fibularis brevis tendon of the heel which is maintained in all higher chameleons. Bradypodion (g), Chameleo (h-j), Furcifer (k, l) and Trioceros (m, n) (‘true chameleons’) sampled in this paper showed an ascending phylogenetic trend toward increased body size and an associated increase in skeletal elements present in the mesopodium, leading to a wrist with increased flexion capability as well as being the first amniote group to form an independently ossified tibiale and intermedium which is only known to be present in amphibians. Thus, in early chameleon lineages there appears to be a skeletal bottleneck relative to outgroup squamates with a subsequent phylogenetic trend of ‘re-evolving’ mesopodial elements present in outgroup taxa. The red gradient shows a trend toward reduction of elements from outgroup taxa to chameleons while the red gradient shows a relative increase in mesopodial skeletal elements in advanced chameleon genera studied. Genera in “<Genus>” are currently recognized taxa but were not examined in this study. Letters within figure correspond to the species with the same letters in Additional file 1: Figure S1
Fig. 18
Fig. 18
Phylogenetic reversal to a ‘typical’ lizard conformation of the autopodia. Despite retaining the cleft, an increase in mesopodial elements leads to increase wrist flexion in higher chameleons. Concomitant with the increase in the number of carpal and tarsal elements, we see a bifurcation where the syndactylous bundles are almost 180° separate in the early diverged lineages (‘leaf chameleons’; Brookesia (a, b), Rieppeleon (c, d), and Rhampholeon (e, f)) with the angle decreasing in Bradypodion (g, h, h’). ‘True chameleon’ syndactylous bundles shift toward the distal midline as is present in the basal squamate plan (with the cleft remaining as the constraint). While Chamaeleo (i-j) show an anteromedial transition of digit bundles, Furcifer (k, l) and Trioceros (m, n), the taxa with greater number of carpal and tarsal elements in this study, exemplify this the best through a greatly reduced angle between syndactylous bundles as digits move closer together along the distal midline. FL: refers to Forelimb with HL: referring to hindlimb. The numbers associated with each lineage on the phylogeny correlate to the same order of illustrations from Fig. 15 and represent formulae for number of elements present in the mesopodium in association with a representative image for autopodial morphology of a preserved specimen. FL: X/X refers to the number of elements in the two mesopodial rows while FL: X/X(X) refers to the two mesopodial rows with the number in parentheses representing the presence or absence of the sesamoid of the fibularis brevis. No centralia are present in chameleons, which would have made the standard 3 rows of mesopodial elements. Genera in “<>” are currently recognized taxa, but were specimens were not obtained in this study
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