Embryology of the naso‐palatal complex in Gekkota based on detailed 3D analysis in Lepidodactylus lugubris and Eublepharis macularius

Abstract

The vomeronasal organ (VNO), nasal cavity, lacrimal duct, choanal groove, and associated parts of the superficial (soft tissue) palate are called the naso‐palatal complex. Despite the morphological diversity of the squamate noses, little is known about the embryological basis of this variation. Moreover, developmental data might be especially interesting in light of the morpho‐molecular discordance of squamate phylogeny, since a ‘molecular scenario’ implies an occurrence of unexpected scale of homoplasy also in olfactory systems. In this study, we used X‐ray microtomography and light microscopy to describe morphogenesis of the naso‐palatal complex in two gekkotans: Lepidodactylus lugubris (Gekkonidae) and Eublepharis macularius (Eublepharidae). Our embryological data confirmed recent findings about the nature of some developmental processes in squamates, for example, involvement of the lateral nasal prominence in the formation of the choanal groove. Moreover, our study revealed previously unknown differences between the studied gekkotans and allows us to propose redefinition of the anterior concha of Sphenodon. Interpretation of some described conditions might be problematic in the phylogenetic context, since they represent unknown: squamate, nonophidian squamate, or gekkotan features.

We used X‐ray microtomography and light microscopy to describe morphogenesis of the naso‐palatal complex in two gekkotans: Lepidodactylus lugubris (Gekkonidae) and Eublepharis macularius (Eublepharidae). Our embryological data confirmed recent findings about the nature of some developmental processes in squamates and revealed previously unknown differences between the studied gekkotans. We also proposed redefinition of the anterior concha of Sphenodon.

1. INTRODUCTION

In squamates, the nasal cavity and the vomeronasal organ (VNO) constitute the peripheral organs of the main and accessory olfactory systems respectively (Halpern, 1992; Martínez‐Marcos & Halpern, 2009). The olfactory sensory epithelium, which forms receptive part of the nasal cavity, projects to the main olfactory bulb, while the vomeronasal sensory epithelium of the VNO projects to the accessory olfactory bulb located medial and posterior to the former (Armstrong et al., 1953; Parsons, 1967; Auen & Langebartel, 1977; Ulinski & Peterson, 1981).

The nasal cavity and VNO originate from the nasal pit, but unlike in other tetrapods the direct connection is lost during embryonic development (Parsons, 1959a). The VNO duct opens on the palate and provides a direct connection between the VNO and the oral cavity. The separation of the VNO from the nasal cavity is associated with the formation of the choanal groove, which represents the ventral remnant of the embryonic nasal cavity (Bellairs & Boyd, 1950; Kaczmarek et al., 2020). The choanal groove may provide the only indirect connection between the VNO duct and the nasal cavity or may be obliterated completely in some squamates (Malan, 1945; Gabe & Saint Girons, 1976). More often it is retained in adults and forms a connection with the lacrimal duct (Bellairs & Boyd, 1950; Kaczmarek et al., 2020).

The nasal cavity of squamates exhibits relatively simple morphology (Figure 1 and see introduction in Kaczmarek et al., 2020). It consists of the tubular vestibulum, main nasal cavity, and outer choanal tube, which may form the nasopharyngeal duct in some taxa (Parsons, 1970). The lateral nasal concha protrudes into the lumen of the main nasal cavity and divides it into the three parts which have different relations to the concha. These are the extraconchal space (lateral and dorsolateral), Stammteil (medial), and inner choanal tube (ventral and ventrolateral) (Fuchs, 1908; Parsons, 1959a, 1970; Kaczmarek et al., 2020) (Figure 1).

FIGURE 1.

3D scheme of the naso‐palatal complex of Tarentola mauritanica (modified from figure 24a in Lemire, 1985; ©Publications Scientifiques du Muséum national d’Histoire naturelle, Paris) and transverse sections of Hemidactylus fasciatus (modified from figures 4d, f and g in Bellairs & Boyd, 1950) through the VNO (I), choanal groove (II) and outer choana (III). Note that levels of the sections are only approximated on the 3D scheme and show different species. Key:a, anterior; Aux, Aulax; chf, choanal fold; chg, choanal groove; dv, VNO duct; ecs, extraconchal space; en, external naris; ich, inner choana; ict, inner choanal tube; lcf, lateral choanal fissure; ld, lacrimal duct; lnc, lateral nasal concha; lng, lateral nasal gland; m, medial; mb, mushroom body; mxf, maxillary fold; och, outer choana; oct, outer choanal tube; scf, subconchal fold; Stt, Stammteil; vc, vomerine cushion; vch, ventral channel; ves, vestibulum; vns, vomeronasal sensory epithelium

The VNO is composed mostly of the dorsal dome of the vomeronasal sensory epithelium, ventral concha (called the mushroom body) and VNO duct (Figure 1) (Wang & Halpern, 1980a; Takami & Hirosawa, 1990; Saito et al., 2010). Vomeronasal chemoreception provides qualitative discrimination and allows for prey or mate trailing (Duvall, 1981; Schwenk, 1995; Cooper, 1997). Pheromones and prey odours, are collected by tongue during its flicks and may reach the VNO lumen through the VNO duct (Young, 1993; Schwenk, 1995; Huang et al., 2006; Filoramo and Schwenk, 2009). It is believed that tongue‐flicking behaviour is triggered by volatile molecules inhaled into the nasal cavity and detected by the main olfactory system (Schwenk, 1995).

Based on their ontogenetic and functional associations, the VNO, nasal cavity, lacrimal duct, choanal groove, and associated parts of the superficial (soft tissue) palate are called the naso‐palatal complex (Kaczmarek et al., 2020).

The morphological phylogeny of squamates suggests increased importance of the chemical sense in Scleroglossa (Cooper, 1996; Pianka & Vitt, 2003; Vitt et al., 2003) which is sister to Iguania (iguanids, agamid and chamaeleonids) (Estes et al., 1988; Gauthier et al., 2012). Within scleroglossans, Gekkota is sister to Autarchoglossa containing among others: skinks, lacertid lizards, anguids, varanids and snakes. Within autarchoglossans, snakes, varanids and teiids are considered to be strongly adapted to vomeronasal chemoreception, because of receptor cell abundance in the vomeronasal sensory epithelium and a highly specialized tongue (Bellairs, 1949; McDowell, 1972; Schwenk, 1993a, 1994; Cooper, 1995a, 1996). In Gekkota the importance of vomeronasal chemoreception can be classified as ‘intermediate’ (Cooper, 1996). In fact, gekkotans are considered to be olfactory specialists, based on abundance of neurons in olfactory epithelium, large lateral nasal concha, extensive distribution of the olfactory epithelium, large main olfactory bulb in relation to accessory olfactory bulb, less specialized tongue morphology, and some behavioural characters (Schwenk, 1993b).

Molecular studies have challenged the morphology‐based phylogeny, suggesting that Iguania are nested within highly chemosensory autarchoglossans and revealing the basal split into Dibamia (or Dibamia + Gekkota) and other squamates (Vidal & Hedges, 2005; Zheng & Wiens, 2016; Pyron, 2017; Burbrink et al., 2020). A molecular scenario implies ‘unprecedented levels of homoplasy in disparate anatomical systems of organisms pursuing very different lifestyles’ (Gauthier et al., 2012).

There are some extensive comparative studies on the morphology of the naso‐palatal complex in adult squamate reptiles (Fuchs, 1908; Malan, 1945; Bellairs & Boyd, 1947, 1950; Pratt, 1948; Stebbins, 1948; Gabe & Saint Girons, 1976; Lemire, 1985; Hallermann, 1994, 1998). These observations indicate some possible groupings corresponding to the large squamate clades or (nonmonophyletic) morphotypes. For example, according to the choanal groove morphology and its relation to the lacrimal duct, a few morphotypes can be distinguished, for example: iguanian like, gekkotan like, lacertid–skink (Bellairs & Boyd, 1950), and helodermatid–varanid (Gabe & Saint Girons, 1976). Some components of the naso‐palatal complexes exhibit a grater variation, for example, the morphology of the vestibulum in Pleurodonta (Stebbins, 1948). On the other hand, some characteristics suggest convergence in a certain part of considered complex. These are a snake‐like features in nonophidian squamates referring to the: intimate connection of the lacrimal duct with the Harderian gland and/or VNO duct (Bellairs & Boyd, 1947, 1950), and the formation of the nasopharyngeal duct (Parsons, 1970). Prominent examples of such situation are some serpentiform Pygopodidae within Gekkota, which may ‘violate’ typical ‘gekkotan‐like morphotype’ (Bellairs & Boyd, 1947; Gabe & Saint Girons, 1976).

In contrast to adult morphology, the embryonic development of the squamate naso‐palatal complex at the anatomical level has received only limited attention (e.g. Fuchs, 1908; Parsons, 1959a; Rudin, 1974; Holtzman & Halpern, 1990; Deiques, 2004; Kaczmarek et al., 2017), focusing predominantly on the internal structures of the snout, not on the palate. In fact, the embryological processes forming the diverse structures in adult squamates remain unclear (Parsons, 1970). However, in a recent study using light microscopy and X‐ray microtomography, the peculiar anatomy of the naso‐palatal complex in the brown anole (Anolis sagrei) was described based on its embryology (Kaczmarek et al., 2020). Thus such processes as reduction of the lateral nasal concha and formation of closed choanal groove have been described.

The aim of this analysis was to check the general conservatism of development of the naso‐palatal complex in nonpygopodid gekkotans which may be expected based on conservatism of adult morphology (Pratt, 1948; Bellairs & Boyd, 1950). In order to do this, we investigated two species of Gekkota: the mourning gecko (Lepidodactylus lugubris) and the leopard gecko (Eublepharis macularius), which represent Gekkonidae and Eublepharidae respectively. There is some evidence from the abundance of the vomeronasal receptors cells (Cooper, 1996) and behavioural experiments (Cooper, 1995b) suggesting that eublepharids rely more on vomerolfaction than other gekkotans. Thus some differences at anatomical level may be expected. The other purpose of this analysis was to fill the gaps in understanding of the common developmental mechanisms leading to the formation of the adult naso‐palatal complex in squamates.

2. MATERIAL AND METHODS

2.1. Animal care and embryo preparation

Eggs of L.lugubris and E.macularius were obtained from animals kept in the breeding room of the Faculty of Natural Sciences of the University of Silesia in Katowice. The environment, housing, and management of adult animals were provided in accordance with the protocol accepted by District Veterinary Officer in Katowice (see Ethics approval and consent to participate). The eggs of L.lugubris are glued by females to the differed vertical surfaces of the terrarium. To avoid damage to the egg shells, the eggs were left and incubated in the terrarium at approximately 23–30°C. The eggs of E.macularius were removed from the terrarium, placed in an incubator (Zoo Med's ReptiBator^®), and incubated at 30°C. In this case, the eggs were half‐buried in vermiculite mixed with water at a 1:1 ratio by weight (Rupik, 2012; Swadźba & Rupik, 2012; Rupik et al., 2016; Kowalska et al., 2017). The eggs of the L.lugubris were collected and the embryos were isolated a few times a year. The embryos of E.macularius were isolated at regular intervals starting immediately after the eggs were laid. The embryos were collected over a period of 4 years. In total 49 embryos were used for this study (Table 1).

TABLE 1.

Numbers of gekkotan embryos used for microtomography (mCT) and light microscopy (LM)

	L. lugubris			E. macularius
Developmental phase of the naso‐palatal complex	Stage a	mCT	LM	Stage b	mCT	LM
Early	3	1	0	28	1	0
	7	1	1	30	1	0
	10	1	1
	12	1	1
Middle	14	2	1	30/31	1	1
	14/16	1	1	31	1	1
	18	1	2	31/32	1	1
	20–22	1	1	34	2	1
Late	26	1	1	35	1	0
	28–30	2	2	37	1	2
	35	1	1	38	1	2
	50–60	2	2	42	1	1
Total		15	14		11	9

^aAccording to Noro et al. (2009).

^bAccording to Wise, Vickaryous, and Russell (2009).

For embryo sedation, the eggs were cooled in a refrigerator for about 20 min and then placed on ice (still in the refrigerator) for about 10 min (see Shine et al., 2015; Rollings et al., 2019). After that, the embryos were isolated, killed by decapitation and immediately transferred to the fixative for the different research methods (Table 1). Developmental stage of the E.macularius embryos was evaluated according to the developmental table for this species based on external morphological characteristics (Wise et al., 2009). The stage of the L.lugubris embryos was evaluated based on the developmental table of another gekkonid, Paroedura picta (Noro et al., 2009). Some embryos were characterized by intermediate morphology (e.g. stages 30/31 of E.macularius or 14/16 of L.lugubris), showed no significant differences across a few developmental stages (e.g. stage 50–60 of L.lugubris), or the morphology of the naso‐palatal complex suggested that additional ‘stage’ might be distinguished (e.g. stage 34_II of E.macularius or stage 14_II of L.lugubris, which were more advanced in development than the other embryos at stages 34 and 14 respectively).

2.2. MCT and 3D reconstructions

For X‐ray microtomography (mCT) the embryos were fixed in Bouin's solution for 24 h and then transferred to 80% ethanol. After rinsing in 80% ethanol, most heads, usually deprived of the mandible, were stained in 0.3% PTA (phosphotungstic acid) in 70% ethanol for 72 h (Metscher, 2009a,2009b). This staining time was insufficient for large scaled head of E.macularius at stage 42. Because of that, the posterior third of the head was removed and the sample was transferred to the fresh staining solution for additional 5 days, with one change after 48 h. Several staining methods were tested, including iodine, and PTA was found to give the most favourable results overall.

The samples were embedded in 0.5% w/v agarose LMT (Metscher, 2011) and scanned using a Xradia MicroXCT‐200. For most scans the source was set at 60 kV and 83 µA. Projection images were taken using the 2×, 4×, 10×, or 20× objective lens depending on the sample size. Tomographic reconstructions were made with XMReconstructor (Zeiss‐Xradia), obtaining final voxel sizes of 2–5 μm.

2.3. Structure interpretation, visualization and segmentation, and alignment of anatomical axes

The nasal cavity and similar structures were defined as epithelium + lumen. However, the segmentations of analysed structure did not include lumens of the organs; thus only the epithelia and cellular plugs sealing the vestibulum of the nasal cavity, VNO duct, and lacrimal duct were labelled. In most cases only the left component of the naso‐palatal complex was segmented. Only in L.lugubris embryo at stage 14/16 the right side was considered (indicated in the figure description), since the quality of the mCT image was much better in that side of the head. Segmentations and visualizations of the analysed structures were made in Amira version 6.4 (Thermo Scientific). The segmented structures were rendered as volumes with the use of Volren option, with different colour maps for each. The volume rendering and transparency of the nasal cavities were adjusted to reveal the sensory olfactory epithelium (nontransparent dark green) and nonsensory, respiratory epithelium (semitransparent light green). The presence of the Bowman's glands was an additional feature allowing differentiation of the sensory olfactory epithelium. The descriptions consider the antorbital space as comprising all three components of the conchal zone of the main nasal cavity rather than as a separate structure.

In general, the bones and the nasal capsule are beyond the scope of this study. Only short descriptions for the cupola Jacobsoni (vomer + septomaxilla + maxilla) (see Rieppel et al., 2008) and the cartilage of the mushroom body were provided. The antero‐posterior axis of gekkotan heads was aligned along the long axis of the maxillary prominences (early embryos) or the upper lip (later embryos) (see Figure 2 in Kaczmarek et al., 2020).

2.4. Light microscopy

Detailed histological description is beyond the scope of this study, but obtained histological serial sections were helpful to interpretation of tomographic data. The material for analysis in light microscope was fixed in Bouin's decalcifying solution (Bancroft & Gamble, 2008) for 48 h. Embryos of L.lugubris starting from stage 18 and embryos of E.macularius starting from stage 34 were additionally decalcified in 10% formic acid for 2 (L.lugubris) or 3 weeks (E.macularius) and the solution was changed after 7 or 10 days respectively. Then the samples were rinsed in distilled water and, like younger embryos, dehydrated and embedded in paraffin. Serial transverse sections (each 7 or 10 μm thick) were cut using a Leica rotary microtome (Leica RM2125RT). The deparaffinized histological sections were stained with Ehrlich's haematoxylin and eosin, and photographed using an OLYMPUS BX60 light microscope with an OLYMPUS DP12 digital camera controlled by Olympus cellSens Standard software (Kowalska & Rupik, 2018; Hermyt et al., 2020).

2.5. Analysis of the results

Differentiation of the naso‐palatal complex was arbitrarily divided into three developmental phases: early, middle, and late. For convenience, most stages of the two studied gekkotans were aligned according to the first appearance of some structures or beginning of the certain developmental processes which were noticed in this study (indicated in Table 2). The events occurred after the connection of the lacrimal duct with the choanal groove were classified either as preadult‐like or adult‐like stages (the latest analysed stages). The alignment of stages between species presented here (see Table 2) does not imply ‘homology’ of L.lugubris and E.macularius developmental stages. In fact, developmental sequences are taxon specific and a normal developmental table prepared for one species should not be used for the other distantly related taxon (Andrews et al., 2013). In this study, the stage alignment was used as a tool to facilitate the between‐species comparison of developmental processes and anatomy of embryonic structures and to condense the results section.

TABLE 2.

Summary of the results of this study for Lepidodactylus lugubris and Eublepharis macularius. Note that the stages of analysed gekkotans are aligned according to selected developmental events (A). Similarities between the same structures for two aligned stages are shown in merged cells

^aPreadult‐like stages (no event used for stages alignment).

^bAdult‐like stage (no event used for stages alignment).

Finally, we presented comparisons of development sequences of selected events of studied gekkotans and representative of Iguania, A.sagrei, for which the detailed data of naso‐palatal complex development were available (Kaczmarek et al., 2020). The events are defined as first appearance of the structure or condition (see Andrews et al., 2013; Skawiński & Borczyk, 2017). The events were ordered according to A.sagrei, but order of events occurring simultaneously is arbitrary.

2.6. Ethics approval and consent to participate

The research were performed in accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes and the Act of 15 January 2015 on the protection of animals used for scientific or educational purposes (Journal of Laws 2015 item 266). In accordance with Directive 2010/63/EU of the European Parliament and of the (Journal of Laws 2015 item 266), the studies do not require the permission of the Local Ethics Committee. The Institute of Biology, Biotechnology and Environmental Protection obtained approval from the District Veterinary Officer in Katowice—Poland (PIW.ZOZ.OZ.5342.5.1/17) and was entered in the Register of the Polish Ministry of Science and Higher Education according to the article 2, paragraph 1, and point 9 Act on the protection of animals used for scientific or educational purposes (Journal of Laws, 2015 item 266). The L. lugubris and E. macularius are not included in the Washington Convention of 1973 (Guidelines for appropriate uses of IUCN red list), which was ratified by Poland in 1991 (Journal of Laws, No. 27 item 112 and latest regulations of Journal of Laws, 2000 No. 66 item 802 and Journal of Laws, 2004 No. 112 item 1183).

3. RESULTS

3.1. Early developmental phase

The nasal pits of L.lugubris are first visible at developmental stage 3 (Figure 2A). On the transverse sections, each of them is relatively shallow and bounded by the relatively weakly developed the lateral and medial nasal prominences (Figure 2A). The nasal pit opens on the lateral surface of the forming snout through the primitive naris, which is surrounded by an epithelial band.

FIGURE 2.

The beginning of early developmental phase of the naso‐palatal complex. (A) L.lugubris at stage 3. (B) E.macularius at stage 28. Key:a, anterior; dnc, diencephalon; e, eye; lnp, lateral nasal prominence; mes, mesencephalon; mnp, medial nasal prominence; pn, primitive naris; tel, telencephalon

In E.macularius the nasal pit is visible at the developmental stage 28, but in this case it is relatively deeper than in L.lugubris (Figure 2B).

The vomeronasal pit of L.lugubris embryo is visible at stage 7. It emerges from the medial wall of the nasal pit, near the primitive naris (Figure 3A–D). At this time the nasal pit is deeper than in developmental stage 3 (Figure 3B,B’). Its main mass is located anterodorsal to the vomeronasal pit (Figure 3A,A’). The primitive naris is now elongated and open on the ventrolateral side of the forming snout (Figure 3B’–D) which corresponds to ventral growth of the nasal prominences. At this developmental stage the lateral nasal prominence becomes better developed than medial nasal prominence (Figure 3B–B’). Moreover, the poorly developed maxillary prominence is located posterior to the primitive naris (Figure 3D).

FIGURE 3.

Early developmental phase of the naso‐palatal complex after formation of the vomeronasal pit in L.lugubris. (A) Stage 7, medial view on the nasal pit. (A’) Magnification of the area from the box in a. (B) Stage 7, anterior view of the head. (B’) Stage 7, transverse section through the nasal pit at the level of the vomeronasal pit. (C) Stage 7, ventral view of the forming palate. (D) Stage 7, lateral view of the forming snout. (E) Stage 12, anterior view of the head. (E’) Stage 12, transverse section through the nasal pit at the level of the vomeronasal pit. (F) Stage 12, ventral view of the forming palate. (G) Stage 12, lateral view of the forming snout. Note: the Stammteil (Stt) at stage 12 is not well defined and may be considered as primordium; red asterisk shows primordial lateral nasal concha. Key:a, anterior; dnc, diencephalon; e, eye; lnp, lateral nasal prominence; mes, mesencephalon; mnp, medial nasal prominence; mxp, maxillary prominence; pct, primitive choanal tube; pn, primitive naris; Stt, Stammteil; tel, telencephalon; vp, vomeronasal pit

Towards the end of early developmental phase of the naso‐palatal complex differentiation the division of the nasal pit into the primordial Stammteil, and the primitive choanal tube becomes visible in L.lugubris at developmental stages 10 (Figure S1) and 12 (Figure 3E,E’). At the same time the primitive choanal tube, which forms the most ventral and posteriormost part of the nasal pit, is not very distinct. At these developmental stages the vomeronasal pit is better developed than at stage 7. Moreover, the medial and lateral nasal prominences continue to grow (Figure 3E’ and Figure S1) and at stage 12 the primitive naris is shifted on the ventral surface of the snout (Figure 3E–G). The lateral nasal prominence is still better developed than the medial one. The primordial lateral nasal concha, emerging from the lateral nasal prominence, may be distinguished at the stage 10 of L.lugubris (red asterisks in Figure S1), but at the stage 12 the extraconchal space is still absent (Figure 3E’).The anteriormost part of the primitive naris becomes narrowed at stages 10 and 12 (Figure 3F and Figure S1). At the developmental stage 12 the maxillary prominence is slightly better developed than at previous developmental stages. Its smaller anterior end seems to terminate just posterior to the primitive naris (Figure 3G).

At the time of vomeronasal pit formation the morphology of the nasal pit in E.macularius of developmental stage 30 (Figure 4A–D) differs from that observed in L.lugubris at stages 7 to 12 (Table 2). The main mass of the nasal pit of E.macularius at stage 30 forms two apexes (short arrows in Figure 4A’). The entire nasal pit of this species is relatively widely open and there is no narrowing of the anteriormost part of the oval primitive naris (Figure 4C) in contrast to L.lugubris at stage 12. However, the presence of the small primordial lateral nasal concha (red asterisks in Figure 4B’), the lack of the extraconchal space, and the ventral location of the primitive naris (Figure 4C,D) are similar in both: E.macularius at developmental stage 30 and L.lugubris at stage 12.

FIGURE 4.

Early developmental phase of the naso‐palatal complex in E.macularius at stage 30 (the vomeronasal pit present). (A) Medial view of the nasal pit. (A’) Magnification of the area from the box in a. (B) Anterior view of the head. (B’) Transverse section through the nasal pit at the level of the vomeronasal pit. (C) Ventral view of the forming palate. (D) Lateral view of the forming snout. Note: the Stammteil (Stt) is not well defined and may be considered as primordium; red asterisk, primordial lateral nasal concha; short arrows, apexes of the nasal pit. Key:a, anterior; dnc, diencephalon; e, eye; lnp, lateral nasal prominence; mes, mesencephalon; mnp, medial nasal prominence; mxp, maxillary prominence; pct, primitive choanal tube; pn, primitive naris; Stt, Stammteil; tel, telencephalon; vp, vomeronasal pit

3.2. Middle developmental phase

The middle developmental phase of the naso‐palatal complex is marked by well‐developed nasal prominences. For better understanding of the processes taking place during this developmental phase, the fusion of the facial prominences and associated changes, which occur in development of the palate, nasal cavity and VNO, will be described first (before and at the time of choanal groove formation). Then the early development of the lacrimal duct is presented.

3.2.1. Palate, nasal cavity, VNO (before the formation of the choanal groove)

The medial nasal prominences are well distinguishable in L. lugubris at developmental stage 14. They approach each other closely and they are separated by deep ventral furrow (Figure 5A,B and see Figure 5D). The entire primitive naris is narrowed, but posterior half of this structure is more widely open to the stomodeum than the anterior one (Figure 5B–D’). The posterior border of the primitive naris, and thus the nasal pit, is formed by the maxillary prominence, which is well visible at this time (Figure 5B). Above the primitive naris, few modifications of the nasal pit can be noted. The anteriormost part of the primitive choanal tube, located just dorsal to the narrowest part of the primitive naris, is filled by the nasal plug (Figure 5D). The larger, remaining part of the primitive choanal tube is well formed at this stage. It is marked by the thinner nonsensory epithelium and is directly connected with the vomeronasal pit (Figure 5D’). Posterodorsal to the nasal plug, the primordium of the lateral nasal concha is well developed (Figure 5D’). The well‐developed nasal concha primordium, emerging from the lateral nasal prominence, corresponds with the appearance of the primordial extraconchal space (Figure 5D’). The vomeronasal pit is well developed and it takes spherical shape in L. lugubris at developmental stage 14 (Figure 5C,D’). Thus, it can now be called the early VNO.

FIGURE 5.

The middle developmental phase of the naso‐palatal complex in L.lugubris at stages 14 and 14_II. (A) Stage 14, anterior view of the head. (B) Stage 14, ventral view of the forming palate. (C) Stage 14, ventral view of the nasal pit. (D) Stage14, transverse section through the nasal pit at the level of the nasal plug. (D’) Stage 14, transverse section through the nasal pit at the level of the early VNO. (E) Stage 14_II, anterior view of the head. (F) Stage 14_II, ventral view of the forming palate. (G) Stage14_II, transverse section through the nasal pit at the level of the nasal plug. (G’) Stage14_II, transverse section through the nasal pit at the level of the early VNO. Note: the Stammteil (Stt), extraconchal space (ecs) and lateral nasal concha (lnc) may still be considered as primordia; red stars, primordia of the vomerine cushion; red arrow, fusion between medial nasal prominence of the frontonasal mass and the lateral nasal prominence. Key:a, anterior; e, eye; ecs, extraconchal space; ev, early VNO; fnm, frontonasal mass; lnc, lateral nasal concha; lnp, lateral nasal prominence; mnp, medial nasal prominence; mxp, maxillary prominence; np, nasal plug, pct, primitive choanal tube; pn, primitive naris; Stt, Stammteil; tel, telencephalon

In the other specimen of L. lugubris at stage 14 (coded here as stage 14_II) the naso‐palatal complex is more developmentally advanced. At this stage, the merging of the medial nasal prominences reduces the ventral furrow and forms the frontonasal mass (Figure 5E–G’). Nevertheless, the remnant of the furrow is still visible ventrally and both medial nasal prominences can be identified. Posterior to the major part of the frontonasal mass, the paired primordium of the vomerine cushion is visible (red stars in Figure 5F). Approximately the anterior two thirds of the primitive naris between the medial and lateral nasal prominences becomes strongly narrowed to the slit‐like opening (Figure 5F). Posterior third of the primitive naris is wider (Figure 5F). Considering the morphology of the primitive naris the nasal pit may be classified now as the early nasal cavity (but see below and Table 2). The region of the primitive choanal tube located anterior to the early VNO is still filled with the nasal plug (Figure 5G and Figure S2).

The occurrence of the nasal prominences fusion, posterior to the nasal plug, is difficult to evaluate based on mCT data (red arrow in Figure 5F,G’ and see Figure S2). This event seems to take place on the right side of the forming snout based on 3D view (red arrow in Figure 5F). Thus the division of the primitive naris into the external naris and primitive outer choana, and the formation of the vestibulum may start at this stage.

The primordial lateral nasal concha is better developed and the primordial extraconchal space is well visible on both sides of the snout (Figure 5G,G’). The primordial extraconchal space extends through the most length of the early nasal cavity. The early VNO is strongly developed and very distinct from the primitive choanal tube (Figure 5G’).

At stage 14/16 of L.lugubris the fusion of the facial prominences (maxillary prominence and medial nasal prominence of the frontonasal mass) forms well‐developed upper lip (Figure 6A,A’). The primitive outer choana narrows anteriorly towards still distinguishable frontonasal mass (Figure 6A’). It is bordered laterally by the maxillary fold, which emerges at this time from the maxillary prominence, and medially by the primordial anterior segment of the palate (anteriorly; yellow star) and primordium of the vomerine cushion (posteriorly; red star) (Figure 6A,A’). Both components of the paired primordium of the vomerine cushion are well fused dorsally, but they are still distinguishable ventrally from each other (Figure 6A,A’), as are both components of the primordial anterior segment of the palate, which becomes visible at this stage (yellow star/s in Figure 6A–B). The developing vomerine cushion and the anterior segment of the palate are weakly distinguishable from each on the palate (Figure 6A,A’).

FIGURE 6.

The middle developmental phase of the naso‐palatal complex in L.lugubris at stages 14/16 and 18. (A) Stage 14/16, ventral view of the palate. (A’) Stage 14/16, ventrolateral view of the palate. (B) Stage 14/16, transverse section through the snout at the level of the early VNO and anterior view of the right nasal cavity. (C) Stage 14/16, lateral view of the right nasal cavity. (C’) Stage 14/16, medial view of the right nasal cavity. (D) Stage 18, ventrolateral view of the palate. (E) Stage 18, transverse section through the snout at the level of the mushroom body of the VNO. (E’) Stage 18, transverse section through the snout at the level of the primitive outer choana. (F) Stage 18, transverse histological section through the VNO duct. (G) Stage 18, lateral view of the nasal cavity. Note:red star, primordium of the vomerine cushion; yellow star, primordium of the anterior segment of the palate; red dashed line, primitive inner choana; white asterisk, choanal diverticulum; blue asterisk, primordial mushroom body. Distribution of the sensory olfactory epithelium may be approximated according to X‐ray opacity (see colour bars). Key:a, anterior; aos, antorbital space; asp, anterior segment of the palate; da, diverticulum of the antorbital space; dv, VNO duct; e, eye; ecs, extraconchal space; en, external naris; ev, early VNO; fnm, frontonasal mass; lnc, lateral nasal concha; mxf, maxillary fold; mxp, maxillary prominence; ne, nonsensory (respiratory) epithelium of the nasal cavity; oe, sensory olfactory epithelium; pict, primitive inner choanal tube; poch, primitive outer choana; poct, primitive outer choanal tube; scf, subconchal fold; Stt, Stammteil; ul, upper lip; vc, vomerine cushion; vch, ventral channel; ves, vestibulum; vf, vomeronasal fenestra; vns, vomeronasal sensory epithelium

The vestibulum and the primordial main nasal cavity are well formed in L.lugubris at stage 14/16 (Figure 6B–C’). The vestibulum originates from the anteriormost part of the primitive choanal tube. At this stage, the vestibulum is vertically oriented (Figure 6B–C’). The primordial main nasal cavity includes the Stammteil, well‐defined extraconchal space, remnant of the primitive choanal tube (located just ventral to the Stammteil) and not very distinct antorbital space, which is located posterior to the lateral nasal concha (Figure 6B,C’). The primitive choanal tube extends beyond the primordial main nasal cavity (Figure 6C,C’) and opens on the palate as a primitive outer choana (Figure 6A’). The extraconchal space extends along the most length of the primordial main nasal cavity (Figure 6C). The subconchal fold originates from the base of the lateral nasal concha. It is located ventrolateral to the early VNO (Figure 6B). Below the subconchal fold moderately deep choanal diverticulum is present (white asterisks in Figure 6B).

The sensory olfactory epithelium constitutes most of the epithelium of primordial main nasal cavity (Figure 6B–C’; for details see Table 2). The early VNO of L.lugubris at developmental stage 14/16 is larger and it is enters the primitive choanal tube approximately at the border between the main nasal cavity primordium and the posterior extension of the primitive choanal tube (Figure 6B–C’). Small primordium of the mushroom body can be distinguished (not shown, but see blue asterisks in Figure 6E,F).

At developmental stage 18 of L.lugubris the primordial duct of the VNO is formed. It can be distinguished from the anteriormost part of the remnant of primitive choanal tube (primitive outer choanal tube; see below) (Figure 6D–F). This part of the primitive choanal tube becomes separated from the primordial main nasal cavity by the fusion of the anteriormost part of the subconchal fold with the derivative of frontonasal mass, located dorsal to the VNO and the anterior segment of the palate (Figure 6E,F). The primordium of the VNO duct is bordered laterally by the maxillary fold and medially by the anterior segment of the palate (Figure 6D). At this time it is sealed by the cellular plug (Figure 6F). The fusion of the subconchal fold to the derivative of frontonasal mass separates also the anteriormost part of the choanal diverticulum, which now may be considered as a primordial ventral channel of the VNO (Figure 6E,F).

The single anterior segment of the palate with the premaxillary papilla (in the middle) is distinguishable ventrally from the single vomerine cushion at this stage (Figure 6D). The vomerine cushion and the maxillary fold border the elongated primitive outer choana (Figure 6D,E’). The primitive outer choana leads directly to the smaller part of the nasal cavity, which is distinct from the well developed at this time the primordial main nasal cavity (Figure 6G). The smaller part of the nasal cavity, directly connected to the VNO, can be called now the primitive outer choanal tube as opposed to the rest primitive choanal tube incorporated to larger primordial main nasal cavity. The part of the primitive choanal tube incorporated to the primordial main nasal cavity is now the primitive inner choanal tube (Figure 6E,E’, g). The primitive inner choana may be distinguished between these two parts of the primitive choanal tube (red dashed line in Figure 6E’). All component of the conchal zone of the main nasal cavity (the Stammteil, extraconchal space and primitive inner choanal tube) pass posteriorly into the antorbital space. The antorbital space remains weakly developed, except its very distinct diverticulum, which is visible at this time (Figure 6G).

The first stage of the E.macularius (30/31) in the middle developmental phase is similar to L. lugubris at stage 14 (Figure S3 and see Table 2). The primitive naris of the E.macularius at stage 30/31 is elongated, but it is relatively widely open in contrast to stage 14 of L.lugubris. Moreover, the primitive choanal tube and the nasal plug of E.macularius embryos at stage 30/31 are not as distinct as in L.lugubris, and the vomeronasal pit does not form early VNO (Figure S3).

The early nasal cavity in E.macularius at middle developmental phase was not found (see the discussion). The embryo identified as stage 31 is characterised by the fusion of the lateral nasal prominence with the medial nasal prominence of the frontonasal mass (red arrow in Figure 7A–B’ and see Figure S2). Moreover, the beginning of the fusion of the nasal prominences is evident and the formed vestibulum may be easily distinguished in the embryo of E.macularius (Figure 7A–B’). Similarly, as in L.lugubris at stage 14_II, the nasal plug is well developed in the vestibulum and the newly formed external naris is sealed. Also as in L.lugubris at stage 14_II, the paired primordium of the vomerine cushion is visible (red stars in Figure 7A). The primordial extraconchal space of E.macularius extends along the posterior two thirds of primordial main nasal cavity (Figure 7B). Moreover, the weakly developed choanal diverticulum is visible at this time (white asterisks in Figure 7C).

FIGURE 7.

Middle developmental phase of the naso‐palatal complex in E.macularius before the formation of the choanal groove. (A) Stage 31, ventral view of the palate. (B) Stage 31, lateral view of the nasal cavity. (B’) Stage 31, medial view of the nasal cavity. (C) Stage 31, transverse section through the snout at the level of the early VNO and the anterior view of the nasal cavity. (D) Stage 31/32, ventral view of the palate. (E) Stage 31/32, transverse sections through the snout at the level of the mushroom body (left) and slightly posterior to it (right). (F) Stage 31/32, dorsal view of the nasal cavity. (F’) Stage 31/32, lateral view of the nasal cavity. (F’’) Stage 31/32, medial view of the nasal cavity. (G) Stage 34, ventral view of the palate. (H) Stage 34, lateral view of the nasal cavity. (I) Stage 34, transverse section through the snout at the level of the primitive outer choana. Note: the Stammteil (Stt), extraconchal space (ecs) and lateral nasal concha (lnc) may still be considered as primordia at stage 31; red arrow, fusion of the lateral nasal prominence and the medial nasal prominence of the frontonasal mass; red arrowhead, nasolacrimal groove; red stars, primordia of the vomerine cushion; red dashed line, primitive inner choana; white asterisk, choanal diverticulum; blue asterisk, primordial mushroom body; green arrowhead, rostral recess of the extraconchal space; yellow arrowheads, choanal fold. Distribution of the sensory olfactory epithelium may be approximated according to X‐ray opacity (see colour bar). Key:a, anterior; asp, anterior segment of the palate; da, diverticulum of the antorbital space; dv, VNO duct; e, eye; ecs, extraconchal space; en, external naris; ev, early VNO; fnm, frontonasal mass; ld, lacrimal duct; lnc lateral nasal concha; mxf, maxillary fold; mxp, maxillary prominence; ne, nonsensory (respiratory) epithelium of the nasal cavity; oe, sensory olfactory epithelium; pct, primitive choanal tube; pict, primitive inner choanal tube; poch, primitive outer choana; poct, primitive outer choanal tube; pp, premaxillary papilla; scf, subconchal fold; Stt, Stammteil; ul, upper lip; ves, vestibulum; vf, vomeronasal fenestra; VNO, vomeronasal organ; vns, vomeronasal sensory epithelium

The primitive outer choana in E.macularius at stage 31/32 is oval rather than strongly elongated and narrowed as at corresponding stage 14/16 of L.lugubris (Figure 7D). The anterior segment of the palate in E.macularius at stage 31/32 resembles the condition of L.lugubris at stage 18, but in contrast to that stage both components of the vomerine cushion are still distinguishable ventrally in E.macularius (Table 2; Figure 7D). The extraconchal space and lateral nasal concha are well developed (Figure 7E) and a small rostral recess of extraconchal space may appears (green arrowhead in Figure 7F,F’).

The subconchal fold and well‐developed choanal diverticulum are present in the embryo of E.macularius at stage 31/32 (white asterisk in Figure 7E). The antorbital space is present as a not very distinct diverticulum (Figure 7F). At this time the early VNO is connected to the primitive choanal tube at the similar level as in L.lugubris at stage 14/16 (Table 2; Figure 7E,F’,F’’). The sensory olfactory epithelium covers most of the main nasal cavity lumen in E.macularius at stage 31/32, as in embryo of L.lugubris at stage 14/16 (Figure 7F–f’’; for details see Table 2).

The embryonic features of the naso‐palatal complex in E.macularius at stage 34 correspond to the many morphological aspects of L.lugubris at stage 18 (see Table 2). In both species the VNO duct can be distinguished from the anteriormost part of the primitive outer choanal tube (Figure 7G,H). The entire primitive outer choanal tube and primitive outer choana of E.macularius at stage 34 becomes elongated (Figure 7G). In contrast to L.lugubris at stage 18, the anterior segment of the palate is obliterated and indistinguishable form the vomerine cushion (Figure 7G). The primordial choanal fold emerges from the maxillary fold (yellow arrowheads in Figure 7G,I). The rostral recess of the extraconchal space is better developed than at the previous developmental stage (green arrowhead in Figure 7H) and an indentation between this structure and Stammteil is visible (Figure 7H).

3.2.2. Palate, nasal cavity, VNO (at the time of appearance of the choanal groove)

The choanal groove of both studied species originates from the anterior part of the primitive outer choanal tube, just posterior to the VNO duct. The primitive outer choanal tube becomes separated from the rest of the nasal cavity by the fusion of the subconchal fold with the part of the vomerine cushion located just medial to this fold (white arrow in Figure 8A,B and see the Video S1). The choanal groove in L.lugubris is first visible at stage 20–22 and in E.macularius at ‘late’ stage 34 (here as 34_II). Anteriorly, the choanal groove is confluent with the VNO duct, while posteriorly its opening is confluent with the remnant of the primitive outer choana, which is called now the outer choana (Figure 8c,d,e,f). At the level of the outer choana the subconchal fold is still distinct (see the Video S1). The choanal groove, just after its formation, is short. In L.lugubris at stage 20–22 it is almost three times shorter than the VNO (Figure 8e) and approximately half the length of the outer choana (Figure 8c). In E.macularius at stage 34_II, the choanal groove is about three times shorter than the VNO (Figure 8f) and about four times shorter than the outer choana (Figure 8d).

FIGURE 8.

Middle developmental phase at the time of formation of the choanal groove in L.lugubris and E.macularius. (A, B) Transverse cutaways at the level of the choanal groove in L.lugubris at stage 20–22 (A) and E.macularius at stage 34_II (b). (C, D) Ventromedial views of the outer choana in L.lugubris at stage 20–22 (C) and E.macularius at stage 34_II (D). Ventromedial (E), dorsal (E’) and lateral views (E’’) of the nasal cavity in L.lugubris at stage 20–22. Ventromedial (F), dorsal (F’) and lateral views (F’’) of the nasal cavity in E.macularius at stage 34_II. (G, H) Transverse sections through the snout at the level of the vestibulum in L.lugubris at stage 22 (G) and E.macularius at stage 34_II (H). Note:thick white arrow, fusion of the subconchal fold with the vomerine cushion; yellow arrowhead, choanal fold; green arrowhead, rostral recess of the extraconchal space; green arrow, initial bud of lateral nasal gland; yellow arrow, initial bud of medial nasal gland. Distribution of the sensory olfactory epithelium may be approximated according to X‐ray opacity (see colour bars). Key:a, anterior; asp, anterior segment of the palate; da, diverticulum of the antorbital space; dv, VNO duct; e, eye; ecs, extraconchal space; en, external naris; ict, inner choanal tube; lnc, lateral nasal concha; mxf, maxillary fold; ne, nonsensory (respiratory) epithelium of the nasal cavity; ns, nasal septum; och, outer choana; oct, outer choanal tube; oe, sensory olfactory epithelium; Stt, Stammteil; vc, vomerine cushion; ves, vestibulum; vf, vomeronasal fenestra

The choanal fold in L.lugubris is first visible at stage 20–22, while at stage 34_II of E.macularius this structure becomes more distinct (yellow arrowheads in Figure 8a–d). After formation of the choanal groove, the shortened primitive inner choana may be called the inner choana. Moreover, the primitive inner choanal tube (part of the main nasal cavity) is now the inner choanal tube and the primitive outer choanal tube may be considered as the outer choanal tube. The inner choanal tube forms a recess in the lateral nasal concha. This recess is called the Aulax (Beecker, 1903; Fuchs, 1908; Pratt, 1948; Parsons, 1970) and it is weakly developed in both compared species (see the Video S1), but it is slightly more visible in embryo of L.lugubris than in E.macularius. The snout as well as the entire nasal cavity and the outer choana in L.lugubris at stage 20–22 is not so elongated (Figure 8C,E–E’’) as in E.macularius at stage 34_II (Figure 8D,F–f’’). Thus the shape of the nasal cavity in the L.lugubris at the time when the choanal groove is first visible is similar to the shape of the nasal cavity at previous stage (see Figure 6G). The vestibulum of the L.lugubris remains vertical (Figure 8E”), while in E.macularius it attains more horizontal orientation (Figure 8F’’). The posteriormost part of the vestibulum forms the initial bud of lateral nasal gland (green arrow in Figure 8G,H). Moreover, in L.lugubris the initial bud of the other nasal gland can be identified. Here we called it the medial nasal gland (yellow arrows in Figure 8G).

The diverticulum of antorbital space in embryo of L.lugubris at stage 20–22 is less distinct than at previous stage, but it is still well visible (Figure 8e’). The antorbital space of E.macularius does not exhibit significant changes from younger embryo at stage 34 (Figure 8f’).

3.2.3. Lacrimal duct

The primordium of the lacrimal duct in L.lugubris is first visible at developmental stage 14/16. It extends as two primordial canaliculi, one of which is located in the tissue of the primordial lower eyelid and it is in contact with its external surface, and the other canaliculus is located in a deep aspect of the groove between the primordium of the lower eyelid and the maxillary prominence (Figure 9A). The canaliculi run close to each other (Figure 9A). Anteriorly they pass into a single primordial lacrimal duct in the groove between the primordium of the lower eyelid and the maxillary prominence (Figure 9A’), but it seems that the duct primordium does not reach the nasolacrimal groove, which marks the border between the maxillary prominence and the lateral nasal prominence (Figure 9B). At stage 18 of L.lugubris, the primordial lacrimal duct is still associated with the surface of the snout and its canaliculi are still hardly distinguishable from each other (Figure 9C,C’). The anterior tip of primordial lacrimal duct (red arrow in Figure 9C’) approaches the primordial ventral channel of the VNO (Figure 9D). The elongated lacrimal duct dives into the underlying mesenchymal tissue and thus becomes independent from the groove between the primordial lower eyelid and the maxillary component of the snout at stage 20–22 (Figure 9E–F). Both lacrimal canaliculi sink into the tissue of the lower eyelid. At this time the anterior tip of the lacrimal duct closely approaches the primordial ventral channel of the VNO (Figure 9F).

FIGURE 9.

Origin of the lacrimal duct in L.lugubris, middle developmental phase of the naso‐palatal complex. (A, A’) Stage 14/16, transverse sections through the snout at the level of lacrimal canaliculi (a) and single part of the primordial lacrimal duct (A’). (B) Stage 14/16, anterolateral view of the head. (C, C’) Stage 18, lateral views of the snout. (D) Stage 18, transverse cutaway at the level of the primordial ventral channel. (E, E’) Stage 20–22, lateral views of the snout. (F) Stage 20–22, transverse cutaway at the level of primordial ventral channel. Note:short red arrow, anterior tip of the (primordial) lacrimal duct; white asterisk, choanal diverticulum. The ventral channel (vch) is at primordial stage and the lacrimal canaliculi (lcc) are indistinguishable on 3D reconstructions. Key:a, anterior; dv, VNO duct; e, eye; ecs, extraconchal space; el, lower eyelid; en, external naris; ev, early VNO; fnm, frontonasal mass; ict, inner choanal tube; lcc, lacrimal canaliculi; lnc, lateral nasal concha; lnp, lateral nasal prominence; mxp, maxillary prominence; pct, primitive choanal tube; pict primitive inner choanal tube; plc, primordial lacrimal duct; scf, subconchal fold; Stt, Stammteil; ul, upper lip; vch, ventral channel; vns, vomeronasal sensory epithelium

The primordium of the lacrimal duct of E.macularius is visible at stage 31/32. Similar to L.lugubris at stage 14/16, it is composed of two canaliculi, which anteriorly pass into the single duct (Figure 10A,A’). As in L.lugubris, the lacrimal canaliculi of the primordial lacrimal duct are associated with the tissue of the primordial lower eyelid. However, in E.macularius, both canaliculi are easily distinguishable from each other, and they are located at the bottom of two relatively deep grooves (Figure 10A,A’). Externally these grooves are visible as peculiar incisions on the surface of the primordium of the lower eyelid (Figure 10A). Anteriorly, the primordial lacrimal duct probably reaches the posterior part of the nasolacrimal duct, but it is difficult to evaluate since this groove is not very distinct at stage 31/32 of E.macularius (Figure 10A). The lacrimal duct, including its canaliculi, is elongated and independent from the surface of the snout at stage 34 of E.macularius (Figure 10B,C). The anterior tip of the lacrimal duct reaches the level of the primordial ventral channel of the VNO (Figure 10B), but it does not approach it closely (Figure 10C). The connection of the well‐developed lacrimal duct and the primordial ventral channel is present at the later analysed stage (stage 34_II) (Figure 10D–E’). Such a connection is not present in L.lugubris at stage 20–22, where only close apposition of the lacrimal duct and the primordial ventral channel can be noted (see Figure 9F).

FIGURE 10.

Origin of the lacrimal duct in E.macularius, middle developmental phase of the naso‐palatal complex. (A, A’) Stage 31/32, anterolateral view of the snout (nasal cavity). (B) Stage 34, anterolateral view of the nasal cavity. (C) Stage 34, transverse cutaway of the snout at the level of the primordial ventral channel. (D) Stage 34_II, anterolateral view of the nasal cavity. (E) Stage 34_II, transverse cutaway of the snout at the level of the primordial ventral channel. (E’) Stage 34_II, transverse histological section at the level of the primordial ventral channel. Note:short red arrow, anterior tip of the (primordium) lacrimal duct. Key:dv, VNO duct; e, eye; ecs, extraconchal space; el, lover eyelid; en, external naris; fnm, frontonasal mass; ict, inner choanal tube; lcc, lacrimal canaliculi; ld, lacrimal duct; lnc, lateral nasal concha; lnp, lateral nasal prominence; mxp, maxillary prominence; ns, nasal septum; pct, primitive choanal tube; pict primitive inner choanal tube; poct, primitive outer choanal tube; Stt, Stammteil; ul, upper lip; vch, ventral channel; ves, vestibulum; vns, vomeronasal sensory epithelium

3.3. Late developmental phase

The late developmental phase starts at the time of the fusion of the lacrimal duct with the choanal groove. Due to increase of morphological complexity of analysed structures, we divided the description of this developmental phase of naso‐palatal complex into the nasal cavity, palate–choanal groove–lacrimal duct, and the VNO.

3.3.1. Nasal cavity

The beginning of late developmental phase in L.lugubris is represented here by stage 26 (Figure 11A–D and see the Video S2). The snout and the entire nasal cavity are now much more elongated (Figure 11A,C,D) compared to the stage 20–22. The vestibulum takes on a more horizontal orientation than previously. The posterior end of the vestibulum is associated with the lateral and medial nasal glands, which are elongated initial buds (Figure 11A,C). Despite the elongation of the entire nasal cavity, the range of the extraconchal space does not differ from the previous stage, and it extends along the posterior two thirds of the main nasal cavity (Figure 11C,D). Anterior to the extraconchal space, the lateral nasal concha is visible as a small anterior extension which becomes better developed in later stages due to the lateral expansion of the inner choanal tube (Video S2). The Aulax is slightly better developed than at stage 20–22 (Figure 11B). The entire antorbital space is now well visible, but its diverticulum is almost indistinguishable (Figure 11C,D). The sensory olfactory epithelium covers the major part of the Stammteil lumen (except its posteriormost, ‘antorbital’, part and small posteroventromedial part), approximately anterior half of extraconchal space and it forms dorsomedial wall of the inner choanal tube (Figure 11B–D).

FIGURE 11.

Beginning (A–D) and the end (E–H) of late developmental phase of the naso‐palatal complex in L.lugubris. (A) Stage 26, anterodorsal view of the snout. (B) Stage 26, transverse section and transverse cutaway through the snout at the level of the Aulax. (C) Stage 26, lateral view of the nasal cavity. (D) Stage 26, dorsal view of the nasal cavity. (E) Stage 50–60, anterodorsal view of the snout. (F) Stage 50–60, transverse section and transverse cutaway through the snout at the level of Aulax. (G) Stage 50–60, lateral view of the nasal cavity. (H) Stage 50–60, dorsal view of the nasal cavity. Note:green arrowhead, rostral recess of the extraconchal space; yellow arrowhead, choanal fold. Distribution of the olfactory sensory epithelium may be approximated according to X‐ray opacity (see colour bars). Key:a, anterior; aos, antorbital space; Aux, Aulax; Bg, Bowman's glands; da, diverticulum of the antorbital space; dv, VNO duct; ecs, extraconchal space; en, external naris; ict, inner choanal tube; lcc, lacrimal canaliculi; lcf, lateral choanal fissure; lnc, lateral nasal concha; mpv, medial protuberance of vestibulum; mxf, maxillary fold; ne, nonsensory (respiratory) epithelium of the nasal cavity; och, outer choana; oct, outer choanal tube; oe, sensory olfactory epithelium; Stt, Stammteil; vc, vomerine cushion; ves, vestibulum

The morphology of the nasal cavity of L.lugubris at stages 28–30 and 35 represents an intermediate condition between those observed at stage 26 and stage 50–60 (Figure S4 and Video S2; for details see Table 2).

The adult‐like morphology of the nasal cavity at stage 50–60 of L.lugubris is shown in Figure 11E–H and see the Video S2). The vestibulum takes on an almost horizontal orientation (Figure 11G). It starts to be patent at this stage, which is due to the reduction of the nasal plug. The peculiar feature of vestibulum at this stage is the presence of the medial protuberance, formed by an epithelial thickening located just anterior to the medial nasal gland (Figure 11E,H). Both nasal glands are well developed and the relatively small anterior segment of the lateral one and larger anterior segment of the medial one are distinguishable (Figure 11E,G,H). The lateral nasal concha is curled ventrally and thus the Aulax is as well developed as at stage 35 (Figure 11F). In contrast to the all previous stages, the embryo of the L.lugubris at stage 50–60 is characterised by a small rostral recess of the extraconchal space (green arrowhead in Figure 11H). Anterior to the extraconchal space, the anterior extension of the lateral nasal concha is well formed due to the dorsolateral expansion of the inner choanal tube (Figure 11G and green asterisk in Video S2). The antorbital space is large and its diverticulum becomes relatively well visible again (but it is not very distinct) (Figure 10G,H and see the Video S2). The Bowman's glands of the sensory olfactory epithelium are very well developed and protruding into the connective tissue (Figure 11E–h). The distribution of olfactory sensory epithelium, however, is greatly reduced, becoming restricted to approximately the anterior two thirds of the Stammteil and anterior third of the extraconchal space (Figure 11F–h).

The beginning of the late developmental phase in E.macularius is stage 35 (Figure 12A–d and see the Video S3). The snout and each component of the naso‐palatal complex are slightly more elongated than at stage 34_II (Figure 12A,C,D). The vestibulum has a similar orientation to previously, but initial bud of the lateral nasal gland is better developed (Figure 12C,D). In contrast to L.lugubris, there is no sign of the medial nasal gland. The range of the extraconchal space becomes reduced. It extends now along the posterior half of the main nasal cavity (Figure 12C,D). The inner choanal tube is relatively well extended laterally, thus the anterior extension of the lateral nasal concha is better developed than in L.lugubris at stage 26 (Figure 12C,D and green asterisk in Video S3). The rostral recess of the extraconchal space is strongly developed at this stage (green arrowhead in Figure 12C,D). Moreover, the indentation between the Stammteil and the rostral recess of extraconchal space is well visible (Figure 12D). The Aulax of E.macularius at stage 35 resembles the condition in the corresponding stage (26) of L.lugubris (Figure 12B and see the Video S3). The antorbital space of E.macularius is present, but it is not so distinct as at stage 26 of L.lugubris, except that its diverticulum is comparatively better developed than in L.lugubris (Figure 12D). The olfactory sensory epithelium forms similar part of the main nasal cavity walls as in L.lugubris at stage 26 (Figure 12C,D; for details see Table 2).

FIGURE 12.

Beginning (A–D) and the end (E–H) of late developmental phase of the naso‐palatal complex in E.macularius. (A) Stage 35, anterodorsal view of the snout. (B) Stage 35, transverse section and transverse cutaway through the snout at the level of the Aulax. (C) Stage 35, lateral view of the nasal cavity. (D) Stage 35, dorsal view of the nasal cavity. (e) Stage 42, anterodorsal view of the snout. (F) Stage 42, transverse section and transverse cutaway through the snout at the level of the Aulax. (G) Stage 42, lateral view of the nasal cavity. (H) Stage 42, dorsal view of the nasal cavity. Note:green arrowhead, rostral recess of the extraconchal space; yellow arrowhead, choanal fold. Distribution of the olfactory sensory epithelium may be approximated according to X‐ray opacity (see colour bars). Key:a, anterior; aos, antorbital space; Aux, Aulax; Bg, Bowman's glands; da, diverticulum of the antorbital space; dv, VNO duct; ecs, extraconchal space; en, external naris; ict, inner choanal tube; lcc, lacrimal canaliculi; lcf, lateral choanal fissure; lnc, lateral nasal concha; mxf, maxillary fold; ne, nonsensory (respiratory) epithelium of the nasal cavity; och, outer choana; oct, outer choanal tube; oe, sensory olfactory epithelium; Stt, Stammteil; vc, vomerine cushion; ves, vestibulum

The morphology of the nasal cavity of E.macularius at stages 37 and 38 represents an intermediate condition between stages 35 and 42 (Figure S5 and Video S3; for details see Table 2).

The adult‐like morphology of the nasal cavity at stage 42 of the E.macularius is shown in Figure 12E–H and see the Video S3). As in L.lugubris at stage 50–60, the vestibulum of adult‐like stage of E.macularius takes an almost horizontal orientation (Figure 12G). The lateral nasal gland is well developed, including its anterior part, which in the adult‐like stage of L.lugubris is not so strongly developed (Figure 12G,H). The medial nasal gland is absent as in previous stages of E.macularius. The extraconchal space emerges from the posterior half of the Stammteil (including antorbital space) (Figure 12G,H), and the rostral recess of extraconchal space is strongly developed as at stage 38 (green arrowhead in Figure 12G,H). The antorbital space and the anterior extension of the lateral nasal concha are strongly developed similar to the adult‐like condition of L.lugubris (Figure 12G,H and Video S3). Due to the growth of entire antorbital space its diverticulum is not as distinct as at stage 38 (similar to the adult‐like stage of L.lugubris) (Figure 12G,H). The Bowman's glands of the olfactory sensory epithelium are greatly developed at this time (Figure 12G,H). The distribution of olfactory sensory epithelium is slightly restricted in comparison to the previous stages, but comparatively larger than in the adult‐like condition of L.lugubris (Figure 12G,H; for details see Table 2).

3.3.2. Palate–choanal groove–lacrimal duct

At the beginning of the late developmental phase of the naso‐palatal complex (stages 26 of L.lugubris and 35 of E.macularius) the anterior segment of the palate is still visible only in L.lugubris (Figure 13A,B). The vomeronasal fenestra is located posterior to the anterior segment of the palate (Figure 13A). The VNO duct is still confluent posteriorly with the choanal groove in both species (Figure 13A,B). In L.lugubris at stage 26, the choanal groove is slightly shorter than the VNO (see Figure 16) and, as previously, approximately half the length of the outer choana (Figure 13A). In E.macularius at stage 35, the choanal groove is approximately half the length of the VNO (see Figure 16) and becomes about two times shorter than the outer choana (Figure 13B). The choanal groove in both studied species is characterised by the primordial inner fold. It emerges from the subconchal fold fused with the vomerine cushion (red asterisks in Figure 13C,D). The primordial inner fold of the choanal groove extends from the inner choana to the level of the connection of the lacrimal duct with the choanal groove (see the Videos S2 and S3). The part of choanal groove homologous to the choanal diverticulum is characterised by horizontal orientation in L.lugubris at stage 26 (Figure 13C), whereas in E.macularius at stage 35 this part of the choanal groove (located posterior to the level of connection between lacrimal duct and the choanal groove) has almost vertical orientation (dashed ellipse in Figure 13D).

FIGURE 13.

Palate–choanal groove–lacrimal duct in L.lugubris (A, C) and in E.macularius (B, D) at the beginning of the late developmental phase of the naso‐palatal complex. (A, B) Ventral views of the palate in L.lugubris at stage 26 (A) and in E.macularius at stage 35 (B). (C, D) Transverse sections through the snout at the level of the choanal groove in L.lugubris at stage 26 (C) and in E.macularius at stage 35 (D). Note:red asterisk, primordial inner fold of the choanal groove; dashed ellipse, vertical part of the choanal groove; yellow arrowhead, choanal fold. Key:a, anterior; asp, anterior segment of the palate; chg, choanal groove; ecs, extraconchal space; ict, inner choanal tube; ld, lacrimal duct; lnc, lateral nasal concha; mxf, maxillary fold; och, outer choana; Stt, Stammteil; vc, vomerine cushion; vf, vomeronasal fenestra

FIGURE 16.

The range of the connections of the lacrimal duct to the choanal groove (from the left side of the snout) in L.lugubris (upper half) and E.macularius (lower half) at late developmental phase of the naso‐palatal complex. Note:long red arrows, the range of the lacrimal duct‐choanal groove connection; short red arrow, anterior tip of the lacrimal duct. Key:a, anterior; dv, VNO duct; mb, mushroom body; vch, ventral channel; vns, vomeronasal sensory epithelium

Due to the further growth of the snout, the relative length of the choanal groove to the VNO and the outer choana is changed. The choanal groove becomes relatively longer at stages 28–30 and 35 of L.lugubris, and at stage 35 its length is almost the same as outer choana (for details see Table 2; Figure 14A and see Figure 16). The elongation of the choanal grove in E.macularius takes place at stages 37 and 38, but it seems that it is not so rapid, and at stage 38 the choanal groove is still approximately half the length of the outer choana (for details see Table 2; Figure 14B and see Figure 16). However, it is worth noting that the definitive border of the outer choana is impossible to evaluate.

FIGURE 14.

Palate–choanal groove–lacrimal duct in L.lugubris (A, C, E) and in E.macularius (B, D, F) at preadult‐like stages; late developmental phase of the naso‐palatal complex. (A, B) Ventral views of the palate in L.lugubris at stage 35 (A) and in E.macularius at stage 38 (B). (C, D) posteroventral views of the choanal groove in L.lugubris at stage 35 (C) and in E.macularius at stage 38 (D). (E, F) Transverse histological sections through the snout at the level of the connection between the choanal groove and the lacrimal duct in L.lugubris at stage 35 (E) and E.macularius at stage 38 (F). Colours of 3D structures as in Figure 13. Note:red asterisks, inner fold of the choanal groove; grey arrow, lacrimal duct entering the tissue of the inner fold of the choanal groove; black arrow, lacrimal duct connected to the middle diverticulum of the choanal groove; yellow arrowheads, choanal fold. Key:a, anterior; apw, anterior palatal wing; asp, anterior segment of the palate; chg, choanal groove; ecc, ectochoanal cartilage; ich, inner choana; ict, inner choanal tube; lcf, lateral choanal fissure; ld, lacrimal duct; mxf, maxillary fold; och, outer choana; p, posterior; pp, premaxillary papilla; scf, subconchal fold; Stt, Stammteil; vc, vomerine cushion; vf, vomeronasal fenestra

At stages 28–30 and 35 of L.lugubris and stage 37 of E.macularius, the anteriormost part of the choanal groove (and posteriormost part of the VNO duct) is characterised by a short ventrolateral fold of the vomerine cushion. It is relatively well visible at stage 35 of L.lugubris (Figure 14A,C) and it is distinct at stages 37 and 38 of E.macularius (Figure 14B,D). We called it here the anterior palatal wing. The anterior end of the inner fold of the choanal groove terminates on the posterolateral part of the anterior palatal wing (Figure 14C). Instead, in E.macularius at stages 37 and 38, the edge of the strongly developed inner fold of the choanal groove is confluent anteriorly with the edge of the anterior palatal wing (Figure 14D). In L.lugubris at stages 28–30 and 35, the inner fold of the choanal groove divides the posterior part of the choanal groove into two diverticula, of which the lower one is homologous to the choanal diverticulum (Figure 14C,E). The condition observed in E.macularius at stages 37 and 38 is even more complicated. The part of the choanal groove homologous to the choanal diverticulum is subdivided into two diverticula. Thus, in the part of choanal groove located posterior to the anterior palatal wing, three diverticula are visible (Figure 14F). From about stage 37 of E.macularius the middle diverticulum of the choanal groove is connected with the anterior part of the lacrimal duct (black arrow in Figure 14F), while in L.lugubris the anterior part of the lacrimal duct intrudes into the tissue of the inner fold of the choanal groove (grey arrow in Figure 14E).

The morphology of analysed components of the naso‐palatal complex in the oldest analysed stages (50–60 of L.lugubris and 42 of E.macularius) resembles in general its morphology in previous stages (35 of L.lugubris and 38 of E.macularius respectively). The relative length of the choanal groove is increased in L.lugubris, but seems to be only slightly changed in E.macularius (for details see Table 2; Figure 15A,B and see Figure 16). The anterior palatal wing in both studied species is well formed (Figure 15A,B). The anterior segment of the palate and the maxillary fold are still well visible at stage 50–60 of L.lugubris (Figure 15A). In E.macularius at stage 42, only the premaxillary papilla is visible and the maxillary fold seems to be less distinct with comparison to L.lugubris (Figure 15B). As previously, the anterior part of the lacrimal duct in L.lugubris intrudes the inner fold of the choanal groove (grey arrow in Figure 15C and see the Video S2). In E.macularius the lumen of the choanal groove and the lacrimal duct are relatively larger (Figure 15D). The anterior part of the lacrimal duct is strongly associated with the middle diverticulum of the choanal groove and the border between these two structures is difficult to evaluate even in the histological sections (Figure 15D and see the Video S3).

FIGURE 15.

Palate–choanal groove–lacrimal duct in L.lugubris (A, C) and in E.macularius (B, D) at the end of the late developmental phase of the naso‐palatal complex (adult‐like stages). (A, B) Ventral views of the palate in L.lugubris at stage 50–60 (A) and in E.macularius at stage 42 (B). (C, D) Transverse histological sections through the snout at the level of the choanal groove in L.lugubris at stage 50–60 (C) and in E.macularius at stage 42 (D). Colours of 3D structures as in Figure 13. Note:red asterisk, inner fold of the choanal groove; grey arrow, lacrimal duct entering the tissue of the inner fold of choanal groove; black arrows, approximated border between the lacrimal duct and the middle diverticulum of the choanal groove; yellow arrowheads, choanal fold. Key:a, anterior; apw, anterior palatal wing; asp, anterior segment of the palate; chg, choanal groove; ecc, ectochoanal cartilage; ld, lacrimal duct; mxf, maxillary fold; och, outer choana; pp, premaxillary papilla; vc, vomerine cushion; vf, vomeronasal fenestra

In L.lugubris the connection of the lacrimal duct with the choanal groove is restricted approximately to the anterior half of the latter through the entire late developmental phase (long red arrows in Figure 16). At the earliest analysed stage of the E.macularius in this phase (stage 35), the lacrimal duct is also connected to the anterior half of the choanal groove, but from about stage 37, the range of connection exceeds the anterior half of the choanal groove (long red arrows in Figure 16; for details see Table 2). Despite the close apposition (or even connection in E.macularius) of the lacrimal duct and the embryonic ventral channel at the middle developmental phase (see Table 2), the connection between the lacrimal duct and the lateral part of the ventral channel of the VNO is not present in both species at late developmental phase. In contrast, the anterior tip of the lacrimal duct of both studied gekkotans (short red arrow in Figure 16) passes over the dorsal part of the choanal groove and gradually moves towards the medial wall of the VNO duct, finally reaching its posterior part at about stage 35 of L.lugubris and at about stage 37 of E.macularius (for details see Table 2). The lacrimal duct is fully patent in adult‐like stages of both species. The opening of the lacrimal duct to the choanal groove in confluent with the opening of the lacrimal duct to the VNO duct (Videos S4 and S5). It is worth mentioning that there is no direct connection between the lacrimal duct and the superficial palate (Figure 16).

3.3.3. VNO

At the late developmental phase the VNO is located ventral to the posterior half of the vestibulum and anteriormost part of the main nasal cavity in L.lugubris (stages 26 and 50–60; see Figure 11A,C,E,G) or ventrally predominantly to the anterior part of the main nasal cavity in E.macularius (Figure 12A,C,E,G). The VNO, as in the previous developmental phase, is composed of the dorsal dome (formed by the vomeronasal sensory epithelium), mushroom body, duct of the VNO and ventral channel (Figure 17 and see the Videos S4 and S5). The VNO duct connects the organ to the choanal groove posteriorly and the oral cavity ventrally. The ventral channel is not very distinct laterally from the rest of the VNO, but extends to the underside of the anterior part of the VNO dorsal dome at stage 26 of L.lugubris (Figure 17A and see the Video S4). This condition correspondences with the mushroom body, which is better developed than at previous stage. The ventral channel of the VNO is similarly developed in E.macularius at stage 35, but it seems to be slightly less distinct (Figure 17B and see the Video S5). The duct of the VNO is partially patent posteriorly in both corresponding stages (stage 26 of L.lugubris and stage 35 of E.macularius) (Videos S4 and S5). At stage 35 of L.lugubris, the ventral channel is well‐developed ventrolaterally and distinct from the rest of the VNO through its entire length. It is also slightly dilated (Figure 17C). The shape of the dorsal dome changes in L.lugubris: its posterodorsal wall becomes flattened at stage 28–30 (Figure S6a), and at stage 35 becomes slightly concave (red asterisks in Figure 17C). In E.macularius at stage 37 the morphology of the ventral channel resembles the condition of L.lugubris at stage 28–30, but it is slightly more distinct in E.macularius (compare Figure S6A and S6B). At stage 38 of E.macularius the ventral channel of the VNO is strongly developed, extended medially under the mushroom body and its lumen may be obliterated (Figure 17D), but usually narrow space is visible on the histological sections. The medial side of the VNO lumen extends ventrally and forms less distinct medial part of the ventral channel (see Figure 16). The duct of the VNO forms very distinct anterior extension which is relatively slim in E.macularius at stages 37 and 38 (Figure 16). In L.lugubris at stages 28–30 and 35, the anterior extension is not very distinct from the rest of the VNO duct, but in both species the anterior extension of the VNO duct does not extend beyond the level of the anterior margin of the VNO dorsal dome (Figure 16).

FIGURE 17.

The VNO at late developmental phase of the naso‐palatal complex in L.lugubris and E.macularius. (A, C, E) Transverse sections through the snout at the level of the VNO duct in L.lugubris at stages 26, 35 and 50–60. (B, D, F) Transverse sections through the snout at the level of the VNO duct in E.macularius at stages 35, 38 and 42. (G, H) Transverse histological sections through the VNO in L.lugubris at stage 50–60 (G) and E.macularius at stage 42 (H). Note:red asterisk, concave externally or flattened wall of the VNO dorsal dome; green asterisk, anterior extension of the lateral nasal concha; short red arrows, columns of the vomeronasal sensory epithelium. Key:dv, VNO duct; ict, inner choanal tube; lng, lateral nasal gland; lta, lamina transversalis anterior; mb, mushroom body; mx, maxilla; ns, nasal septum; smx, septomaxilla; vc, vomerine cushion, vch, ventral channel; ves, vestibulum; vns, vomeronasal sensory epithelium; vom, vomer; Stt, Stammteil

At the adult‐like stages of both gekkotans (stage 50–60 of L.lugubris and stage 42 of E.macularius), the lateral portion of the ventral channel is highly developed. Its lumen increases in volume along with the entire lumen of the VNO (Figure 17E,F). The medial portion of the ventral channel becomes visible in adult‐like stage of L.lugubris and is similarly distinct as in E.macularius at stage 38 (Figure 16 and see the Video S4). In adult‐like stage of E.macularius the medial part of the ventral channel becomes better visible (Figure 16 and see the Video S5). The well‐developed ventral channel corresponds to the well‐developed mushroom body, especially in E.macularius (Figure 17F). The VNO duct in both species is fully patent. The massive anterior extension of the VNO duct at stage 50–60 of L.lugubris is more distinct than at stages 28–30 and 35 (Figure 16). The slim anterior extension of the VNO duct at stage 42 of E.macularius is even more distinct than at stage 38 (Figure 16). The concave part of the dorsal dome in L.lugubrisis well visible at stage 50–60 (red asterisks in Figure 17E and see the Video S4), while in E.macularius this part of the dorsal dome is only slightly flattened at stage 42 (red asterisks in Figure 17F and see the Video S5). The striking difference of the vomeronasal sensory epithelia between two adult‐like stages of studied gekkotans is visible on histological sections. In E.macularius the cells of the basalmost part of the vomeronasal sensory epithelium are organised into the short columns, which are not present in L.lugubris (compare Figure 17G and H).

The presumptive cartilage of the mushroom body is possibly marked by condensed mesenchymal cells in E.macularius at stage 35 (based only on mCT images) and in L.lugubris at stage 26 (Figure 17A,B). The elements of the cupola Jacobsoni (the vomer, septomaxilla and maxilla) are first visible in L.lugubris at stage 28–30 (Figure S6A, C) and in E.macularius at stage 35 (Figure 17B). The cartilage of the mushroom body can be first observed in L.lugubris at stage 28–30 and at stage 37 of E.macularius (Figure S6C, D).

3.4. Analysis of developmental sequences

Two gekkotans, L.lugubris and E.macularius, and one iguanian, A.sagrei, were considered in the analysis of developmental sequences of selected events of the naso‐palatal complex differentiation (Figure 18). In all studied species, the early and late developmental phases represent the most invariant parts of developmental sequence. In all studied species the formation of the nasal pit (event 1) precedes all other events. Some changes, however, can be noted. The late phase of development exhibits greater variation in developmental sequence than the early one. The nasal plug elimination (event 23) and patent lacrimal duct (event 24) are visible at the same time and represent the last events in all analysed species (Figure 18).

FIGURE 18.

Sequences of selected developmental events of the naso‐palatal complex for A.sagrei (Iguania) and two gekkotans, L.lugubris and E.macularius. One developmental event is not present in E.macularius (event 6). Cells with more than one event show events that occur simultaneously (order of events is then arbitrary). Smaller numbers indicate developmental stages of normal developmental tables. The arbitrarily defined phases of development indicated

The middle developmental phase is characterized by the greatest variation in developmental sequence among studied species. Many events occur simultaneously (Figure 18).

4. DISCUSSION

The present study shows the embryonic development of the naso‐palatal complex of two gekkotans, L.lugubris (Gekkonidae) and E.macularius (Eublepharidae) in unprecedented 3D detail. As far as we know it is the first such analysis for these species and the first such description for representatives of Gekkota utilizing both X‐ray microtomography and light microscopy. This study discusses the most significant findings in a broad comparative framework, with special reference to the other squamates and representative of its sister group, Sphenodon punctatus.

4.1. Nasal pit, early nasal cavity and initial fusion of the facial prominences

The nasal pits were present in both species, L.lugubris and E.macularius at the earliest analysed stages (Figure 2). In all Tetrapoda, they develop from the nasal placodes, and each is bounded by the lateral and medial nasal prominences (Fuchs, 1908; Parsons, 1959a, 1967; Rudin, 1974). In L.lugubris at the end of the late developmental phase the epithelium of the nasal pit extends anterodorsally in relation to the primitive naris. This condition is similar to that described in A.sagrei (Kaczmarek et al., 2020). In E.macularius the epithelium of the nasal pit forms two apexes: anterior and dorsal. The anterior extension of the nasal pit was sometimes called the apikaler Blindsack and in some squamates it may be expanded well anterodorsally (Rudin, 1974).

The condition of early nasal cavity has been described in A.sagrei in the middle phase of the naso‐palatal complex differentiation (Kaczmarek et al., 2020). The early nasal cavity is characterized by narrowing of the anterior part of the primitive naris, located between the nasal prominences, to a slit like structure. This condition may occur in L.lugubris at stage 14_II (Figure 5F). The narrowed part of the primitive naris marks the presumptive external naris (anteriorly) and the area of future fusion between nasal prominences (posteriorly). In A.sagrei the early nasal cavity and initial fusion of the facial prominences are not temporally overlapping events (Figure 18). In contrast, the initial fusion of the facial prominences in L.lugubris may start abruptly after or even before the end of early nasal cavity formation (see stage 14_II in Table 2). In E.macularius such early nasal cavity condition was not found, even though embryos at stage 30/31 and at stage 31 (see Table 2) were available in the studied material. This may suggest that the formation of the early nasal cavity in E.macularius is a quick process and that the fusion of the nasal prominences may occur at the end of early nasal cavity formation, as in L.lugubris.

Initial fusion of the facial prominences is involved in the division of the primitive naris into the external naris and choana (called here the primitive outer choana) (Parsons, 1959a), formation of the vestibulum (Pratt, 1948), and is crucial for development of the intact upper lip and formation of the primary palate (Abramyan et al., 2015; Abramyan & Richman, 2018). Geometric morphometric analyses have shown that the shape of amniote facial prominences is the most conserved during this period (Young et al., 2014). However, qualitative observations show that some variation may occur (see below).

In both studied species the initial fusion between the facial prominences takes place between the lateral nasal prominence and medial nasal prominence of the frontonasal mass. In A.sagrei the formation of the primary palate also involves the maxillary prominence (Kaczmarek et al., 2020). Eventually, also in studied gekkotans the maxillary prominence joins to the nasal prominences to form the upper lip with the frontonasal mass. Interestingly, in the stage of early nasal cavity in A.sagrei the maxillary prominence closely approaches the frontonasal mass, just before the fusion of the facial prominences. At this time, approaching maxillary prominence of A.sagrei is characterised by the maxillary fold, which is not present in the studied gekkotans even during the initial fusion of the facial (medial and lateral nasal) prominences (Figure 18). Thus the maxillary prominence of A.sagrei can be considered as relatively more developmentally advanced during initial fusion of the facial prominences and even just before this event. This might imply that the initial fusion of the facial prominences takes place relatively earlier in gekkotans, or that the fusion of these prominences in A.sagrei is delayed due to ‘waiting’ for maxillary prominence. Interpretation of such heterochrony requires knowledge about the pattern of the facial prominences fusion in other squamates, but might be problematic because of the mentioned discordance between molecular and morphological phylogeny (see Koch & Gauthier, 2018).

Studies using optical projection tomography (OPT) showed that the fusion between facial prominences initiating the formation of the primary palate varies across amniotes (Abramyan et al., 2015; Abramyan & Richman, 2015). In the turtle Emydura subglobosa the nasal prominences have been observed to fuse first, similar to the studied gekkotans. In mice and the Nile crocodile (Crocodilus niloticus) ‘the majority of the initial fusion’ takes place between these two prominences (Abramyan et al., 2015). In contrast, the initial fusion of facial prominences in chick (Gallus gallus) engages the maxillary prominence and the frontonasal mass. A study based on OPT in three other species of lizard (Chamaeleo calyptratus, Pogona vitticeps and Aspidoscelis uniparens) (Abramyan et al., 2015) suggested the same pattern of initial fusion as we found in studied gekkotans. However, it seems that they did not distinguished the ‘classical’ fusion from the nasal plug formation between lateral and medial nasal prominences, which in fact takes place anterior to this fusion (this study and see e.g. Pratt, 1948). Thus we are unable to evaluate this condition in the mentioned taxa. The nasal plug may develop from the epithelial seam of the approaching nasal prominences (Albawaneh et al., 2020) and the presence of such closure is common in the embryonic vestibulum of amniotes (Parsons, 1959b; Kumoi et al., 1993; Buchtová et al., 2007; Abramyan et al., 2015; Abramyan & Richman, 2015; Albawaneh et al., 2020). However, the formation of the nasal plug at least in some squamates starts before the actual fusion of the facial prominences (this study; Kaczmarek et al., 2020). Parsons (1959) noticed that in turtles the external naris becomes closed some time after its formation, when the vestibulum is relatively well developed. Moreover, it seems that newly formed external naris of squamates is comparatively narrower than in other amniotes (mammal, turtles, birds and crocodilians) (see Figure 7A in this study and see figures 1 and 2 in Abramyan et al., 2015), which probably facilitate its closure by the nasal plug.

In contrast to the external naris, the choana of most nonavian reptiles remains open throughout development (Abramyan & Richman, 2015). In mammals, and probably in crocodilians (Abramyan et al., 2015), the choanae are closed by the bucconasal membrane, which undergoes rupture in later development (Parsons, 1959a).

4.2. Vestibulum and ‘external nasal’ glands

The morphology of the vestibulum exhibits great variation in adult squamates (Stebbins, 1948). For example, in Gekkota this part of the nasal cavity is usually moderately long, but it may be elongated as in Stendactylus and Tarentola (Pratt, 1948; Gabe & Saint Girons, 1976; Lemire, 1985). In L.lugubris the characteristic medial protuberance of the vestibulum can be found in the oldest analysed stage. Such a peculiar feature seems to be unknown or overlooked in other Gekkota.

The vestibulum of adult squamates is putatively responsible for removal of dust from the respiratory tract (Pratt, 1948). This function is improved by the epithelium hyperplasia of vestibular wall, spongy sinusoidal tissue, which surrounds the vestibulum as ‘sleeve’, and the lateral nasal gland producing mucoid secretion (see Pratt, 1948; Stebbins, 1948). Moreover, in some squamates the lateral nasal gland may act as ‘salt gland’ and be responsible for osmoregulatory function (Saint Girons & Bradshaw, 1987). The homology of the external nasal glands of living sauropsids is well established, but similar glands are present in amphibians and mammals (Witmer, 1995). The morphology of sauropsidian external nasal gland exhibits some variation. In Sphenodon it is weakly developed (Pratt, 1948; Gabe & Saint Girons, 1976). In turtles, the external nasal glands are generally well developed and in most cases forms ‘single middorsal plate’ (Parsons, 1959b, 1970; Parsons & Stephens, 1968). In this group, it may enter the vestibulum (Parsons, 1959b) or main nasal cavity (Parsons & Stephens, 1968; Parsons, 1971). In extant archosaurs it enters the posterior part of the vestibulum (Parsons, 1959b), and in crocodilians it takes a dorsal position to the nasal capsule, while in birds its location may vary and even be moved to the supraorbital region (Witmer, 1995). In squamates the lateral nasal gland usually enters the posterior part of the vestibulum and is located lateral to the main nasal cavity at least partially taking place in the tissue of the concha (conchal space) (Pratt, 1948; Parsons, 1970). The lateral nasal gland is present in both studied gekkotan species. The anterior part of lateral nasal gland, located ventrolateral to the entrance of the main duct to the vestibulum, can be distinguished in both species at late developmental stages, but it is much better developed in E.macularius. However, the most striking difference between L.lugubris and E.macularius in the morphology of the nasal cavity is the presence of an additional nasal gland, which we described in L.lugubris as a medial nasal gland. As far as we know, such a well‐developed medial nasal gland has never been described in any other gekkotan or any other squamates (Malan, 1945; Pratt, 1948; Stebbins, 1948; Bellairs & Boyd, 1950; Gabe & Saint Girons, 1976; Lemire, 1985; Witmer, 1995). The only indication of such a structure was found in embryo of Varanus bengalensis as a small ventral outgrowth of the vestibulum and it was believed to be a vestigial structure (Shrivastava, 1963). The presence of such ‘ventral blind pocket’ of the vestibulum has been noted in adult Varanidae by Malan (1945), but not by Bellairs (1949) and Lemire (1985). The homology of the medial nasal gland of L.lugubris and the ventral vestibular pocket of varanid lizard is unclear. A small ventral or medial nasal gland in a form of unbranched tube is present in Sphenodon (Hoppe, 1934; Parsons, 1970; Gabe & Saint Girons, 1976), but is considered to be vestigial (Pratt, 1948). It seems to be homologous to the medial nasal gland of turtles, which may be highly developed in some species (Parsons, 1959b, 1970). The medial nasal gland is absent in crocodilians and birds (Parsons, 1959b, 1970; Witmer, 1995). The medial nasal gland of L.lugubris seems to be relatively better developed than in Sphenodon, and its presence has not been noted in any other gekkotan. Thus is reasonable to consider the presence of the medial nasal gland in L.lugubris as an autapomorphy. Alternatively it could be considered as a reversal (homoplasy of Sphenodon condition). Further detailed comparative study of gekkotans and detailed histological examination will be required to explain the nature of this feature.

4.3. Frontonasal mass, vomerine cushion and anterior segment of the palate

The frontonasal mass forms due to merging of the medial nasal prominences in both studied species. The posterior extensions of the frontonasal mass form paired primordia of the anterior segment of the palate, which was described also in A.sagrei (Kaczmarek et al., 2020). The anterior segment of the palate becomes obliterated relatively quickly in E.macularius and becomes indistinguishable from the vomerine cushion. In L.lugubris the anterior segment of the palate is retained in adult‐like stage and the border between this structure and vomerine cushion is sharp. However, it is difficult to determine whether the adult‐like anterior segment of the palate corresponds entirely to the embryonic structure or only to its anterior part, since the adult‐like structure is located anterior to the VNO duct. This is not the case in earlier embryos in which the anterior segment of the palate borders the VNO duct medially (compare Figures 6d and 15a). The presence of the anterior segment of the palate in other adult squamates is unclear. However, it is likely that the remnants of this structure represent the premaxillary papillae (see Fuchs, 1908).

4.4. Choanal groove and the nasopharyngeal duct

The mammalian and crocodilian secondary palate is formed by a median fusion of the medial outgrowths of the maxillary prominences (Ferguson, 1981, 1988; Bush & Jiang, 2012), which is associated with the evolution of the well‐formed nasopharyngeal duct connecting the nasal and oral cavities (Parsons, 1967, 1970; Witmer, 1995). The medial soft tissue outgrowths of the maxillary prominences are usually termed as the palatal shelves (Richman et al., 2006; Buchtová et al., 2007). However, the homology of the mammalian palatal shelves and squamate choanal folds needs to be confirmed (see Fuchs, 1908; Haller, 1921; Kaczmarek et al., 2020). In squamates, the median fusion of the choanal folds does not occur (Bellairs and Boyd, 1950), but the connection of the nasal and oral cavities may be restricted by the formation of the choanal groove (Fuchs, 1908) and various kinds of the nasopharyngeal duct (Malan, 1945; Parsons, 1970).

The formation of the choanal groove seems to be a squamate synapomorphy, since this structure is not present in adult Sphenodon (Hallermann, 1998). In contrast, Bellairs and Boyd (1950) reported that this structure is present in this taxon, but it is very short. Nevertheless, the choana (the primitive outer choana) in Sphenodon extends almost along the entire length of the main nasal cavity (Fuchs, 1908; Hoppe, 1934; Parsons, 1959b; Gabe & Saint Girons, 1976). In most iguanians, the choanal groove is short and long choana resembles the condition of Sphenodon (Malan, 1945; Hallermann, 1994). In the two studied gekkotans the choanal groove retains the connection with the VNO duct at the last analysed stages. The same condition is characteristic for adults representatives of Iguania and Gekkota (Bellairs and Boyd, 1950; Parsons, 1970). In contrast, in Autarchoglossa the choanal groove is reduced anteriorly and may be even absent, like in snakes. Thus, in some cases the fusion separating the nasal cavity from oral cavity may involve two levels: the (primitive) inner and (primitive) outer choana (Malan, 1945; Bellairs & Boyd, 1950). The autarchoglossan condition is believed to be a result of secondary obliteration of the entire or anterior part of the choanal groove (Hallermann, 1998).

The choanal groove has been found in snake embryos (Bellairs & Boyd, 1950; Kaczmarek et al., 2017), with the iguanian or gekkotan‐like condition recapitulated in this group. Thus it is reasonable to think that also in other nonophidian autarchoglossans, a fully formed choanal groove extending from the VNO duct to the outer choana is present at some point in their embryonic development. The choanal groove in the studied gekkotans and A.sagrei is formed by the fusion of the subconchal fold (originating from the lateral nasal prominence) and the vomerine cushion (this study; Kaczmarek et al., 2020). This event causes the shortening of the primitive inner choana. In consequence, the anterior part of the primitive outer choanal tube becomes separated from the main nasal cavity (this study; Kaczmarek et al., 2020). The separated part of the primitive outer choanal tube, located just posterior to the VNO duct, is the choanal groove, which may provide only indirect connection between the VNO and the nasal cavity (Pratt, 1948; Bellairs & Boyd, 1950).

Other interpretations suggest that the choanal fold, instead of the lateral nasal prominence, is involved in the fusion with the vomerine cushion during formation of the choanal groove (Bellairs & Boyd, 1950; Parsons, 1970; Hallermann, 1998). In such a scenario, the fusion engages the maxillary prominence (choanal fold). In fact, the same explanation for the grass snake (Natrix natrix) was presented in our previous study (Kaczmarek et al., 2017). Indeed, the presence of the subconchal fold in the grass snake seems difficult to evaluate (see Beecker, 1903; Kaczmarek et al., 2017). Moreover, the formation of the nasopharyngeal duct (Parsons, 1959b) and highly developed dorsal dome of the VNO (Bellairs, 1949) may potentially affect the formation of the ophidian choanal groove. Thus, further investigations on autarchoglossans are required to explain the nature of choanal groove formation and reduction in these squamates. Nevertheless, the obliteration of the choanal groove with no doubts, at least in the grass snake, involves the maxillary prominence (possibly the choanal fold) which merges with the vomerine cushion.

As the choanal groove, the outer choanal tube in squamates derives from the primitive outer choanal tube (Kaczmarek et al., 2020; this study). The outer choanal tube of adult squamates is variously referred as the choanal tube (Bellairs & Boyd, 1950), part of the choanal tube (Parsons, 1970), part of the Choanengang (Parsons, 1959b) or ‘descending limb of the choanal tube’ (absteigender Schenkel des Choanenganges; Fuchs, 1908). The well‐formed ophidian nasopharyngeal duct seems to be homologous to the squamate outer choanal tube, which elongates due to ‘active proliferation’ (Bellairs & Boyd, 1950) and/or becomes separated ventrally from the oral cavity by the fusion of the choanal fold and the vomerine cushion (see Fuchs, 1908; Bellairs & Boyd, 1950; Parsons, 1959b, 1970). In nonophidian squamates, for example, skinks, chameleons, Dibamus, amphisbaenids and Xantusia, various forms of open or closed nasopharyngeal duct may exist (Parsons, 1970; Hallermann, 1998). This duct is homologous to the outer choanal tube or orbitonasal trough which is more or less separated from the oral cavity by the medial extension of choanal folds or elongation of the vomerine cushion (Bellairs & Boyd, 1950; Parsons, 1970). In the studied gekkotans the nasopharyngeal duct is absent and the lack of this structure is typical squamate condition (Parsons, 1967). However, the presence of the nasopharyngeal duct in Gekkota has been reported for Ptenopus garrulus (Gekkonidae) (Bellairs, 1948; Bellairs & Boyd, 1950) and some Pygopodidae: in Lialis and Aprasia, but not in Delma (Underwood, 1957).

4.5. Lacrimal duct

The nasolacrimal groove, located between the lateral nasal prominence and the maxillary prominence, is not very distinct in L.lugubris and E.macularius. The major part of the primordial lacrimal duct seems to be located posterior to this groove, similar to A.sagrei (Kaczmarek et al., 2020). This is not a typical condition for many other tetrapods, in which the lacrimal duct originates from this groove (Bellairs & Boyd, 1950; Ferguson, 1985; Witmer, 1995; de la Cuadra‐Blanco et al. 2006). The differences of the lacrimal duct place origin may reflect the differences in the length of the forming snout and size of the eyes. In later development, the lacrimal duct sinks down below the surface of the snout, and its anterior tip approaches the primordial ventral channel of the VNO. A temporary contact between the walls of these two structures can be found in E.macularius (Figure 10E,E’). This contact eventually becomes lost, and the lacrimal duct starts to form a definitive connection with the choanal groove. Finally the anterior tip of the lacrimal duct reaches the medial wall of the VNO duct. This condition constitutes a squamate synapomorphy (Hallermann, 1998). However, the anteriormost part of the lacrimal duct of anguids is terminated just posteromedial to the VNO duct and opens on the superficial palate (Bellairs & Boyd, 1950; Parsons, 1970).

The association of the lacrimal duct and the choanal groove is a characteristic feature of most nonophidian squamates (e.g. Malan, 1945; Lemire, 1985). The gradual increase of the range of the connection between these structures is a common squamate embryological process (this study; Kaczmarek et al., 2020). In chamaeleonids (Iguania), skinks and many lacertids the connection between both structures is extensive, and they form the structure sometimes called the lachrymo‐choanal gutter, since the distinction there between the lacrimal duct and choanal groove is difficult to evaluate (Bellairs & Boyd, 1950). The lacrimal duct and the choanal groove are probably extensively connected in many iguanians, but the border between these two components seems to be relatively easy to distinguish (see Malan, 1945; Bellairs & Boyd, 1950; Lemire, 1985; Kaczmarek et al., 2020). It has been found that in gekkotans the connection of these structures does not exceed the anterior half of the choanal groove (Gabe & Saint Girons, 1976) and in some pygopodids such a connection may even be absent (see below). The condition of restricted connection of lacrimal duct and the choanal groove, which has been retained in the last analysed stage of L.lugubris seems to confirm the previous description for gekkotans. However, in stage 42 of E.macularius the connection between the lacrimal duct and the choanal groove is very extensive. As far as we know such an extensive connection has never been reported for Gekkota, even in studies including representatives of Eublepharidae.

Considering the restricted connection of the lacrimal duct and the choanal groove of most Gekkota as a squamate plesiomorphy, the extensive connection of these structures might support the molecular clade Unidentata (sister to Gekkota or Gekkota +Dibamia; see Zheng & Wiens, 2016; Burbrink et al., 2020) and represent a peramorphic character. Unfortunately, interpretation of this character is problematic. The choanal groove in the sister group of squamates, represented by Sphenodon, is absent or extremely short (Bellairs and Boyd, 1950). In Sphenodon the lacrimal duct enters the lateral wall of the choanal tube in the anterior part of the nasal cavity, just opposite (or slightly posterior) to the entrance of the VNO (Broom, 1906; Hoppe, 1934; Parsons, 1959a). Nevertheless, the restricted connection of the nasal cavity and the lacrimal duct (see Figure 13f in Bellairs & Boyd, 1950), corresponds to the restricted connection of the lacrimal duct and the choanal groove in gekkotans (but not in E.macularius).

A further difficulty with interpreting the range of lacrimal duct–choanal groove connection might be the length of the choanal groove. Despite the extensive connection of the choanal groove and lacrimal duct in E.macularius, the choanal groove is approximately half the length of the outer choana. In the adult‐like stage of L.lugubris the connection between the lacrimal duct and choanal groove seems to be not so extensive, but the entire length of the latter is the same as the outer choana. The relative length of the outer choana, and presumably the range of the subconchal fold‐vomerine cushion fusion, may vary across squamates (see Lemire, 1985). The secondary reduction of the anterior part of the choanal groove in autarchoglossans (Bellairs & Boyd, 1950) and the length of the snout (see Metzger & Herrel, 2005; Stayton, 2005) should also be considered. On the other hand, some evidence suggests that the length of the choanal groove does not affect the range of its connection with the lacrimal duct, and this in turn may suggest strong functional significance. For example in adult‐like stage of L.lugubris the choanal groove is long, while its connection is relatively short, but the choanal groove in A.sagrei is also long and its connection with the lacrimal duct is extensive. Moreover, at least in one pygopodid, Lialis, the choanal groove is well formed and confluent with the VNO duct, but the lacrimal duct is connected exclusively to the VNO duct (Bellairs & Boyd, 1950).

In contrast to most nonophidian squamates, in which the orbital end of the lacrimal duct is composed of two lacrimal canaliculi opened into the conjunctival or sub‐brillar space, the lower lacrimal canaliculus of pygopodids is closely associated with the duct of the Harderian gland (Lialis and Pygopus), or the Harderian gland discharges directly to the lacrimal duct (Aprasia) (Bellairs & Boyd, 1947; Underwood, 1957). Similar, but not identical modifications of the posterior end of the lacrimal duct and/or close association of its anterior end with the VNO duct occur in other squamates (monitor lizards, Xantusia, snakes and other snake‐like squamates such as amphisbaenids and at least one dimabid) in which the choanal groove is usually reduced or absent (Malan, 1945; Bellairs & Boyd, 1947, 1950; Bellairs, 1949; Parsons, 1970; Hallermann, 1998). Such anatomical specializations of the lacrimal duct allow for direct flow of Harderian gland secretion from the orbital region to the VNO duct. This may represent an adaptation to a burrowing lifestyle, nocturnality or desert habitat (Bellairs & Boyd, 1947). A similar effect probably could be achieved in Anolis characterised by closed choanal groove (Kaczmarek et al., 2020).

It is widely accepted that the lacrimal duct of squamates and other tetrapods is involved in vomeronasal chemoreception, by delivering the secretion of the Harderian gland into the VNO directly or to its vicinity (Rehorek, 1997; Rehorek et al., 2000; Rossie & Smith, 2007). This is not true for turtles and some plethodontid salamanders in which the lacrimal duct is absent (Bellairs and Boyd, 1947; Siegel et al., 2018) and for crocodilians and birds, in which the VNO is lost (Parsons, 1959a). It has been confirmed that in snakes (Thamnophis) the female pheromones are soluble in the homogenate of the Harderian gland (Huang et al., 2006). The functional significance of the extensive connection between the lacrimal duct and open choanal groove seems to be unclear. Pratt (1948) suggested that in squamates with a well‐developed choanal groove (iguanians, gekkotans, but possibly also in lacertids and skinks) ‘a continuous stream of the lachrymal fluid’ inside the choanal groove, caused by ciliary currents, may deliver odours to the VNO duct (see also Hallermann, 1994). Filoramo & Schwenk (2009) proposed that the chemical‐bearing fluids on the tongue tips are delivered to the VNO fenestrae hydraulically. Whether or not this hypothesis is true, ciliary movements along the choanal groove might be too slow for the dynamics of tongue flicking. In fact, the sequential tongue flicks (which presumably deliver chemicals to the VNO with each flick) are often occur within a fraction of a second (e.g. Gove and Burghardt, 1983; Herrel et al., 1998). Moreover, in teiids the choanal groove ends at significant distance behind the VNO duct, but the lacrimal duct opens into the lateral choanal fissure posteriorly (and to the VNO duct anteriorly) (Bellairs & Boyd, 1950).

As was shown above the interpretation of lacrimal duct–choanal grove connection in the phylogenetic and functional context may be problematic. Thus, future comparative and physiological studies in different representatives of adult squamates appear desirable.

4.6. Vomeronasal Organ

The vomeronasal organ (VNO) of tetrapods develops from a medial outpocketing of the nasal pit (Fuchs, 1908; Parsons, 1959a,1959b; Rudin, 1974). Due to the further growth, the vomeronasal pit of studied gekkotans attains early VNO form. The separation of the VNO from the nasal cavity is preceded by the formation of the mushroom body and then the VNO duct, which appears at the same time as the primordial ventral channel (Table 2). The indirect connection with the nasal cavity, through the choanal groove, is retained in adult Gekkota and Iguania (e.g. Malan, 1945; Bellairs & Boyd, 1950; Lemire, 1985). The direct connection between the VNO and the nasal cavity is retained in adult mammals (Bertmar, 1981; Sánchez‐Villagra, 2001; Takami, 2002) and Sphenodon (Broom, 1906; Hoppe, 1934), while the vomeronasal sensory epithelium covers a certain part of the nasal cavity in amphibians and turtles (Takami, 2002; Dawley, 2017; Quinzio & Reiss, 2018). The VNO is reduced or absent in adult chameleons (Haas, 1947; Pratt, 1948) and some mammals (Smith & Bhatnagar, 2009; Yohe et al., 2018). It is absent in extant archosaurs, but the vomeronasal pit appears during embryonic development in birds and crocodilians (Parsons, 1959a).

The adult‐like VNO in L.lugubris and E.macularius is characterized by a well‐defined mushroom body which corresponds with a well‐developed ventral channel (Figure 17E,F). Both structures are believed to be functionally important. The mushroom body is considered to be involved in the suction mechanism transferring the chemical molecules, dissolved in ‘lacrimal fluid’, from the palatal opening of the VNO (vomeronasal fenestra) into the lumen of the organ (Young, 1993; Filoramo & Schwenk, 2009). Pratt (1948) suggested that the ventral (spiral) channel drains the fluid from the lumen of VNO to the VNO duct, from which the fluid runs back to the oral cavity. Moreover, in some squamates this part of the VNO has a secretory function due to the presence of the goblet cells (Gabe and Saint Girons, 1976; Takami, 2002). A well‐developed dorsal dome of the VNO was found in both gekkotans. In L.lugubris the posterodorsal wall is concave rather than just flattened as in E.macularius. This variation may reflect the difference in the pressure which is exerted on the posterodorsal wall of the VNO by the anterior part of the growing main nasal cavity.

Nevertheless, the most significant difference in morphology of the dorsal dome of the VNO between studied gekkotans is a presence of epithelial columns in stage 42 of E.macularius in the basal part of the vomeronasal sensory epithelium (Figure 17h). Such peculiar columns of the vomeronasal sensory epithelium interspersed by connective tissue and blood vessels are characteristic for snakes. However, in most snakes the undulation of the basal lamina is significant and columns extend along almost the entire height of the dorsal dome, leaving ‘outside’ only the thin apical layer of supporting cells (Wang & Halpern, 1980a,1980b; Takami & Hirosawa, 1990), although in some taxa the height of the columns may be relatively shorter. In some ‘scolecophidians’ (Indotyphlops braminus, Rena dulcis) the columns extend along the basal third of the vomeronasal sensory epithelium (Gabe & Saint Girons, 1976), similar to the late stage of E.macularius. In henophidian snakes Gabe and Saint Girons (1976) described the columns extending along the basal half of the epithelium (Casarea dussumieri and Cylindrophis ruffus), the basal two thirds (Lichanura trivirgata and Xenopeltis unicolor), and the basal four fifths (in Morelia spilota). It is likely that conditions in which the columns does not extend along the most of the height of the vomeronasal sensory epithelium represent various plesiomorphic conditions of snakes. Such conditions may be recapitulated during embryonic development in caenophidian snakes. In fact, the gradual increase in height of the columns relative to the entire height of the vomeronasal sensory epithelium has been observed in embryos of Natrix natrix (Kaczmarek et al., 2017) and Thamnophis (Parsons, 1959b; Holtzman & Halpern, 1990). The ‘snake‐like’ columns in the basal part of the vomeronasal sensory described in E.macularius may represent the first report of such regular structures for nonophidian squamates.

In lizards the presence of columnar compartments in the vomeronasal sensory epithelium is unclear. It is likely that significant undulation of the basal lamina and intrusion of the connective tissue towards the apical part of the vomeronasal sensory epithelium is not present in Iguania (Gabe and Saint Girons, 1976; Sapoznikov et al., 2016; Kaczmarek et al., 2020). Neither connective tissue nor blood vessels were found in this study in adult‐like stage of L.lugubris at the level of the vomeronasal sensory epithelium. In Tiliqua (Scincidae) ‘the intrusion of columns of connective tissue that carry blood vessels almost to the epithelial surface’ was noted (Kratzing, 1975). Unfortunately, based on the images provided in that study, we are unable to evaluate if such intrusion of connective tissue forms ‘snake‐like’ regular columns. The other descriptions for the VNO dorsal dome of gekkotans and nonophidian autarchoglossans only indicate the presence of capillaries (Gabe & Saint Girons, 1976; Saito et al., 2010). Considering reports mentioned above, it is possible that in lizards the penetration of the vomeronasal sensory epithelium by connective tissue, without column formation, may be comparable to the condition in some mammals and Sphenodon (Gabe and Saint Girons, 1976; Takami, 2002). The presence of the columns of the vomeronasal sensory epithelium in Gekkota and in snakes along with the absence of these structures in other nonophidian autarchoglossans suggests that this trait evolved independently in snakes and in E.macularius or some gekkotan clade (Eublepharidae?).

4.7. Nasal conchae

The nasal conchae of amniotes has had different definitions, but in this article they were classified as any projection into the main nasal cavity (Parsons, 1970). The lateral nasal concha develops from the lateral nasal prominence (Kaczmarek et al., 2020) and it is strongly developed in adult‐like stages of both analysed gekkotans. Thus all basic components of the conchal zone of the main nasal cavity are well distinguishable: the extraconchal space, Stammteil and inner choanal tube. Posterior to the lateral nasal concha, all these components form the antorbital space which is well developed at adult‐like stages of both studied species. The location of the extraconchal space corresponds to the region of the well‐developed lateral nasal concha. Anteriorly, where the extraconchal space is absent, the lateral nasal concha passes into its anterior extension. The spatial extent of extraconchal space differs in studied gekkotans at late stages. In L.lugubris it emerges from the posterior two thirds of the main nasal cavity (Figure 11G,H) whereas in E.macularius the extraconchal space emerges from the posterior half of the main nasal cavity (Figure 12G,H). It can be stated that in L.lugubris the embryonic condition is retained in adult‐like stage, while in E.macularius the range of the extraconchal space is reduced (see Table 2). In contrast, the distribution of the sensory olfactory epithelium seems to be reduced in L.lugubris, while the extensive distribution of this epithelium is retained from earlier stages in E.macularius (see Table 2).

In some iguanians the lateral concha may be absent, but this is apparently the result of secondary loss (Stebbins, 1948; Kaczmarek et al., 2020). In contrast to the single concha of Squamata, Sphenodon is characterized by two nasal conchae, anterior and posterior (Hoppe, 1934; Parsons, 1959b). The most comprehensive description of the nasal cavity in Sphenodon was provided by Hoppe (1934). The part homologous to the extraconchal space called the dorso‐lateraler Muschelraum (dorsolateral conchal space) is present at the level of the posterior concha. The extension of the choanal tube (=Choanengang) beneath the anterior concha is called the dorsal pocket of the choanal tube (dorsale Tasche des Choanenganges) (upper row in figure 19 based on Parsons, 1959a), while the part of the nasal cavity under the posterior concha is called the ventraler Muschelraum (ventral conchal space) and is sometimes considered as a part of the choanal tube (Fuchs, 1908; Parsons, 1959a, 1970).

FIGURE 19.

Proposed homology of the anterior concha of Sphenodon and squamate structures. Upper half of the figure shows the sets of transverse sections through the nasal cavity of ‘late’ Sphenodon embryo based on Figure 36 in Parsons, 1959 (credit: Museum of Comparative Zoology and Harvard University): the first row is based on original description, while the second row shows our interpretation. Lower half of the figure shows the sets of transverse sections through the nasal cavity of E.macularius (this study) at stage 34 (third row) and 42 (fourth row). Silhouette of Sphenodon from http://phylopic.org/. Key:a, anterior; ac, anterior concha; chg, choanal groove; ct, choanal tube (sensu lato); dcs, dorsolateral conchal space; dpct, dorsal pocket of the choanal tube; dv, VNO duct; ecs, extraconchal space; ict, inner choanal tube; ld, lacrimal duct; lnc, lateral nasal concha; lng, lateral nasal gland; mnc, main nasal cavity; och, outer choana; oct, outer choanal tube; p, posterior; pc, posterior concha; pict, primitive inner choanal tube; poch, primitive outer choana; poct, primitive outer choana tube; Stt, Stammteil; VNO, vomeronasal organ

The homology between the lateral nasal concha of squamates and the posterior concha of Sphenodon is widely accepted (Parsons, 1959b; Witmer, 1995). However, the homology of the anterior concha of Sphenodon still remains unclear. Hoppe (1934) proposed that it is homologous to the ‘Lippe am Choanengang’, described by Beecker (1903). The latter structure is clearly the subconchal fold described in this study, and in adult‐like stages it is located at the level of (primitive) inner choana, between the (primitive) inner choanal tube and (primitive) outer choanal tube (more specifically the choanal diverticulum or lateral choanal fissure). Hoppe (1934) based his view about the homology of the anterior concha of Sphenodon and the squamate ‘Lippe am Choanengang’ (=subconchal fold) on the fact that the former structure is restricted ventrally by the dorsal pocket of the choanal tube, which he believed was homologous to the choanal diverticulum of squamates (=Winkeltasche) (Figure 19). Parsons (1959a) rejected the homology of the anterior concha of Sphenodon and the ‘Lippe am Choanengang’, stating that both structures are similar only in adult specimens and the orientation of anterior concha of Sphenodon changes from vertical to horizontal during embryonic development, while the ‘Lippe am Choanengang’ ‘forms in place as horizontal ridge’. Witmer (1995) agreed with this interpretation and considered anterior concha of Sphenodon as neomorphic. Moreover, even Hoppe (1934) indicated that the similarity between the anterior concha of adult Sphenodon and the ‘Lippe am Choanengang’ of adult squamates is clear only at the level of posterior part of the nasal cavity, and anteriorly the anterior concha of Sphenodon runs obliquely to reaches the level of the vestibulum. Indeed, considering the drawings of Parsons (1959a) showing the posterior transverse section of the ‘late’ Sphenodon embryo, the similarity between the anterior concha and squamate subconchal fold is striking (fourth column in Figure 19 and see the subconchal fold in Figure 6k in Kaczmarek et al., 2020). Thus we suppose that the homology of the squamate subconchal fold and the anterior concha of Sphenodon is true at least for the posterior part of the latter structure, whereas the anterior part of the anterior concha of Sphenodon seems to be homologues to the anterior extension of the lateral nasal concha described here for both gekkotans (first column in Figure 19). Moreover, the anterior concha and the subconchal fold seem to be visible on the single transverse section of Sphenodon, but (Parsons, 1959b) ignored that (second column for Sphenodon in Figure 19). Such explanation implies that both ‘parts’ of the anterior concha of Sphenodon, homologues of squamate subconchal fold and anterior extension of the lateral nasal concha, are confluent with each other (third column for Sphenodon in Figure 19). In squamates the subconchal fold becomes fuses with the vomerine cushion (this study) and the dissociation of the subconchal fold and the anterior extension of the lateral nasal concha structures seems to be result of the choanal groove formation, which is absent in Sphenodon (Hallermann, 1998).

The proposed homology of the anterior concha of Sphenodon and the squamate structures requires confirmation by embryological data of Sphenodon.

4.8. Developmental sequences

We found common developmental features for gekkotans and A.sagrei (possibly conservative or homoplastic traits depending on phylogenetic scenario), for only E.macularius and L.lugubris (which may constitute gekkotan synapomorphies), and for one gekkotan and A.sagrei (potential homoplasies) (Figure 18). Nevertheless, more taxa are necessary to test the phylogenetic signal in developmental sequence of naso‐palatal complex and to evaluate the impact of temporal event shifts on adult morphology.

The conservatism observed in the early developmental phase may correspond to a phylotypic stage, which occurs in the middle part of the development and is characterized by the greatest conservation (Kalinka & Tomancak, 2012). Indeed, unlike in other reptiles, the squamate phylotypic stage is associated with the time of oviposition (Andrews & Mathies, 2000; Andrews et al., 2013). The invariant sequences observed in the late developmental phase might be explained by the trade‐off between the need for preparation to live outside the egg and some developmental constraints. The vestibulum and lacrimal duct should be functional at hatching, thus elimination of cellular plugs (events 23 and 24 in Figure 18) seems to be crucial for hatchlings. On the other hand, the earlier elimination of plugs could affect previous development. The same may be true for Bowman's glands function (event 22 in Figure 18).

5. CONCLUSIONS

This study of naso‐palatal complex development in two gekkotans representing two different families provides a detailed anatomical description of the embryonic structures based on histology and 3D images. Developmental data confirmed two recent findings from our study on A.sagrei with respect to processes that seem to be conserved at least for most nonophidian squamates: formation of the choanal groove due to the fusion of the subconchal fold (originating from lateral nasal prominence) with the vomerine cushion, and emergence of the anterior segment of the palate. Some structures described may be important in a functional context, for example, the inner fold of the choanal groove and the anterior palatal wing. Our data and literature review allowed for redefinition of anterior concha of Sphenodon, which in fact seems to be the combination of two different structures: the anterior extension of the posterior concha (=anterior extension of the squamate lateral nasal concha) and subconchal fold.

We found an unexpected level of variation between adult‐like stages of the studied nonpygopodid gekkotans. Some of these features are problematic in the phylogenetic context, since they are uncommon for nonophidian squamates or squamates as a whole. The columns present in the basal part of the vomeronasal sensory epithelium were found in late stage of E.macularius. Such regular structures of the basal part of the vomeronasal sensory epithelium have been found in some ‘scolecophidians’ and in snake embryos of caenophidians, but probably never in nonophidian squamates. The extensive connection of the lacrimal duct and the choanal groove in E.macularius represents an unusual gekkotan condition, which, however, co‐occurs with the relatively short choanal groove. The presence of a well‐developed medial nasal gland, which was found in late stages of L.lugubris, probably has never been noted before for any other representative of squamates. Interpretation of this condition is difficult and will require future comparative studies, and the homology of this structure with the ventral vestibular pocket of varanid lizards is as yet uncertain.

Our analysis of developmental sequences of the naso‐palatal complex revealed possible heterochronies, which may be useful for future phylogenetic analysis and explaining the differences of squamate adult morphology.

AUTHOR CONTRIBUTIONS

PK conducted most of the laboratory work and segmented analysed structures (mCT). BM performed mCT scanning. PK, WR and BM analysed the data and wrote the paper. All authors read and approved the final manuscript.

Supporting information

Fig. S1.Early developmental phase of the naso‐palatal complex in L. lugubris at stage 10. Compare with Fig. 3. Note : the Stammteil (Stt) may be considered as primordium. Key: a, anterior; e, eye; lnp, lateral nasal prominence; mnp, medial nasal prominence; mxp, maxillary prominence;pct, primitive choanal tube; pn, primitive naris; Stt, Stammteil; tel, telencephalon; vp, vomeronasal pit.

Fig. S2.Transverse histological and tomographic sections through the nasal plug and the fusion of the nasal prominences in L. lugubrisat stage 14_II and E. maculariusat stage 31. Key: ecs, extraconchal space; en, external naris; ev, early VNO; fnm, frontonasal mass; lnp, lateral nasal prominence; pct, primitive choanal tube; Stt, Stammteil; ves, vestibulum.

Fig. S3.Early developmental phase of the naso‐palatal complex in E. macularius at stage 30/31. Compare with Fig. 4.Note : the Stammteil (Stt), extraconchal space (ecs) and lateral nasal concha (lnc) may be considered as primordial; short arrowsshowthe apexes of the nasal pit. Key: a, anterior; e, eye;ecs, extraconchal space; lnc, lateral nasal concha; lnp, lateral nasal prominence; mnp, medial nasal prominence;mxp, maxillary prominence; pct, primitive choanal tube; pn, primitive naris; Stt, Stammteil; tel, telencephalon; vp, vomeronasal pit.

Fig. S4.Late developmental phase of the naso‐palatal complex in L. lugubris at stages 28–30 (A–D) and 35 (E–H). (A) Stage 28–30, anterodorsal view of the snout. (B) Stage 28–30, transverse section and transverse cutaway through the snout at the level of the Aulax. (C) Stage 28–30, lateral view of the nasal cavity. (D) Stage 28–30, dorsal view of the nasal cavity. (E) Stage 35, anterodorsal view of the snout. (F) Stage 35, transverse section and transverse cutaway through the snout at the level of Aulax. (G) Stage 35, lateral view of the nasal cavity. (H) Stage 35, dorsal view of the nasal cavity. Note: yellow arrowhead, choanal fold. Distribution of the olfactory sensory epithelium may be approximated according to X‐ray opacity (see color bars). Key: a, anterior; aos, antorbital space; Aux, Aulax; Bg, Bowman’s glands; da, diverticulum of the antorbital space; dv, duct of the VNO; ecs, extraconchal space; en, external naris; ict, inner choanal tube; lcc, lacrimal canaliculi; lnc, lateral nasal concha; mxf, maxillary fold;ne, non‐sensory (respiratory) epithelium of the nasal cavity; oct, outer choanal tube; oe, sensory olfactory epithelium; Stt, Stammteil;ves, vestibulum.

Fig. S5.Late developmental phase of the naso‐palatal complex in E. macularius at stages 37 (A–D) and 38 (E–H). (A) Stage 37, anterodorsal view of the snout. (B) Stage 37, transverse section and transverse cutaway through the snout at the level of the Aulax. (C) Stage 37, lateral view of the nasal cavity. (D) Stage 37, dorsal view of the nasal cavity. (E) Stage 38, anterodorsal view of the snout. (F) Stage 38, transverse section and transverse cutaway through the snout at the level of the Aulax. (G) Stage 38, lateral view of the nasal cavity. (H) Stage 38, dorsal view of the nasal cavity. Note: green arrowhead, rostral recess of the extraconchal space; yellow arrowhead, choanal fold. Distribution of the olfactory sensory epithelium may be approximated according to X‐ray opacity (see color bars). Key: a, anterior; Aux, Aulax; Bg, Bowman’s glands; da, diverticulum of the antorbital space; dv, duct of the VNO; ecs, extraconchal space; en, external naris; ict, inner choanal tube; lcc, lacrimal canaliculi; lcf, lateral choanal fissure; lnc, lateral nasal concha; mxf, maxillary fold;ne, non‐sensory (respiratory) epithelium of the nasal cavity; och, outer choana;oct, outer choanal tube; oe, sensory olfactory epithelium; Stt, Stammteil;vc, vomerine cushion; ves, vestibulum.

Fig. S6.The VNO at late developmental phase of the naso‐palatal complex in L. lugubrisat stage 28–30 (A, C) and E. maculariusat stage 37 (B, D). (A, B) Transverse sections through the snout at the level of the VNO duct in L. lugubris at stages 28–30 (A), and E. maculariusat stage 37 (B). (C, D) Transverse histological sections through the cartilage of the mushroom body in L. lugubrisat stage 28–30 (C) and E. maculariusat stage 37 (D). Note: red asterisk, flattened wall of the VNO dorsal dome; green asterisk, anterior extension of the lateral nasal concha. Key: cmb, cartilage of the mushroom body; dv, duct of the VNO; lta, lamina transversalis anterior; mb, mushroom body; mx, maxilla; ns, nasal septum;smx, septomaxilla; Stt, Stammteil; vc, vomerine cushion;vch, ventral channel; vns, vomeronasal sensory epithelium; vom, vomer.

Video S1.The formation of the choanal groove based on E. macularius at stage 34_II. Note: white arrow, fusion of the subconchal fold and the vomerine cushion; red dashed line, inner choana; white dashed line, outer choana; yellow arrowhead, choanal fold. Keys as in Figures.

Video S2.Late developmental phase of the naso‐palatal complex in L. lugubriswith special references to the anterior extension of the lateral nasal concha (green asterisk) and the antorbital space. Note: yellow arrowhead shows the choanal fold. For detailed descriptions of sections at the level of the lateral nasal concha see Fig. 11B, F and Supporting Information Fig. S4B, F.Keys as in Figures.

Video S3.Late developmental phase of the naso‐palatal complex in E. maculariuswith special references to the anterior extension of the lateral nasal concha (green asterisk) and the antorbital space. Note: yellow arrowhead shows the choanal fold. For detailed descriptions of sections at the level of the lateral nasal concha see Fig. 12B, F and Supporting Information Fig. S5B, F. Keys as in Figures.

Video S4.Late developmental phase of the naso‐palatal complex: VNO‐choanal groove‐lacrimal duct in L. lugubrisat stages 26 and 50–60. For detailed descriptions of sections at the level of the VNO see Fig. 17A, E.

Video S5.Late developmental phase of the naso‐palatal complex: VNO‐choanal groove‐lacrimal duct in E. maculariusat stages 35 and 42. For detailed descriptions of sections at the level of the VNO see Fig. 17B, F. Keys as in Figures.

SuppInfo

Kaczmarek P, Metscher B, Rupik W Embryology of the naso‐palatal complex in Gekkota based on detailed 3D analysis in Lepidodactylus lugubris and Eublepharis macularius . J Anat.2021;238:249–287. 10.1111/joa.13312

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.