Research Article |
Corresponding author: Constanze Bickelmann ( constanze.bickelmann@mfn-berlin.de ) Academic editor: Johannes Penner
© 2018 Wessel van der Vos, Koen Stein, Nicolas Di-Poï, Constanze Bickelmann.
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.
Citation:
van der Vos W, Stein K, Di-Poï N, Bickelmann C (2018) Ontogeny of Hemidactylus (Gekkota, Squamata) with emphasis on the limbs. Zoosystematics and Evolution 94(1): 195-209. https://doi.org/10.3897/zse.94.22289
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Squamate reptiles constitute a major component of the world’s terrestrial vertebrate diversity, 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 staging table of the geckoHemidactylus, housed at the Museum für Naturkunde, is established. Twelve post-ovipositional stages are erected, monitoring morphological embryological transitions in eye, ear, nose, heart, limbs, pharyngeal arches, and skin structures. Ecomorphological specializations in the limbs include multiple paraphalanges, hypothesized 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 III and IV and pedal digits III, IV, and V. Development, composition, and growth of limb elements is characterized in detail via µCT, histochemistry, and bone histological analysis. 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 geckoHemidactylus and its ecomorphological specializations in the adult autopodia, enlarges our knowledge of morphological trait evolution and of limb diversity within the vertebrate phylum.
Gecko, in-ovo development, staging table, limb development, bone histology, µCT, immunohistochemistry, ossification, ecomorphological specialization
Gekkota (Squamata; lizards, snakes and amphisbaenians sensu
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 (
Besides these general gekkotan traits, one gekkotan genus shows additional ecomorphological specializations in both fore- and hind limbs: Hemidactylus (Fig.
Here, we present a morphological staging table for Hemidactylus based on external embryonic developmental 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, µCT, and immunohistochemistry. Enlarging the database of well documented developmental character traits of organisms allows for a more detailed and comprehensive knowledge of vertebrate morphological diversity. The described ecomorphological specializations in the autopodia of the adult phenotype make Hemidactylus an ideal candidate for eco-evo-devo studies (
AC – Comparative Anatomy, Musée d‘Histoire Naturelle, Paris, France; CA – Cold Archive, Museum für Naturkunde Berlin, Leibniz Institute for Evolution and Biodiversity Science, Berlin, Germany; MK – Museum Koenig, Bonn, Germany; ZMB – Museum für Naturkunde Berlin, Leibniz Institute for Evolution and Biodiversity Science (former Zoologisches Museum Berlin), Berlin, Germany.
The study organism Hemidactylus is a yet unidentified species based on a pregnant female which was collected in Sudan in 2011 (Fig.
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 post oviposition | Snout vent length (range in mm) | Forelimb measurement (mean in mm) | Hind limb measurement (mean in mm) | Number of studied specimens |
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A | 4–5 | 9.3–11.5 | n/a | n/a | 3 |
B | 6 | 17.4–20.1 | 0.2 | 0.3 | 2 |
C | 9–14 | 14.1–17.6 | 0.7 | 0.6 | 4 |
D | 11–30 | 18.9–24.7 | 1.5 | 1.4 | 11 |
E | 8–30 | 21.0–24.0 | 1.8 | 1.8 | 7 |
F | 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 | 3.7 | 3.5 | 6 |
I | 35–56 | 29.0–34.2 | 4.9 | 4.8 | 10 |
J | 40 | 32.8–34.2 | 5.3 | 6.5 | 2 |
K | 40–58 | 36.0–39.4 | 6.2 | 6.7 | 3 |
L | 50–60 | 37.5–41.9 | 6.8 | 6.9 | 2 |
µCT scans were performed using a Phoenix nanotom X-ray|s at the Museum für Naturkunde Berlin (Suppl. material
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 accretion rates.
Humerus ZMB 87078 | Humerus CA2017_014 | ||
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Life history data | Adult, Born 10.12.15, deceased 22.03.17 | Stage I development | |
Total days lived | 468 | 25 (unborn) | |
Days since onset ossification | 493 | 0 | |
Humerus shaft width (µm) | 587.6 | 161.0 | |
posterior cortex | anterior cortex | ||
Non-remodeled Cortical thickness (µm) | 139.4 | 91.2 | 0.1 |
No. of lamellae in cortex | 40 | 29 | 1 |
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 represents stage D in (H–J). Note the immunohistochemistry 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; cc – cartilage capsule; cf – choroid fissure; en – external nares; ep – eye pigmentation; fp – frontal nasal process; ha – hyoid arch; lb – limb bud; ll – lateral lines; mda – mandibular arch; mxa – maxillary process; oc – optic cup; of – olfactory pit; op – otic pit; pa – pharyngeal arches; st – stylopod; tc – thorac cavity; zu – zeugopod.
Overview of embryonic developmental stages G to L, erected in this study, including µCT 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 1mm. Abbreviations: at – adhesive toepads; ca – cornea; cw – claw formation; l – lens; np – nose pit; ot – otic capsule; s – scansors; sp – swollen pads; ub – urogenital bud; ws – wrinkly skin.
µCT 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 µm. Abbreviations: dc – distal carpals; m – metacarpals; p –pisiform; pp – paraphalanges; R – radius; r – radiale; rp – reduced phalanges; U – ulna; u – ulnare; 1-5 – phalanges 1-5; I-V – digits I-V.
Forelimb of juvenile Hemidactylus, stained with azan (A, B) and scanned by µCT (C). (A) and (C) are of the hatchling ZMB 87077 (cross section no. in A is A5_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 µm, and scale bar in (C) is 550 µm.
We used two histo-technological procedures: (i) azan stained microtome paraffin sections and (ii) ground petrographic sections.
For azan staining, limbs were embedded (Leica EG 1160 and Shandon Hypercenter XP) in paraffin, and sectioned with a microtome (Leica 2000R). Sections were treated following the standard protocol by Heidenhain. In detail, sections were washed or immersed in xylene paraffin (ten minutes), xylene (twice five minutes), aniline alcohol (five minutes), wash in aquadest, azocarmine (25 minutes), 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.
For ground sections (Fig.
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 µCT scans (G). Abbreviations: eb – endosteal bone; f – femur; h – humerus; hl – Howship lacunae; mc – medullary cavity; pb – periosteal bone; r – radius; u – ulna. Scale bars in (A, B) equal 200 µm, in (C–E) 100 µm, in (F) 500 µm, and 1 mm in (G).
Photographs of embryos were taken with a Leica M205C camera. Photomicrographs for histomorphometric analysis 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 (
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 xylene and rehydrated through ethanol steps into 1X PBS. Antigen retrieval was accomplished by citrate buffer treatment (pH = 6.0). Protease XXV treatment (Thermo Scientific) 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).
A morphological staging table of Hemidactylus establishing twelve post-ovipositional developmental stages is presented here (Figs
Lamellar bone histology and endosteal remodeling in other squamate lizards. (A) Cross section of an Iguana 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 µm, and in (C) 500 µm.
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. Focused on limb development, darkening shades of gray indicate (i) both limb buds present after oviposition, (ii) interdigital tissue starts retreating, (iii) 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.
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Muthukkarruppan et al. ( |
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Hemidactylus sp. | Paroedura pictus | Tarentola annularis | Eublepharis macularius | Nothobachia ablephara | Calyptommatus sinebrachiatus | Tropidurus torquatus | Anolis sagrei | Calotes versicolor |
0 dpo | Stage 27 | |||||||
1 dpo | Stage 28 | |||||||
Stage A | 2 dpo | St 28 | St 1 | |||||
Stage B | 3-4-5 dpo | St 29 | St 1 | St 2-3 | St 28 | 3 | Stage 29 | |
6-7 dpo | S29 | St 2 | St 29 | 4-5 | ||||
Stage C | 8-9-10 dpo | St 30 | St 4 | St 30 | 6 | Stage 30 | ||
S30 | ||||||||
12 dpo | St 3 | Stage 31 | ||||||
Stage D | 14 dpo | St 31 | St 4 | St 5 | St 31 | |||
S31 | St 5 | St 32 | ||||||
St 6 | Stage 32 | |||||||
16 dpo | St 32 | |||||||
Stage E | 18 dpo | S32 | St 33 | St 6 | St 7 | St 33-34 | 7-8 | Stage 33 |
Stage F | 20-22 dpo | S33 | St 34 | St 7 | St 8 | St 35 | 9 | Stage 34 |
St 8 | St 9-10 | |||||||
Stage G | 24 dpo | S34 | St 9 | St 11 | St 36 | 10 | Stage 35 | |
26 dpo | St 35 | 11 | ||||||
Stage H | 28 dpo | S35 | St 36 | St 37 | Stage 36 | |||
Stage I | 30 dpo | S36 | St 37 | St 38 | 12-13-14 | |||
Stage 37 | ||||||||
Stage 38 | ||||||||
Stage J | 35 dpo | S37 | St 38 | St 39 | 15-16-17 | Stage 39 | ||
St 39 | ||||||||
S38 | St 40 | Stage 40 | ||||||
Stage K | 40-45-50 dpo | S39 | St 41 | St 12 | St 40 | 18 | Stage 41 | |
Stage L | 55-60 dpo | St 42 | St 41-42 | 19 | Stage 42 |
Ear: The otic pit is present and located dorsally to the hyoid arch (Fig.
Eye: The eye is composed of an optic cup that has not yet fully enclosed the lens (Fig.
Heart: The heart shows the distinct s-shape resulting from the curvature of the developing ventricle. It protrudes 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 rim with its curvature pointing to the cranial side and the two points of the rim facing ventrally, towards the heart (Fig.
Pharyngeal arches: The mandibular pharyngeal arch is prominent, with a small bud from the maxillary that has just started to bud out (Fig.
Ear: The cartilage capsule dorsal to the otic pit is enlarged (Fig.
Eye: The optic cup has almost enclosed the lens, except 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.
Head: The mesencephalon protrudes prominently in the cranial direction.
Heart: The heart and its placement in the thoracic cavity have not changed in comparison to the previous stage (Fig.
Limbs: Both fore- and hind limbs appear as small buds (Fig.
Nose: The rim of the olfactory pit is still partially covered by a membrane.
Pharyngeal arches: The maxillary process has not proceeded its development compared to the previous stage. The mandibular and hyoid arches, including the gap between them (Fig.
Ear: The otic pit is enlarged and extends antero-posteriorly 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.
Limbs: Both limb buds are proximo-distally longer than wide. The apical ectodermal ridge (AER) is distinct (Fig.
Nose: The external nares have developed, replacing the olfactory pit (Fig.
Pharyngeal arches: The maxillary process is linked to the dorsal side of the fronto-nasal process. The mandibular 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.
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.
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.
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 hyoid arch are now indistinguishable from one another (Fig.
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 primordial 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.
Nose: Deep furrows between the nasal and maxillary prominences are developing further and show invaginations in this region.
Pharyngeal arches: The prominence of the mandibular and hyoid arches is now located anterior to the lens.
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.
Limbs: Recession of the interdigital tissue is extensive (Fig.
Nose: The olfactory pit has disappeared. The fronto-nasal process is fully fused (Fig.
Pharyngeal arches: The pharyngeal arches do not show the distinction between the different arches anymore (Fig.
Ear: No further development in comparison to the former stage (Fig.
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 mesencephalon, have disappeared. The head resembles that of a hatchling. Internally, only the calcified endolymphatic sacs and statolithic masses are visible in the µCT scans (Fig.
Limbs: The autopodia show further recession of the interdigital 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.
Nose: The nose has become a pit laterally at the rostral end of the snout (Fig.
Jaw: Upper and lower jaws are fully formed.
Urogenital bud: The urogenital bud is located medially between the hind limbs (Fig.
Eye: There are four distinct changes that are present in the eye at this stage: (i) The lens has changed from a matte white color to a more shining white color. (ii) The iris starts to cover the margins of the lens. (iii) The black pigmented 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 irregularities. The mesencephalon is still identifiable. Skull bones, such as parietal and surangular, start ossifying (Fig.
Limbs: The digits are almost free of interdigital tissue, and the distal phalanges start showing claw formation (Fig.
Eye: The iris surrounding the lens starts to display a wavy pattern but is still confined along the margin (Fig.
Head: More or less all skull bones are ossifyed (Fig.
Limbs: Claws have developed on all digits. Adhesive toepads are more prominent at this stage: one can discriminate the wavy skin and swellings on the toepads (Fig.
Skin: The skin starts to get wrinkly, a precursor stage prior to scale formation of the skin (Fig.
Eye: Within the eye, the cornea is developing, and the eye socket begins to enclose the eye (Fig.
Head: The mesencephalon has started to retract, exposing 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.
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 retreating into the body (Fig.
Eye: The eye is located inside the socket, and the scale ridge surrounding it starts to develop (Fig.
Head: Fully scaled.
Limbs: Ossification of the long bones continues (Fig.
Skin: All scales are pigmented. Scales are present on the upper and lower jaw.
Eye: There is 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.
Limbs: Phalanges and metacarpals and -tarsals continue ossifying, with the epiphyses not yet being ossified (Fig.
Skin: The scales, present on tail, back, limbs, and head show differences in pigmentation and color (Fig.
The phenomenon of limb reduction is of great interest in evolutionary biology (e.g.
Between fore- and hind limbs, no heterochronic differences in size and shape were monitored at any developmental stage (Figs
Histochemical staining and µCT scanning show that the paraphalangeal elements of Hemidactylus are present in three different shapes (Figs
The long bones of one-year-old adult individuals show a lamellar bone matrix with no vascular spaces (Fig.
From an adult individual of known age (ZMB 87078, 468 days old, 493 days since onset of ossification, Fig.
Where apposition.rate is the daily accretion rate (in µm/day), lamellar.accretion.rate is the number of lamellae deposited per day, Wadult.hum is the width of the adult humerus (in µm), Wonset.oss is the humeral width at the onset of ossification (in µm), N°days.onset.oss is the age of the adult individuals, in days, since the onset of ossification, Wprimcortex is the thickness of the primary cortex in the adult individual, and N°lamellae.primcortex is the number of lamellae counted in the primary cortex of the adult individual (Measurements in Tab.
The in-ovo development of the geckoHemidactylus was defined into twelve stages post-oviposition based on morphological embryonic characters (Figs
Bone histology shows rapid accretion and endosteal remodeling 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 fibulae of wild Varanus niloticus (
Our detailed analysis of ecomorphological specializations in the limbs of Hemidactylus also reveal that, in contrast to earlier observations (
Reduced phalanges ossify at the same time schedule as the other phalangeal elements in Hemidactylus (Figs
CB designed the study. CB and WvdV collected gecko embryos. WdV carried out experiments in the morphology & histology lab. CB compiled µCT datasets. KS carried 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.
Johannes Müller (Berlin) provided the parental Hemidactylus 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 Julia 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 µCT 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 timorensis, Tupinambis teguixin, and Iguana iguana at the histology 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 critical 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).
SOM Table
Data type: Adobe PDF file
Explanation note: μCT scanning details for specimens used in this study and depicted in Figures