Development of the squamate naso-palatal complex: detailed 3D analysis of the vomeronasal organ and nasal cavity in the brown anole Anolis sagrei (Squamata: Iguania)

Background Despite the diverse morphology of the adult squamate naso-palatal complex – consisting of the nasal cavity, vomeronasal organ (VNO), choanal groove, lacrimal duct and superficial palate – little is known about the embryology of these structures. Moreover, there are no comprehensive studies concerning development of the nasal cavity and VNO in relation to the superficial palate. In this investigation, we used X-ray microtomography and histological sections to describe embryonic development of the naso-palatal complex of iguanian lizard, the brown anole (Anolis sagrei). The purpose of the study was to describe the mechanism of formation of adult morphology in this species, which combines the peculiar anole features with typical iguanian conditions. Considering the uncertain phylogenetic position of the Iguania within Squamata, embryological data and future comparative studies may shed new light on the evolution of this large squamate clade. Results Development of the naso-palatal complex was divided into three phases: early, middle and late. In the early developmental phase, the vomeronasal pit originates from medial outpocketing of the nasal pit, when the facial prominences are weakly developed. In the middle developmental phase, the following events can be noted: the formation of the frontonasal mass, separation of the vestibulum, appearance of the lacrimal duct, and formation of the choanal groove, which leads to separation of the VNO from the nasal cavity. In late development, the nasal cavity and the VNO attain their adult morphology. The lacrimal duct establishes an extensive connection with the choanal groove, which eventually becomes largely separated from the oral cavity. Conclusions Unlike in other tetrapods, the primordium of the lacrimal duct in the brown anole develops largely beyond the nasolacrimal groove. In contrast to previous studies on squamates, the maxillary prominence is found to participate in the initial fusion with the frontonasal mass. Moreover, formation of the choanal groove occurs due to the fusion of the vomerine cushion to the subconchal fold, rather than to the choanal fold. The loss or significant reduction of the lateral nasal concha is secondary. Some features of anole adult morphology, such as the closure of the choanal groove, may constitute adaptations to vomeronasal chemoreception.

(XRM) were used. Histological serial sections were used to facilitate interpretation of tomographic data and to perform general histological description, and tomographic reconstructions were used to perform 3D visualizations of analyzed structures.

Methods
Animal care and embryo preparation The eggs of the brown anole were obtained from animals kept in the breeding room of the Faculty of Natural Sciences of the University of Silesia in Katowice. One male and two females were housed in the terrarium of dimensions L 35 cm x W 35 cm x H 60 cm. The terrarium contained cork tubes, a structural background and live plants to allow climbing and provide hiding places. Moist coconut ber substrate was used in the terrarium, which at the same time constituted the substrate for egg incubation. A 35 W spot bulb and a compact uorescent UVB bulb (Exo Terra, REPTILE UVB100) were provided. The temperature in the basking place was approximately 31-33°C, while in the cool area it was approximately 23-25°C. During the night, the temperature in the entire terrarium was about 23-25°C. Spraying the terrarium with water was performed usually twice a day to maintain proper humidity. Additionally, two water dishes were kept inside the terrarium. Anoles were fed with crickets two or three times a week. Each time insects were dusted with powder composed of Sera Reptimineral C vitamins and sepia calcium powder in a 1:3 volume ratio. The eggs were collected 4 times a year from the terrarium substrate. Embryos for the present study were collected during a period of 3 years. During this time 43 embryos were sacri ced and used for the different research methods (Table S1). Prior to isolation the embryos inside the eggs were sedated by cooling in a refrigerator for about 20 min and then placed on ice (still in the refrigerator) for about 15-20 min (see: [51,52]). Then, the embryos were isolated, decapitated and immediately transferred to the xative (see below). The age of the embryos was calculated according to the developmental table for Anolis, based on external morphological characteristic [53].

Light microscopy
For histological study, the embryos (31 specimens in total) were xed in Bouin's solution for 48 h. Embryos older than stage 10 were decalci ed in 10% formic acid for 2 weeks, then rinsed in distilled water and, like younger embryos, dehydrated and embedded in para n. The heads were serially cut at 7-10 μm using a Leica rotary microtome (Leica RM2125RT). Para n sections were stained with H&E [54,55]. The histological sections were sequentially photographed, using an OLYMPUS BX60 light microscope with an OLYMPUS DP12 digital camera. The images were saved as TIFF les using the Olympus cellSens Standard software. The images in the stack were aligned using TrakEM2 plug-in in ImageJ software [56]. The aligned stack slices were used for comparisons to the XRM data.
X-ray microtomography and 3D reconstructions Whole-sample 3D images were made using non-destructive contrast-enhanced XRM (microCT) [59][60][61]63]. The embryos (12 specimens in total) were xed in a 1:1 mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°C [57,58]. After rinsing in phosphate buffer the samples were rinsed in distilled water and then stained in 3.75% w/v in Lugol solution for 48 h to enhance the X-ray contrast of soft tissues [59,60]. After staining, the samples were rinsed in water and dehydrated to 70% ethanol for storage. To ll the gap in the developmental sequence, one sample (an embryo at stage 8) was obtained from material designated for light microscopy. After xation in Bouin's solution and dehydration to 80% ethanol, it was stained in 0.3% PTA in 70% ethanol for 72 h for contrast enhancement [60,63]. Samples were scanned in 70% ethanol or in 0.5% w/v agarose LMT [61]. The scan parameters and XRM imaging systems for each sample are shown in Table S2. Tomographic reconstructions were made with XMReconstructor (Zeiss-Xradia) and results were exported as TIFF images. Segmentations and visualizations of the analyzed structures were made in Drishti v.2.6.3 and v.2.6.4 [62].
Structure interpretation and anatomical axes The nasal cavity was de ned in similar way as VNO and other structures: the epithelium + lumen, rather than lumen only (Fig. S1A). Segmentations included epithelium of analyzed structures and cellular plugs. The segmentations did not include lumens (the endocasts of the lumens are not displayed as colored spaces in 3D images) (Fig. S1A). The nasal capsule or bones were not considered to be an integral part of the nasal cavity. The antero-posterior axis of the embryonic head, at different developmental stages, was aligned according to the long axis of the maxillary prominences or maxilla (upper lip) (Fig. S1B). The segmented structures were rendered as volumes. Individual colormaps were used for each of analyzed structures. In some cases the rendering and transparency of the nasal cavities were adjusted to show the sensory olfactory epithelium (non-transparent green) and non-sensory (respiratory) epithelium (semitransparent green) (see [60,63]). Such adjusted rendering and transparency revealed also the nasal plug, which exhibited relatively high X-ray density after contrast staining (in stages 14 and 17 labelled as yellow green).

Results
Development of the naso-palatal complex including the nasal cavity and VNO involves many different structures of the anterior part of the embryonic head. Therefore, for a better description of analyzed processes the results were divided into 3 developmental phases: early, middle, and late. The results of this study are summarized in Table 1.
Early developmental phase (stages 2-5/6) In this developmental phase the main events involve differentiation of the nasal and vomeronasal pits.
The facial prominences (lateral nasal, medial nasal, and maxillary) are relatively weakly developed. The stage in which the nasal placode is present was not found in this study. At the beginning of this phase (stages 2-3), shallow nasal pits are located lateral to the developing telencephalon and anteroventral to the primordia of the eyes ( Figs. 2A-B'). Each of them is bounded medially by the medial nasal prominence and laterally by the lateral nasal prominence (Figs. 2A, B).
Round primitive nares open ventrolaterally and each of them is surrounded by an epithelial band of the nasal pit ( Figs. 2A, B'). The medial wall of the nasal pit closely approaches the developing telencephalon ( Fig. 2B). At the end of this developmental phase (stages 5-5/6) the primitive naris is clearly elongated and, due to the ventral growth of the nasal prominences (especially the lateral one), it opens on the ventral surface of the forming snout (Figs. 2C-G). The lateral border of the nasal pit is still formed by the lateral nasal prominence exclusively. The medial border of the nasal pit is formed by the medial nasal prominence and the primordium of the vomerine cushion (red stars in Fig. 2C). Both primordia of the vomerine cushion are separated from each other by a signi cant distance. The lateral nasal prominence and the anterior part of the maxillary prominence are separated by the nasolacrimal groove (red arrowhead in Fig. 2C). The maxillary prominences are relatively better developed at this time (Figs. 2C, D'). At these stages, the main mass of the nasal pit constitutes its anterior part, probably due to the pressure exerted by the developing eye ( Fig. 2D'). This part of the nasal pit is mostly composed of thick epithelium (Figs. 2E, F), which, at least in the larger part, constitutes the differentiating sensory olfactory epithelium, since it is associated with the olfactory nerve (white asterisk in Figs. 2D and D'). The occurrence of this epithelium is restricted to the part of the nasal pit, which is now the primordium of the Stammteil (Figs. 2D-H'). The differentiating Stammteil passes into the extraconchal space primordium, which becomes visible at about stage 5/6, when it is slightly developed in the dorsolateral part of the nasal pit (Figs. 2E, F). The medial wall of the lateral nasal prominence forms the primordium of the lateral nasal concha (red asterisk in Fig. 2E). At developmental stages 5-5/6, the part of the nasal pit connecting the primordial Stammteil with the primitive naris forms the primitive choanal tube (Figs. 2E-H'). The differentiating sensory olfactory epithelium of the Stammteil decreases in thickness ventrally and posteriorly passes into the non-sensory epithelium of the primitive choanal tube. In the anterior-posterior axis the changes in the thickness of the nasal pit epithelium occurs more abruptly on the surface formed by the medial wall of the lateral nasal prominence (Figs. 2E-G). The primitive choanal tube runs posteriorly beyond the level of the primordial Stammteil to form the posterior extension of the nasal pit (Figs. 2C and G-H'). In this part of the nasal pit, the thickness of epithelium forming it is reduced in some parts to a single layer of cuboidal or cylindrical cells (Fig. 2G). The vomeronasal pit may appear at stage 3 on the medial wall of the nasal pit, but in two of three studied specimens at stage 4 this structure was absent. At stages 5-5/6, the vomeronasal pit is well distinguishable and it enters the primitive choanal tube (Figs. 2F, H and H'). The thickness of the vomeronasal sensory epithelium is equal to the thickness of the thickest part of the differentiating sensory olfactory epithelium (Figs. 2E, F).
Middle development (developmental stages 6-10) The differentiation of the nasal cavities and VNOs are strongly related to the growth and fusions of the facial prominences forming the entire snout and the super cial palate. Alongside that, the lacrimal duct develops, but at the end of this phase it constitutes a separate structure. Thus it was described separately.
Development of the super cial palate, nasal cavity and VNO At stage 6 the facial prominences are easily visible (Fig. 3A). The medial nasal prominences are con uent dorsally, but ventrally there is a relatively deep median furrow separating them (white arrowheads in Figs. 3A and B). The posterior extensions of the medial nasal prominences, forming two primordia of the anterior segment of the palate, become visible (yellow stars in Fig. 3A). They are located anteromedial to the ipsilateral primordia of the vomerine cushion (red stars in Fig. 3A). The lateral nasal prominence is still separated from the maxillary prominence by the nasolacrimal groove (red arrowhead in Fig. 3A). The maxillary prominence is composed of a larger posterior part, swelled medially, and smaller anterior portion (white asterisk in Fig. 3A). Anterior parts of the medial and lateral nasal prominences approach each other, narrowing the anteriormost part of the primitive naris (Figs. 3A, B). Posterior to this region, the nasal prominences, medial and lateral, are still at a signi cant distance to each other, leaving the primitive naris widely open (Fig. 3A). The posteriormost part of the nasal pit, formed by the primitive choanal tube, extends beyond the level of the nasolacrimal groove (red arrowhead) and the primordium of the vomerine cushion (red star), achieving almost the level of the border between the anterior and posterior portion of the maxillary prominence (Fig. 3A). For this reason, the posterior part of the nasal pit is bounded laterally by the anterior portion of the maxillary prominence.
The vomeronasal pit may be called the early VNO since it takes a spherical shape (Figs. 3C-D'). It is still connected with the nasal pit through the primitive choanal tube (Fig. 3D). The primitive choanal tube is higher than at the previous stage, and its anterior part may be considered as a primordium of the vestibulum. At this time it starts to be lled by the nasal plug containing cells with spherical nuclei (Fig.  3B), but the cylindrical cells of epithelial edges of the nasal prominences are distinguishable (Fig. 3B). The distribution of the presumptive sensory olfactory epithelium seems to be restricted to the Stammteil and the anterior part of the extraconchal space. It correlates with the part of the nasal pit characterized by higher X-ray density, but the vestibular part of the primitive choanal tube, lled by the nasal plug, is also relatively dense at this stage (Figs. 3C'', D'). At stage 7, the medial nasal prominences are merged and form a single median structure called the frontonasal mass (Fig. 4A). The posterior extensions of the medial nasal prominences, forming the paired primordium of the anterior segment of the palate, become almost obliterated. The lateral nasal prominence and the frontonasal mass approach closely to each other through their entire lengths, reducing the anterior part of the primitive naris to a narrow slit (grey dashed lines in Figs. 4A and A'). This part of the primitive naris marks the presumptive external naris, which is directed ventrally (Fig. 4B). Although fusion between the medial nasal prominence of the frontonasal mass and lateral nasal prominence does not occur, the anteriormost part of the primitive naris is non-patent, because it is lled by the nasal plug (Fig. S2A). Medial to the nasolacrimal groove (red arrowhead) and ventral to the primordium of the vomerine cushion, the maxillary prominence approaches closely the frontonasal mass (white arrow in Figs. 4A and A'). The posterior half of the primitive naris, located behind the approaching tips of the maxillary prominence and the frontonasal mass, widens gradually (Figs. 4A, A'). Posterior to the primitive naris, the maxillary fold, emerging from the lateral surface of the maxillary prominence, is visible (Fig. 4A).
The growth of the facial prominences causes that the primordium of the vestibulum and the remaining part of the primitive choanal tube become deeper and more distinct than at stage 6 (Figs. 4C-D'). Thus, the early VNO increases the distance to the primitive naris ( Fig. 4D). At this developmental stage the extraconchal space is much more distinct (Figs. 4C, C' and see Fig. S2B). Due to the signi cant changes in the shape of the nasal pit at stage 7 it may be considered as an early nasal cavity. There are no signi cant changes in the distribution of the presumptive sensory olfactory epithelium (Figs. 4C'', D'). In the sensory olfactory and vomeronasal sensory epithelium, the division into the basal cells (L1) with spherical nuclei and central layer (L2) of cells with slightly more darkly stained and elongated nuclei is relatively weakly visible (Figs. S2A-C). The most apical part of the epithelium (L3) is very thin and the brightest layer. It contains only a few nuclei, usually undergoing mitosis (Figs. S2A-C).
At stage 8 the fusion of the maxillary prominence and lateral nasal prominence with the frontonasal mass (including a part of the primordial anterior segment of the palate) divides the primitive naris into the external naris and the primitive outer choana (Figs. 5A-B' and Movie S1: 12 s-16 s). Below the fusion, a relatively deep groove between fused prominences can be observed (two white arrows in Figs. 5B and B'). The elongated external naris is bounded by the lateral nasal prominence and frontonasal mass (Fig. 5C).
Due to the signi cant ventral growth of the frontonasal mass, the external naris takes a lateral position in comparison to the presumptive external naris, which is directed ventrally at the previous stage ( Fig. 5C and compare with Fig. 4B). At the same time the lateral nasal prominence takes a dorsal rather than a lateral position in relation to the external naris (Fig. 5C). The nasolacrimal groove (red arrowhead) passes into the much longer groove existing between the developing lower eyelid and the maxillary prominence (red arrows in Fig. 5B). The paired primordium of the anterior segment of the palate is visible again (yellow stars in Fig. 5B). Between the ventrolateral ridge of the vomerine cushion primordium and the maxillary fold, the primitive outer choana is located (Figs. 5B, B'). It is a relatively wide opening, but it gradually narrows anteriorly (Fig. 5B').
The vestibulum is well de ned and separated from the posterior part of the primitive choanal tube (Figs. 5D, E) due to the mentioned fusion of the maxillary and lateral nasal prominence with the frontonasal mass. The external naris as the entire vestibulum is still sealed by the nasal plug (Figs. 5F, F').
The part of the primitive choanal tube, located just dorsal to the fusion becomes incorporated into the primordium of the main nasal cavity (Fig. 5A). The main nasal cavity primordium passes into the posterior part of the primitive choanal tube (Figs. 5D, E). From this stage, the primordium of the main nasal cavity is well de ned (Figs. 5D-F), but the extraconchal space is restricted approximately to the posterior half of this part of the nasal cavity (Fig. 5D). The extraconchal space is strongly developed there and extends well ventrally, demarcating the lateral border of the lateral nasal concha, which emerges from the lateral nasal prominence (Fig. 5A). A small antorbital space may be distinguished in the primordial main nasal cavity, and it is located just posterior to the lateral nasal concha (Figs. 5G, H).
The sensory olfactory epithelium seems to be restricted to the Stammteil and the medial wall of the antorbital space (Figs. 5F-G). The connection between the ellipsoidal early VNO and the posterior part of the choanal tube is narrowed (dashed ellipse in Fig. 5G). Ventrolateral to the early VNO a relatively shallow choanal diverticulum of the primitive choanal tube may be distinguished. It is located between the maxillary fold and the base of the lateral nasal concha (white star in Figs. 5D and G). At developmental stage 9 the maxillary prominences and the frontonasal mass are almost completely fused and form the upper lip (Fig. 6A). The well-developed anterior segment of the palate is formed by the merging of both components of its paired primordium. The premaxillary papilla is visible at the posterior edge of the segment (Fig. 6A). The maxillary fold closely approaches the anterior segment of the palate and together they form the walls of the non-patent primordial duct of the VNO (Figs. 6A, A'). Posterior to the primordial VNO duct, the primitive outer choanae of both sides demarcate the lateral borders of the elongated vomerine cushion (Fig. 6A). At this stage, the choanal fold (yellow arrowheads) emerges from the maxillary fold (Fig. 6A). It narrows the anterior part of the primitive outer choana, but its posteriormost part remains widely open to the oral cavity (Figs. 6A, A'). The external naris is well established on the lateral side of the snout (Fig. 6B). The nasolacrimal groove is hardly distinguishable (red arrowhead in Fig. 6B).
The vestibulum takes a more tubular form, but it is still oriented more vertically rather than horizontally (Figs. 6C, D) and it remains sealed by the nasal plug (Figs. 6D', D''). In some specimens of this stage the small primordium of the lateral nasal gland was present, emerging from the vestibulum as an initial bud, just anterior to the anterior end of the primitive inner choanal tube.
The extraconchal space attains a clearly vertical orientation in relation to the antero-posterior axis of the snout and becomes restricted to the posterior third of the main nasal cavity primordium (Figs. 6C, E). The ridge formed by the lateral border of the extraconchal space is con uent ventrally with the posterior part of the choanal diverticulum (white asterisks in Fig. 6C). The antorbital space becomes less distinct (Fig.  6E). The distribution of the sensory olfactory epithelium seems to be the same as previously (Figs. 6D', D'' and F, G). The subconchal fold ( = "Falte" [64] or "Lippe am Choanengang" [65]) becomes distinguishable from the lateral nasal concha. It is restricted ventrally by the choanal diverticulum (white star) and dorsally by the lateral expansion of the primitive inner choanal tube (red arrow in Figs. 6F and G; and see below).
The primitive choanal tube is signi cantly drawn out posteriorly beyond the level of the antorbital and extraconchal space (Figs. 6D, E). The posterior extension of the primitive choanal tube together with the ventral part of this structure located below the subconchal fold can now be called the primitive outer choanal tube (Fig. 6C). The rest of the primitive choanal tube, which is a ventral part of the primordial main nasal cavity, can now be called the primitive inner choanal tube. The primitive inner choana is located between the primordial main nasal cavity and the primitive outer choanal tube. The anterior and, at the same time, more horizontal part of the primitive inner choana (more horizontal part of blue dashed line in Fig. 6C) is de ned by the subconchal fold and the lateral ridge of the vomerine cushion (red asterisks in Fig. 6G). The posterior part of the primitive inner choana takes a vertical orientation. It is located behind the extraconchal space, between the main nasal cavity primordium and the outer choanal tube (more vertical part of blue dashed line in Fig. 6C).
At this developmental stage, the VNO is still connected to the nasal cavity. It is open on the medial wall of the primitive outer choanal tube just ventral to the primitive inner choana and medial to the choanal diverticulum (Figs. 6F and G). The primordial VNO duct can be distinguished from the anteriormost part of the primitive outer choanal tube (Figs. 6C-D'' and F). At this time the close apposition of the maxillary fold and the anterior segment of the palate reduces the lumen of the primordial VNO duct, which is lled by a narrow cellular plug (Fig. 6F). In the anterior part of the VNO, the small anlage of the mushroom body is observed (blue asterisks in Fig. 6F). It emerges from the oor of the VNO lumen. It is covered by much thinner epithelium than the sensory epithelium of the dorsal dome (Fig. 6F).
At stages 9/10 and 10 the bud of the lateral nasal gland, emerging from the posterior part of the vestibulum, increases in size and thus becomes more distinct (dashed circle in Fig. 7A). The entire nasal cavity takes a form similar to that observed at the beginning of the next developmental phase. The differentiating mushroom body, reducing the lumen of the VNO, is well visible at these stages, but it is not strongly developed (Fig. 7B). At stage 9/10 the fusion of the subconchal fold with the vomerine cushion ( The histology of the sensory epithelia of the nasal cavity and VNO does not exhibit signi cant changes from that observed at development stage 7 (Fig. 7B).
Origin of the lacrimal duct The primordium of the lacrimal duct is rst visible at stage 8, when the groove between the developing lower eyelid and the maxillary prominence is relatively well distinguishable (two long red arrows in Fig.  8A). The lacrimal duct primordium arises from the deep non-patent part of this groove (Figs. 8A', B), which is con uent anteriorly with the nasolacrimal groove (red arrowhead in Fig. 8A). At least the posterior part of the nasolacrimal groove seems to also give rise the lacrimal duct (Fig. 8A). In further development (stage 9), the primordium of the lacrimal duct attains the form shown in Fig. 8C. Two lacrimal canaliculi depart from it and grow into the tissue of the primordial lower eyelid towards its inner surface. Posteriorly, the lacrimal duct located between the lacrimal canaliculi is still a part of the groove between the developing lower eyelid and the maxillary prominence (Figs. 8C-D). At the level of the connection with anterior canaliculus the lacrimal duct dives into the underlying mesoderm. The freed anterior part of the lacrimal duct (single short red arrow in Fig. 8C) runs anteromedially towards the anteriormost part of the choanal diverticulum, located just posterior to the VNO duct (see Figs. 6F and G). Despite the close apposition of the anterior tip of the primordial lacrimal duct to the choanal diverticulum at stage 9, there is no connection between the lacrimal duct primordium and the choanal groove even at the end of this developmental phase (stage 10). The primordium of the lacrimal duct and lacrimal canaliculi are composed of non-patent cellular cord in which a single layer of external cells may be distinguished (Fig. 8E). From stage 9/10 the lacrimal duct becomes completely separated from the groove between the eyelid and the maxillary part of the palate (Fig. 8E). Moreover, the posterior (Fig. 8E) and anterior lacrimal canaliculi clearly reach the inner surface of the eyelid.
At the end of the middle phase of development all structures considered in this study are present, but they do not attain their adult morphology and topographical relationships.
Late developmental phase (developmental stages [11][12][13][14][15][16][17][18][19] Main changes at the beginning of the late developmental phase At developmental stages 11-12, the vestibulum takes a more horizontal orientation than at the previous stage, but its posterior part is still located clearly dorsally in relation to the anterior one (Fig. 9F). The anterior part of the vestibulum turns laterally to achieve the lateral surface of the snout (Fig. 9G). The ventral vestibular channel is restricted to the posterior portion of the vestibulum, and posteriorly it is con uent with the inner choanal tube (Fig. 9F). The lateral nasal gland is composed of the non-patent primordium of the main duct, which is directly connected to the posterior end of the vestibulum, and the spherical bud divided by a few shallow clefts (Fig. 9G).

Main nasal cavity
The extraconchal space is restricted to the posterior end of the main nasal cavity (Figs. 9F, G). In this region, the lateral nasal concha is well developed (Fig. 9D) Table 1).
At developmental stage 14, the general morphology of the nasal cavity is very similar to that observed at stage 12, but the lateral nasal gland is better developed (Figs. S3A, B; Table 1 . However, the nasal plug can still be well developed at this stage or only its remnants may be visible (Fig. 11B). At these stages the vestibulum is surrounded by a "sleeve" containing a well-developed sinusoidal capillary network associated with smooth muscle bers (spongy sinusoidal tissue) (Fig. 11B). The lateral nasal gland can be classi ed as compound tubular (Fig. 11C).

Main nasal cavity
The lumen of the main nasal cavity increases its volume in such a manner that the entire nasal cavity seems to be dilated in comparison to the previous stage (Figs. 11A and C-D). The lateral edge of the extraconchal space is almost obliterated (Fig. 11A). The antorbital space is no longer visible. The location of the olfactory sensory epithelium is the same as at stage 17. It is marked by relatively high X-ray density (Figs. 11A', A'') and the Bowman's glands (Fig. 11C'). The respiratory epithelium also exhibits relatively high X-ray density (Figs. 11A', A''), which seems to be linked with the presence of well-developed goblet cells. The olfactory sensory epithelium becomes thinner than at previous stages and it is now reduced to a few rows of cells (Figs. 11D, E and compare with Figs. S4A and 9D). The division into the basal layer (L1) containing cells with spherical nuclei and the central layer (L2) containing cells with elongated and slightly more darkly stained nuclei is retained (Fig. 11E). The non-sensory epithelium of the inner choanal tube is ciliated. Moreover, the numerous goblet cells and crypts are visible there (Fig. 11F).
Outer choanal tube and choanal groove In all pre-hatched embryos which were studied (stage 18) the choanal groove was open to the oral cavity across its entire length. The opening of this structure was reduced by the choanal fold, which at that time approaches the lateral ridge of the vomerine cushion (  (Fig. 13D). Alternatively, at least some considered part of the lacrimal duct expansion (white arrows in Fig. 13D) may constitute the dorsal diverticulum of the choanal groove, since it is covered by ciliated epithelium containing goblet cells (red star in Fig. 12B). The anterior tip of the lacrimal duct is moved more anteriorly along the ventral part of the medial wall of the VNO duct (Fig. 13E). In all but one studied specimens that are at stage 18, the lacrimal duct as well the lacrimal canaliculi were completely patent through their entire length (Figs. 13F, G). Thus a long slit-like opening extends along the contact region between the lacrimal duct and the choanal groove and anteriorly continues as a slit between the lacrimal duct and the VNO duct (turquoise arrows in Fig. 13E).
Development of the VNO At the beginning of the late developmental phase (stages 11-12) the VNO is located ventromedial to the vestibulum of the nasal cavity at the level of its posterior part (Fig. S5A). Later in development (from about stage 17) it is located ventromedial to the middle of the length of the vestibulum (Figs. S5B, C).
The main mass of the VNO is formed by thick sensory epithelium, which becomes gradually shifted medially to the VNO duct (Figs. 14A, B and C). Despite the fact that the entire nasal cavity is much larger than the VNO, the difference in size between the sensory part of the nasal cavity and the dorsal dome of the VNO is not very large (Figs. S5A-C). Moreover, at developmental stage 18, the sensory olfactory epithelium is about half as thick as the sensory epithelium of the VNO (compare Fig. 14C (Fig. 14B, C). The septomaxilla is located dorsal to the VNO, while the vomer is located ventral and ventromedial to the organ. The gap between the vomer and septomaxilla is lled by the nasal septum. The maxilla approaches the lateral side of the VNO, but the lamina transversalis anterior (and the cartilage of the mushroom body) separates these two structures (Figs. 14B, C).

Discussion
The present study provides the rst description of the development of the naso-palatal complex in a member of the Dactyloidae. At the same time it is one of the most comprehensive anatomical descriptions of the squamate naso-palatal complex, taking into account morphogenesis of both the super cial palate and internal structures of the snout, such as the nasal cavity and the VNO. For a better description of the analyzed developmental events we refer to some structures which have previously been overlooked, namely the subconchal fold and the anterior segment of the palate. They are involved in the formation of the choanal groove and the duct of the VNO respectively. Moreover, we propose new terminology for the maxillary fold and different parts of the choanal tube. The former seems to be "forgotten" in contemporary literature.

Early developmental phase
The paired nasal pits in tetrapods, originating from the nasal placodes, are located on the lateral sides of the developing head. Each of them is bounded by the lateral and medial nasal prominences [6,17,20]. The results of our study showed that the nasal pit in the brown anole embryos is present very early, at stage 2.
The vomeronasal pit appears at stages 3-5 (Figs. 2F, H and H'), emerging from the medial wall of the nasal pit. The primordial Stammteil, extraconchal space, and choanal tube were distinguishable in the nasal pit at the end of the early developmental phase of the naso-palatal complex. Moreover, the maxillary prominence terminates just posterior to the nasal pit at this time (Fig. 2C).
The beginning of the developmental sequence of analyzed structures in the brown anole (the origin of the vomeronasal pit from preexisting nasal pit, and the later anterior extension of the maxillary prominence) seems to be similar to that described in other Tetrapoda [6,17,20].
Initial fusion and formation of the vestibulum The medial nasal prominences fuse and form the single frontonasal mass at stage 7 of the brown anole.
It is generally believed that the fusion of the nasal prominences result in the formation of the vestibulum of the nasal cavity and separation of the external naris from the primitive outer choana [16,17,69].
However, the importance of the maxillary prominence in this process may be underestimated since the studies utilizing optical projection tomography (OPT) showed that the fusion between facial prominences, which initiates the formation of the primary palate, varies across amniotes. In turtle (Emydura subglobosa) the medial nasal prominence fuses with the lateral nasal prominence, but in chick the initial fusion with the medial nasal prominence involves the maxillary prominence only [67]. In crocodilians and mammals the initial fusion involves all facial prominences [68]. A similar situation was found in our study in the brown anole, despite the signi cant differences in shape of the facial prominences. Abramyan et al. [67] reported that in lizards (bearded dragon, veiled chameleon, and whiptail lizard), the initiation of the primary palate formation does not engage the maxillary prominence as in turtle, but the external naris becomes closed by the fusion of the nasal prominences and reopens in later development. However, it seems that the complete fusion described by Abramyan et al. [67] in lizards is a combination of two events: the formation of the nasal plug (anteriorly) and the "classical" fusion (posteriorly; if present), where the epithelial seam is replaced by the mesenchymal bridge (see [68,70]). Thus we are unable to say whether the condition observed by Abramyan et al. [67] in lizards is truly different from that observed by us in the brown anole, in which all facial prominences initiate the fusion of the primary palate. The description provided by Parsons [69] indicates that obliteration of the external naris, and thus formation of the nasal plug, may occur relatively later in development of turtle (Chrysemys) than in snakes and other squamates. This is consistent with our observations on the brown anole, since the formation of the nasal plug starts before the initial fusion of the facial prominences and formation of the vestibulum. In fact, at the time of separation of the external naris and the primitive outer choana, which occurs at stage 8 of the brown anole (Movie S1), the external naris and the vestibulum are sealed by the well developed nasal plug. Similar "delay" in formation of the nasal plug, which has been noted in turtle, may occur in birds (see [71]). These facts may explain the description of Abramyan et al. [67], who did not nd the closed external naris in turtle and chick in stages at the time of initial fusion of facial prominences.
The presence of the embryonic nasal plug is a common transient embryonic feature of amniotes. It has been found in snakes [66,69,72], lizards [18,73], turtle [69], Sphenodon [74], birds [71,72] and mammals [72,75] including human [76,77]. The nature of the nasal plug is poorly understood. It is described as mass of epithelial cells [75], mass of isodiametric cells [21], peridermal plug [72,78], or structure formed by keratinocytes [77]. It has been proposed that it forms between the nasal prominences from the epithelial seam, which does not undergo disintegration [71,72], or as a proliferation of the "invaginated" oral epithelium [78], which in fact may be considered as walls of the presumptive vestibulum. It seems to us that these explanations are not mutually exclusive. In fact, the presumptive vestibulum containing the nasal plug at stages 6 and 7 of the brown anole (Figs. 3B and S2A) resembles a multilayered medial edge epithelial seam in the human embryonic soft palate (see [79]), although in the anole the basal layer, which forms the walls of the future vestibulum, can easily be distinguished. Distribution of the nasal plug is restricted to the vestibulum in later embryonic life [21,71,72].
The functional signi cance of the nasal plug formation is unknown. Weber [72] concluded that the nasal plug is functionless and, in contrast to the auditory meatal plug, does not protect undifferentiated sensory epithelium. As he stated, the amniotic uid may still reaches the lumen of the main nasal cavity through the open choana (see also [67]). Moreover, despite the fact that recanalization of the vestibulum takes place shortly before hatching in the brown anole (this study) and in other extant sauropsids [72], in some mammals including human, the reduction of the nasal plug occurs relatively early [71,72,76].
Nevertheless, we suspect that the formation of the nasal plug may be crucial for vestibulum formation.
Recent studies on chick have suggested that the canalization of the vestibulum and the external naris is caused by "combination of cellular remodeling, apoptosis, as well as non-apoptotic necrosis" [71]. In the brown anole the vestibulum takes the relatively simple form of a long tube, oriented almost horizontally at late stages (17-19; Figs. 10A, 11A). Elongated vestibulum has been found in the other studies on the brown anole [35], in Anolis carolinensis [33], A. cristatellus, and possibly in A. pulchellus, A. evermanni, and A. stratulus [34]. In contrast, the vestibulum of A. roquet (described there as A. alligator) has been found to be short [16]. The shortened vestibulum seems to be present also in A. equestris (see Fig. 12B in [6]). As was shown, the morphology of this structure may exhibit some variations in these lizards. The great variation of this structure was also found in other squamates. For instance, a shortened vestibulum occurs in snakes and Gerrhonotus (Anguidae), but it may be well developed and take an almost vertical orientation as in Phrynosoma [33] (Iguania). It has been suggested that the elongation of the vestibulum in squamates is associated with deserticolous habitats [16] and it has been suspected that such feature as well as "less direct communication with the exterior" may decrease the evaporation rate [33]. It is worth mentioning that the vestibulum of adult squamates excludes inspired foreign bodies, such as soil particles, from the respiratory tract. This is possible due to hyperplasia of its epithelium (which occurs in some forms) and mucoid secretion of the lateral nasal gland, which discharges between the vestibulum and anterior part of the main nasal cavity [5,17]. The function of cleansing the nose may be improved by the length and shape of the vestibulum, and by the presence of a network of sinusoids associated with the reticulum of smooth muscle bers (spongy sinusoidal tissue, cavernous tissue) surrounding this part of the nasal cavity, which probably close the vestibulum during intumescence [21,22,33].

Formation of the choanal groove and homology of the choanal folds
The term choanal groove is often used to describe the groove on the palatine bone in the region of the choana [38,80]. Here we use this term exclusively for a soft tissue structure [5,16,21] as de ned above. The formation of the choanal groove in squamates is associated with the separation of the VNO from the nasal cavity and separation of the main nasal cavity from the oral cavity [6]. The results of our studies showed that in the brown anole embryos all three events occur due to the closure of the primitive inner choana by the fusion of the subconchal fold with the vomerine cushion. Just prior to formation of the choanal groove, the duct of the VNO can be distinguished. The choanal groove, as well the VNO duct, constitute the persistent portion of the primitive outer choanal tube. Our interpretation differs from the previous descriptions of choanal groove formation which indicated that this structure develops by the fusion of the maxillary prominences (or choanal fold) with the vomerine cushion [6,21]. It was also suggest that the same fusion event is responsible for the obliteration of the primitive outer choanal tube just posterior to the VNO duct, resulting in the shortening of the adult choanal groove. In fact, the choanal groove is shortened anteriorly in many adult squamates and does not reach the VNO duct [5,6].
It seems that the formation and the obliteration of the choanal groove constitutes two different processes in squamates. As we showed, in the brown anole the former involves the fusion of the vomerine cushion with the subconchal fold, which originates from the lateral nasal concha. The lateral nasal concha originates in turn from the lateral nasal prominence (see also [81]). With the formation of the upper lip, the lateral nasal prominence and the nasolacrimal groove become hardly distinguishable from the rest of the snout (Fig. 6B). This condition may explain previous misinterpretation about the participation of the maxillary prominence, rather than lateral nasal prominence, in formation of the choanal groove.
Despite the absence of the choanal groove -in most snakes [6,36,40] it has been shown to occur in the grass snake embryo (and probably in embryos of other snakes) and extends from the VNO duct to the outer choana [19]. Thus it is reasonable to assume that also in non-ophidian autarchoglossans, in which the choanal groove does not reach the VNO duct [36], the anterior part of the choanal groove becomes obliterated during a separate process. It seems that in contrast to the formation of the choanal groove its obliteration may indeed engage the maxillary prominence (choanal fold), as has been suggested [6,21].
In Gekkota and Iguania, the choanal groove is con uent with the duct of the VNO. We found that in the late stages of the brown anole the choanal groove extends along most of the length of the main nasal cavity. The outer choana and the outer choanal tube are located well posteriorly (Fig. 11A and Figs. 12A,   A'). Interestingly, at stage 19 most of the choanal groove was found to be separated from the oral cavity, by the fusion of the medial edge of the choanal fold with the vomerine cushion (Figs. 12A', B). Our ndings agree with the previous description of the brown anole [35]. The closed choanal groove has been noted also in A. cristatellus and possibly in A. pulchellus, A. evermanni, and A. stratulus [34]. Bellairs & Boyd [6], who based their description on A. equestris, A. lineatopus and A. valencienni, stated that closure of the choanal groove is restricted to the anterior part of the choanal groove, "a little behind the level of the organ of Jacobson." As far as we know the formation of the closed channel in a place of choanal groove has never been noted in other squamates and such condition is likely a synapomorphy of Dactyloidae.
The presence of the choanal groove was considered to be a synapomorphy of squamates [40]. In fact, in Sphenodon, the only extant member of Rhynchocephalia, commonly considered to be a sister group to Squamata [28,[82][83][84], the nasal cavity widely communicates with the oral cavity through the long outer choana [5,16,17]. Bellairs & Boyd [6] suggested that only a short choanal groove is present anteriorly. However, a long outer choana is also present in many representatives of Iguania [21,33].
The oor of the choanal groove and the lateral margin of the outer choana is formed by the choanal fold, which is supported by the ectochoanal cartilage [6,21]. The squamate choanal folds (sometimes called the palatine processes) seem to be considered as a structures homologous to the crocodilian and mammalian palatal shelves [see [3][4][5], which in these taxa form "true" secondary palate and well developed nasopharyngeal duct [69,86,87]. In mammals the palatal shelves arise as vertically-oriented structures, then become reoriented horizontally and fuse with each other over the tongue to form the secondary palate, which anteriorly fuses to the primary palate and the ventral part of the nasal septum [87,88]. Crocodilian palatal shelves grow horizontally from the beginning (except their posteriormost parts) and they merge to the nasal septum (anteriorly) and to each other (posteriorly) [86,89].
A review of the German literature published around 1900 indicates that at least two kinds of medial softtissue outgrowths of reptilian maxillary prominence can be distinguished [5,81,90]. This is consistent with our results, since we were able to nd the maxillary fold and the choanal fold originating from it. Based on comparative embryological data, Fuchs [5] postulated that the squamate choanal folds are not homologous to the mammalian palatal shelves. On the contrary, he suggested that mammalian palatal shelves are homologous to the mediale Seitenfalten (here: the maxillary fold). Moreover, it was suggested that the homolog of the choanal fold is present in mammalian embryos. It is located dorsal to the ("secondary") palatal shelve [5,81] and may be called as "primitive palatal shelve" (primitive Gaumenfalte) [91]. Moreover, Fuchs [5] suggested that crocodilian secondary palate is formed by the homologs of choanal folds not the maxillary folds.
The presence of two rather than one medial soft-tissue outgrowth of the maxillary prominence in reptiles seems to be overlooked in current literature. Alternatively, the choanal fold could be considered as a part of the maxillary fold, since the former seems to emerge from the latter in the brown anole (compere Figs. 5B and 6A). However, both structures are relatively distinct from each other from the beginning of the late developmental phase of the naso-palatal complex (see Fig. 9H).
The initial vertical orientation of the mammalian palatal shelves has been discussed by Ferguson [86,89,92]. In fact so-called "palatal shelves" of sauropsids grow horizontally from the beginning. He stated that such condition is associated with the large muscular tongue. The alternative explanation may in fact support the view of Fuchs [5] and the elevation of maxillary folds (mediale Seitenfalten) to the horizontal orientation may constitute the mammalian synapomorphy.
In birds the palatal shelves approaches closely to each other, but do not fuse as in crocodilians [93].
Interestingly in turtles the similar medial outgrowths of the maxillary prominence (palatal shelves or alternatively the choanal folds) are not formed in some taxa [85], but the main nasal cavity is well separated from the oral one, probably due to posterior extension of the primary palate [21]. The homology between the amniote medial soft-tissue outgrowths of the maxillary prominence requires con rmation in future comparative studies.
Outer choanal tube and nasopharyngeal duct Fuchs [5] recognized two parts (limbs) of the choanal tube: horizontal ("horizontaler Schenkel des Choanenganges") and descending ("absteigender Schenkel des Choanenganges"). A similar division was applied by Parsons [21], who distinguished a "more or less horizontal dorsal portion lying ventral to the concha" and a ventral portion which "from near the lateral end of this dorsal portion" "extends ventromedially to the choana". At the same time, he did not consider the choanal tube as a part of the conchal zone. Bellairs [22] and Bellairs & Boyd [6] distinguished the ventral conchal zone and restricted the term "choanal tube" to the descending limb of the choanal tube sensu Fuchs [5]. In most nonophidian squamates the horizontal limb of the choanal tube (or ventral conchal zone) leads to the descending limb of the choanal tube or choanal tube sensu Bellairs & Boyd [6] through the inner choana. The descending limb of the choanal tube runs ventromedially and opens to the oral cavity by the outer choana [6,22,64]. Here we employed division of the choanal tube into the inner and outer choanal tube (Figs. 1C, D), which correspondence to the two parts of this structure distinguished by Fuchs [5] and Parsons [21].
For embryonic structures we also introduced the term "primitive choanal tube" to de ne the structure connecting the primordial Stammteil to the oral cavity from about stage 5 of the brown anole (Figs. 2E-H). In later development of this species this structure gives rise to the vestibulum, the primitive outer choanal tube, and the primitive inner choanal tube. The primitive outer choanal tube gives rise to the primordial VNO duct, choanal groove, and the outer choanal tube. With the closure of the part of the primitive inner choana (stage 9/10) and the formation of the choanal groove, the primitive inner choanal tube may be called the inner choanal tube. Because the structure de ned here as the outer choanal tube is usually recognized to be homologous to the nasopharyngeal duct of snakes and some lizards [6,21], we consider that only the inner choanal tube belongs the main nasal cavity. In snakes the outer choanal tube forms the nasopharyngeal duct separated ventrally from the oral cavity by the fusion of the vomerine cushion and choanal fold [21].
It is worth noting that the nasopharyngeal duct of snakes and some lizards is not homologous with the nasopharyngeal duct of mammals and crocodiles [21,94]. The ophidian nasopharyngeal ducts of both sides join together in their posterior course and enter the oral cavity into the median palatal trough (orbitonasal trough) [6,17,19,21]. In this case the inner and outer choana become widely separated [6].
Some forms of the nasopharyngeal ducts (paired or single median) were also found in non-ophidian squamates (Xantusia, Dibamus chameleons, some skinks). This structure is formed by the medial extension of choanal folds or elongation of the vomerine cushion [21,33,39,40,67]. However, in most cases the nasopharyngeal ducts of lizards is incomplete ventrally and some authors did not distinguish it as a "snake-like" nasopharyngeal duct [6,21,33,39,40,68]. Stebbins [33] stated that in Anolis carolinensis "the nasopharyngeal duct situated well posteriorly" is present. It seems that Parsons [21] did not consider the condition of Anolis as the presence of the nasopharyngeal duct. Instead he noted that: "the choana is short and well posterior". A similar interpretation was adopted in this study; thus the outer choanal tube was not considered to form the nasopharyngeal duct in the brown anole.
Lateral nasal concha and main nasal cavity The nasal concha constitutes any projection into the nasal cavity of amniotes, but de nition of this structure varies among different authors [20]. In most squamates only a single concha is present, called the lateral nasal concha. It forms a space for the lateral nasal gland [17,21]. The lateral nasal concha of squamates is homologous to the posterior concha of Sphenodon [94]. Due to the presence of the concha, 3 parts of the conchal zone of the nasal cavity in squamates can be distinguished: the extraconchal space, the Stammteil, and the choanal tube (the inner choanal tube). The primordium of the lateral nasal concha in the brown anole appears at the end of the early developmental phase (stage 5/6), when it emerges from the lateral nasal prominence (Fig. 2E) and thus before the separation of the external naris and the primitive outer choana at stage 8. The same developmental sequence was described in Thamnophis [69].
Previous studies indicated that the lateral nasal concha is absent in anoles [2,16,21,33]. We found that reduction of the concha is associated with the shift of the extraconchal space to the posteriormost part of the main nasal cavity and change of its orientation to vertical position. In later development, the extraconchal space becomes dilated and incorporated into the Stammteil (Figs. 10A, B and Figs. 11A, C). However, we found that even in the latest developmental stages (18)(19) of the brown anole the remnant of the concha, called here the anterior extension of the lateral nasal concha, is still noticeable in the region of the lateral nasal gland, between the Stammteil and the inner choanal tube (Figs. 11A, D). It is likely that it undergoes further reduction in later ontogenesis. The reduction or lack of the concha was also found in other members of the Iguania including Acrodonta and Pleurodonta. Within the latter, the lateral nasal concha is absent not only in anoles, but also in the taxa classi ed as "Uma-morphotype" containing: Uma, Callisaurus, Holbrookia, Sceloporus, Uta, and Phrynosoma [33], which all belong to the Phrynosomatidae [95]. Depending on a phylogenetic analysis it could be inferred that the lack of the lateral nasal concha occurred independently in the Phrynosomatidae and the Dactyloidae (see [28]) or the loss of this structure might have taken place in the common ancestor of the Phrynosomatidae and a clade containing Dactyloidae and Polychrotidae (see [96]). Still, little is known about the state of this character in other groups of Pleurodonta. In "Dipsosaurus-morphotype" lizards (Dipsosaurus, Iguana, Crotaphytus, Sauromalus, Ctenosaura) [33], some anguids and in monitor lizards the concha is attached dorsally or dorsolaterally. In such cases the extraconchal space was suggested to be absent, and instead the subconchal recess is distinguishable [21]. However, in another study these two structures were considered to be homologous [22].

VNO
The primordium of the VNO appears as a vomeronasal pit emerging on the medial wall of the nasal pit. It appears before the fusion of the nasal prominences [17,69]. The vomeronasal pit is also described in crocodilian and bird embryos, but eventually disappears early in development and the VNO is not present in adult specimens [17]. In crocodilians the vomeronasal pit is no longer noticeable after the fusion of the lateral and medial nasal prominences [69]. In the brown anole, the vomeronasal pit was rst found at developmental stages 3-5. Considering the level of association of the VNO with the nasal cavity and degree of its development in adult specimens a few types of the VNO may be distinguished. The VNO of amphibians and turtles is a part of the nasal cavity and the non-sensory part cannot be distinguished [97][98][99]. The mammalian VNO is connected to the nasal cavity and in some forms also to the oral cavity, and it consists of more or less developed sensory epithelium, which is thicker than the non-sensory one [4,97]. A non-sensory vestige of the VNO is present in humans and chimpanzees [100], but in some bats the VNO may be rudimentary or even absent [101]. In Sphenodon the VNO takes a lens-shape form and there is no mushroom body. The VNO lumen is connected to the anterior part of the nasal cavity near the long outer choana [69,102]. However, some descriptions suggest that the VNO of this taxon communicates with both the oral cavity and the short choanal groove [6].
In adult squamates the VNO has no direct connection with the nasal cavity and its duct enters the oral cavity exclusively as in most squamate groups or the oral cavity and the choanal groove as in iguanians and gekkotans. In skinks and lacertids the choanal gutter is formed by the close association of the choanal groove with the lacrimal duct and is connected to the VNO duct by the lacrimal component [6]. In the brown anole separation of the VNO from the nasal cavity occurs at developmental stage 9/10. At this stage the mushroom body is well distinguishable, but relatively small (Fig. 7B).
In adult squamates the vomeronasal sensory epithelium is generally strongly developed and the nonsensory epithelium covers the mushroom body [2,14,103]. The VNO can be reduced in some arboreal Iguania, especially in chameleons in which the mushroom body or even the entire organ is absent [16,39,69]. It was suggested that the anole VNO is: "present but less well-differentiated than in most lizards" [2]. In fact we found that the VNO of the brown anole at stages 18 and 19 possesses a poorly developed mushroom body and weakly developed ventral channel (see Fig. 14). Histological study revealed medial localization of the dorsal dome in relation to VNO duct. Interestingly, the adult vomeronasal sensory epithelium has well-visible pigment aggregated within its central layer (Fig. 14C).
Similarly as in brown anole embryos three layers of developing vomeronasal sensory epithelium were also found in other light microscopic studies [2,17,19] (e.g. Fig. 14A). The ultrastructural studies indicated that this epithelium contains different populations of cells [15,103,104]. Based on previous data and our studies we can suppose that the basal layer (L1) is a population of developing bipolar neurons and undifferentiated cells, the central layer (L2) is composed of supporting cells while the apical one (L3) includes the dendrites of bipolar neurons and protrusions of the supporting cells.
The sensory epithelium of the ophidian VNO is characterized by a peculiar columnar organization [15,17,19,103,104]. The columns are located basal to the thin layer of the supporting cells, and contain bipolar neurons and undifferentiated cells. Columns extend through most of the height of the entire vomeronasal sensory epithelium. Such columnar compartments are separated by the invaginated basal lamina and connective tissue containing the capillary network. Similar columnar organization may be present in non-ophidian squamates, but they are not such well developed as in snakes [21]. For instance, Kratzing [14] stated that columns of connective tissue with a capillary network intrude the sensory epithelium of the VNO in Tiliqua, while in Takydromus only small, sparsely distributed blood vessels were found in vomeronasal sensory epithelium [105]. The penetration of vomeronasal sensory epithelium by connective tissue, without formation of columns, was found in some mammals: rodents and rabbit [97].
The columns of neurons have never been described for adult anoles. Neither columnar organization of VNO sensory epithelium nor blood vessels within bipolar and undifferentiated cells were detected in the present study.
The only indication of intrusion of connective tissue into the vomeronasal sensory epithelium might be the occurrence of the pigment granules within the layer of supporting cells, especially frequent in late stages of the brown anole (17)(18)(19), but such explanation seems to be unlikely. At stages 18 and 19 of the brown anole, fewer pigment granules occurred also within the basal layer containing bipolar neurons. The presence of connective tissue with associated pigment cells at the base of the sensory epithelium as well as between epithelial columns of bipolar neurons was found in newly hatched and adult Thamnophis [43]. The presence of pigment cells in the connective tissue around the base of the VNO was noted previously in anoles, as in other squamates [2,21,97]. Nevertheless, the lack of even sparsely distributed blood vessels within the bipolar cell layer in the VNO of the brown anoles suggests that the intrusion of connective tissue into the vomeronasal sensory epithelium does not occur. Moreover, the association of such pigmentation with the supporting cell layer suggest that pigment granules observed here are homologous to the olfactory pigment of supporting cells, which has been found in some vertebrates, but usually reported for mammals and birds [106,107]. It has been suggested that the carotenoid component of such pigment may be involved in olfactory function in terrestrial vertebrates, in which olfactory pigment is present also in cells of Bowman's glands [107]. Interestingly, we did not observe similar pigmentation in the sensory olfactory epithelium of the brown anole and the pigment granules have not been noted in vomeronasal sensory epithelium in other vertebrates [107].
The mushroom body in squamates [14,15,103,104], the oor of the VNO in Sphenodon [16], and the lateral or dorsolateral wall of the organ in mammals [100,108,109] are covered with ciliated non-sensory epithelium. In general the luminal surface of mature vomeronasal sensory epithelium in tetrapods is covered with microvilli of dendrites and protrusions of supporting cells, and there are no cilia characteristic for the dendritic surface of olfactory sensory epithelium. We found that in the brown anole at late developmental stages (18 and 19) the apical surface of the vomeronasal sensory epithelium is covered with microvilli resembling the cilia (Fig. 14C''). Similar images from light microscopy have been obtained for Takydromus (see Fig. 2 in Saito et al. [105]). Our preliminary studies based on transmission electron microscopy revealed that the apical surface of the vomeronasal sensory epithelium in late stages of the brown anole is covered by long and thin microvilli and only sparsely distributed cilia can be observed (unpublished observations).

Lacrimal duct
Most tetrapods, except some plethodontid salamanders [110] and turtles [6], possess a lacrimal duct. In mammals, birds, crocodilians, Sphenodon and most amphibians, it extends from the lower eyelid and discharges into the nasal cavity near the external naris or more caudally, closer to the entrance to the VNO [20,[111][112][113][114][115]. In squamates the lacrimal duct is usually directly associated with the VNO [40]. Moreover, in most non-ophidian squamates it is connected with the choanal groove and, in some cases, also with the nasal cavity at the level of the outer choana [6,21,22]. Close association of the VNO and the lacrimal duct was also found in the Gymnophiona [20,116]. It is widely accepted that the lacrimal duct is involved in direct or indirect delivery of secretions of the orbital gland, including the Harderian gland secretion, into the VNO lumen [e.g. 24,42,114], except for crocodilians and birds, in which the VNO is transitory embryonic feature. It means that the secretion of the Harderian gland is involved in vomeronasal chemoreception, rather than eye lubrication [23,24,42,117]. In fact the ow of the Harderian secretion gland or orbital uid to the VNO was experimentally tested in snakes [118] and frogs [111,115]. The other studies on snakes found that female pheromones in Thamnophis are soluble in the Harderian gland homogenate [9].
It seems that the origin of the lacrimal duct from the nasolacrimal groove ( = naso-optic furrow), located between the maxillary prominence and the lateral nasal prominence, is evolutionarily conserved in tetrapods [6,89,94,119]. In the brown anole, the primordium of the lacrimal duct was rst present at stage 8. Interestingly, we found that only a small anteriormost part of the duct seems to originate from the nasolacrimal groove. The major part of the duct in the brown anole develops from the groove located between the primordial lower eyelid and the maxillary prominence (Figs. 8A, A' and Movie S1). Such a peculiar condition could be explained by the fact that the eyes in this species seem to be very large in relation to the snout. In consequence, the developing eye may cover the posterior extent of the lateral nasal prominence and reduces the length of the nasolacrimal groove. This hypothesis needs to be con rmed by comparative morphometric studies, but the occurrence of the huge eyes of the brown anole in relation to the other squamates seems to be very clear (compare Fig. 1, stage 8 in [53], Fig. 4, stage 32 in [120], Fig. 2h in [121], and Fig. 2, stage 4 in [122].
The orbital end of the lacrimal duct of tetrapods usually forms two canaliculi or sometimes one [23,113,123]. Unfortunately, many studies concerning development of the nasolacrimal apparatus are based on relatively late developmental stages, when the lacrimal duct is well developed [e.g. [112][113][114].
Thus, still little is known about development of the lacrimal canaliculi. Here we show that the lacrimal canaliculi of the brown anole become distinguishable just after the formation of single lacrimal duct primordium.
The lacrimal duct of Sphenodon discharges exclusively into the nasal cavity [20,124]. In contrast to that condition, the lacrimal duct of most adult non-ophidian squamates establishes a relatively extensive connection with the choanal groove [6,34,125]. Here we show the steps of formation of such extensive connection in the brown anole, which is established by the lacrimal duct through its rostral plate (see Figs. 13A-D). In gekkotans, except pygopodids, the connection of the lacrimal duct and the choanal groove seems to be restricted to the anterior part of the latter [21,34]. The rostral end of the squamate lacrimal duct is usually connected to the VNO duct or enters the VNO lumen directly in some forms [6]. The lacrimal duct of monitor lizards, pygopodids, some amphisbaenids, and at least one dibamid, reaches the VNO duct directly, and there is no communication with the choanal groove [6]. The same condition occurs in adult snakes in which the choanal groove is absent [6,19,118]. Moreover, in pygopodids, amphisbaenids, and snakes, the lacrimal duct establishes a more intimate connection with the duct of the Harderian gland via lacrimal canaliculi or the Harderian gland discharges directly into the lacrimal duct [23,42].
In the latest developmental stages of the brown anole (18 and 19), the lacrimal duct is not connected to the outer choanal tube, but the posterior expansion of the rostral plate of the lacrimal duct runs backward toward the outer choana (Fig. 13D). This expansion is lined with the epithelium characteristic for the choanal groove (with the presence of cilia and goblet cells) (Fig. 12B); thus it could be considered as a part of the choanal groove rather than the lacrimal duct. In fact Bellairs & Boyd [6] suggested that "transition from strati ed to ciliated epithelium" may approximate the border between the fused lacrimal duct and the choanal groove. However "ciliated respiratory type" epithelium was found in the anterior part of the shorter lacrimal duct in monitor lizards [22], goblet cells in the epithelial lining of the saccus lacrymalis in the lacrimal duct are present in crocodilians [112], and the goblet cells in addition to cilia have been found in the human lacrimal duct [123]. Moreover, the changes in anatomy of the lacrimal duct observed in successive stages at late developmental phase suggest that the posterior part of the rostral plate of the lacrimal duct gradually grows posteriorly (see Figs. 13A-D) and thus the region under consideration does not represent the part of the choanal groove. If our interpretation is correct, then it could be assumed that the posterior expansion of the rostral plate of the lacrimal duct (white arrows in Fig. 13D) of the brown anole is involved in a different function than the rest of the lacrimal duct, and constitutes a functional extension of the choanal groove.
Importance of the olfactory system and functional remarks Pratt [16] suggested that reduction or loss of functionality of olfactory systems in such forms as Anolis, arboreal agamids and chameleons corresponds to the "massive orbital development". The sensory part of the nasal cavity was described as "almost non-sensory", while the VNO was described as "reduced and completely non-sensory". The Dactyloidae originates from an arboreal ancestor [32] and most extant species contain toe pads [126] which serve an adhesive function [127]. However, many extant forms are not strictly associated with a fully arboreal lifestyle [29][30][31][32]. The brown anole occupies a "trunk-ground" niche [29,32]. Pratt's [16] notion about the non-sensory character of the anole VNO was based on the anole described there as Anolis alligator (which probably is now A. roquet [95] and represents a trunkcrown ecomorph [128,129]). In fact, behavioral evidences, including that involving tongue extrusion, indicates that the accessory olfactory system may still be important in males of the brown anole for detection of female pheromones [130]. Increases in the tongue ick rates were found in A. carolinensis during male-male encounters and in individuals which were transferred to novel habitats and exposed to foliage or air movements [131]. The tongue extrusions were also observed in the eld for A. trinitatis [132]. The interpretation of such behavior as evidence for the importance of the accessory olfactory system may be problematic. The role of tongue extrusion in anoles, especially substrate licking, may mediate gustation rather than vomeronasal function, since taste buds of the tongue tip are found to be generally abundant in Iguanidae ("present" in A. carolinensis and "abundant" in A. bonairensis) [133]. However, some ndings suggest that lingual gustation in squamates cannot replace vomerolfaction and thus be responsible for chemical discrimination of prey or conspeci cs [12].
Morphological studies also showed that the VNO is functional at least in A. garmani, A. grahami and A. lineatopus [2], which are classi ed as crown-giant, trunk-crown and trunk-ground ecomorphs respectively [32,128,129]. Some features of anole adult morphology may constitute adaptations to vomeronasal chemoreception. For instance, the closure of the choanal groove may e ciently deliver the secretion of the Harderian gland to the VNO duct and its vicinity. A similar condition is present in snakes in which the choanal groove is absent, but the anterior end of the lacrimal duct is exclusively connected with the VNO duct [6]. The presence of a functional VNO in anoles representing different ecomorphs (including crowngiants and trunk-crowns) may suggest that Pratt's [16] interpretation for A. roquet was not correct and the arboreal lifestyle in anoles may be associated with the reduction in size of the sensory epithelium of the VNO (and accessory olfactory bulb) rather than with complete loss of its chemosensory function.
Moreover, the generalization of Pratt [16] is falsi ed by the fact that well-developed olfactory systems in arboreal geckoes are present [36,134].
It was suggested that reduction of olfactory organs in such representatives of Iguania as chameleons and agamas (and probably Anolis) is secondary, and the condition of Iguanidae, which are characterized by relatively well-developed chemosensory abilities, is ancestral [36]. In fact, our studies may indirectly con rm this notion at least for Pleurodonta, since the morphology of the adult brown anole probably does not represent the ancestral condition. The loss/signi cant reduction of the concha, characteristic for Anolis and the "Uma-morphotype", but not for "Dipsosaurus-morphotype" including Iguanidae, seems to be secondary, since at the beginning of the middle developmental phase the concha and extraconchal space were easily distinguishable in the brown anole. Nevertheless, members of the Iguania in contrast to the rest of the squamates (Scleroglossa) are characterized by a less developed VNO [36], lower tongueick rate [135] and lower abundance of vomeronasal and olfactory receptor cells [135]. The weakly or moderately developed chemosensory abilities in Iguania are di cult to explain, since the phylogenetic position of this group is still a matter of debate [49]. Such a Sphenodon-like condition may be considered either as plesiomorphy according to morphological studies placing the Iguania as sister to the other squamates [e.g. 83], or reversal according to molecular phylogeny, which suggests the nested position of Iguania [e.g. 28,82].

Conclusions
The present study provides the rst detailed description of the development of the naso-palatal complex

Consent for publication
Not applicable.
Availability of data and material The image data used in this study are part of an ongoing research project and therefore have not yet been made pubic but are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The lead author was supported by a scholarship from the National Science Centre (NCN), Poland (2018/28/T/NZ4/00182). This project was also partially performed in the NanoFun laboratories conanced by the European Regional Development Fund within the Innovation Economy Operational Programme POIG.02.02.00-00-025/09.

Authors' contributions
PK conducted most of the laboratory work and segmented analysed structures (XRM). KJ and BM performed XRM scanning. PK, WR and BM analyzed the data and wrote the paper. All authors read and approved the nal manuscript.   Supplementary Information indicate the concavities in rendered structures. Colours of 3D structures as in Fig. 9. Scale bars 250 μm.