The present study provides the first 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 soft 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 ignored, 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 other part of the choanal tube and the maxillary fold. The latter 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 prominence [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. 3F, 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 starts to participate in the formation of the nasal pit at this time (Fig. 3C). The developmental sequence of analyzed structures in the brown anole is 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 frontonasal mass at stage 7 of the brown anole. This structure is characteristic for the other squamates, but it is present also in embryos of turtles and birds [17, 62–64]. The fusions of the frontonasal mass (medial nasal prominence) and the lateral nasal prominence result in formation of the vestibulum of the nasal cavity and separation of the external naris from the primitive outer choana [16, 17, 19]. In contrast, we found that the maxillary prominences, together with the lateral nasal prominence, participate in this process in the brown anole. In this case, separation of the external naris and the primitive outer choana occurs at stage 8 (Additional file 1). The nasal plug is well formed at this time. It fills the space between the lateral nasal prominence and the frontonasal mass and causes the “temporal closure” of the external naris (and thus the vestibulum). Then, the “reopening” of the external naris and the vestibulum may be easily explained by the elimination of the nasal plug, which starts at stage 18 of the brown anole and it seems to be a common phenomenon in tetrapods, since the nasal plug was also found in mice and humans [65–67].
However, there are some other interpretations of this developmental event. Abramyan et al. [63] reported that in some lizards (bearded dragon, veiled chameleon, and whiptail lizard), unlike other amniotes such as crocodilians, chick, and mouse, the external naris becomes closed by the fusion of the frontonasal mass and the lateral nasal prominence and reopens in later development. However, in the light of our histological and mCT studies and previous findings [65–67], it seems that the fusion described by Abramyan et al. [63], at least in most of its extent, does not represent “classical” fusion, which involves the formation of the bilayered epithelial seam (see [64]). Thus, the closure of the external naris may be explained by the formation of the nasal plug anteriorly to the “classical” fusion of the lateral nasal prominence with the frontonasal mass.
In the brown anole the vestibulum takes the relatively simple form of a long tube, oriented almost horizontally at late stages (17–19; Fig. 11A and Fig. 12A). It was found that the vestibulum in some anoles is relatively short [16, 21], which indicates that 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, but it may be well developed and take an almost vertical orientation as in Phrynosoma [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 [16, 33]. The function of this part of the nasal cavity may be improved by the presence of a network of sinusoids associated with the reticulum of smooth muscle fibers (spongy sinusoidal tissue, cavernous tissue) surrounding the vestibulum and probably by closing it during intumescence [21, 22, 33].
Formation of the choanal groove and choanal fold
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 early 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 first one 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 [68]). With the formation of the upper lip, the lateral nasal prominence becomes hardly distinguishable from the rest of the snout. This condition may explain previous misinterpretation about the participation of the maxillary prominence in formation of the choanal groove. In fact, from stage 9, the border between the lateral nasal prominence and the maxillary prominence may be evaluated on the basis of the location of the nasolacrimal duct (externally) and the extent of the lateral nasal concha/subconchal fold (in sections) (see Fig. 4H and Figs. 6K and L).
Despite the absence of the choanal groove – in most snakes [6, 34, 38] 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 squamates, in which the choanal groove does not reach the VNO duct, 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]. Unexpectedly, it was found that the choanal groove in the grass snake undergoes obliteration from posterior to anterior direction [19]. This might be due to the peculiar morphology of the posterior part of the palate, and the formation of the snake-like nasopharyngeal duct. In Gekkota and Iguania, this structure is confluent with the duct of the VNO. We found that in 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. 12A and Figs. 13A and 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 tip of the choanal fold with the vomerine cushion, and it forms a patent tube of the choanal groove (Figs. 13A’ and B). A similar condition was found in anoles by Bellairs & Boyd [6], but according to their description, such closure was restricted to the anterior part of the choanal groove: “a little behind the level of the organ of Jacobson”.
The presence of the choanal groove was considered to be a synapomorphy of squamates [38]. In fact, in Sphenodon, the only extant member of Rhynchocephalia, commonly considered to be a sister group to Squamata [28, 69–71], 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 some representatives of Iguania [21, 33].
The term choanal groove is often used to describe the groove on the palatine bone in the region of the choana [36, 72]. Here we use this term exclusively for a soft tissue structure [5, 16, 21] as was define above. The floor of the choanal groove is formed by the choanal fold, which is supported by the ectochoanal cartilage [6]. In crocodilians and mammals the separation of the main nasal cavity from the embryonic oral cavity and the formation of the secondary palate occurs through the midline fusion of the palatal shelves [63, 73]. It was suggested that the choanal folds (sometimes called the palatine processes) are homologous to palatal shelves [62, 64, 74]. However, because the other outgrowth of the maxillary prominence (= mediale Seitenfalten [5]; called here the maxillary fold) of squamates is usually ignored in current literature, we do not apply this terminology for the choanal fold until the homology is established between all of these structures.
Lateral nasal concha and main nasal cavity
The nasal concha constitutes any projection into the main nasal cavity of amniotes, but definition 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 [75]. 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, here called the inner choanal tube. Previous studies indicated that the lateral nasal concha is absent in anoles [2, 16, 21, 33]. However, we found that even in the latest developmental stages (18–19) of the brown anole the lateral nasal concha is still noticeable in the anterior part of the nasal cavity. It is present in the region of the lateral nasal gland, but it is almost obliterated (Fig. 12D). It is likely that it undergoes further reduction in later ontogenesis. The concha in the brown anole appears before the separation of the external naris and outer choana at the end of the early developmental phase, when it emerges from the lateral nasal prominence (Fig. 3E). The same developmental sequence was described in Thamnophis [76]. At stages 7 and 8 of the brown anole, the lateral nasal concha as well 3 subdivisions of the conchal zone, including the extraconchal zone, are very well defined (Fig. 5F and Fig. 6D). We found that reduction of the concha is associated with the shift of the extraconchal zone to the posteriormost part of the main nasal cavity and change of its orientation. In fact, in the brown anole at stage 12, the extraconchal zone is restricted to the posterior end of the nasal cavity, where it forms a vertical “wing” (Fig. 9F). In later development, the extraconchal zone becomes dilated and incorporated into the Stammteil (Fig. 11A and Fig. 12A). For this reason the posterior part of the lateral nasal concha is completely obliterated at stage 17.
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 classified as “Uma-type” containing: Uma, Callisaurus, Holbrookia, Sceloporus, Uta, and Phrynosoma [33], which all belong to the Phrynosomatidae [77]. According to 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 [78]). Still, little is known about the state of this character in other groups of Pleurodonta. In “Dipsosaurus-type” 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].
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 non-ophidian 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 latter runs ventromedially and opens to the oral cavity by the outer choana [6, 22, 61]. Here we employed division of the choanal tube into the inner and outer choanal tube (Fig. 1C), 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 define the structure connecting the primordial Stammteil to the oral cavity from about stage 5 of the brown anole (Figs. 3E–H). In later development of this species this structure gives rise to the vestibulum (at the stage 8) (e.g. Figure 6B), the primitive outer choanal tube, and the primitive inner choanal tube (the last two were relatively well distinguishable from stage 9, when the subconchal fold becomes well developed) (e.g. Figure 6H). The primitive outer choanal tube gives rise to the primordial VNO duct (stage 9) (Figs. 6H and K), choanal groove and the outer choanal tube (both from stage 9/10) (Figs. 7C and D). 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 (Figs. 7C and D). Because the structure defined 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, 75]. 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 medial elongation of the vomerine cushion [21, 33, 37, 38, 63]. 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, 37, 38, 64]. 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 be the nasopharyngeal duct.
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, 76]. 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 the brown anole, the vomeronasal pit was first 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 [79–81]. 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, 79]. A non-sensory vestige of the VNO is present in humans and chimpanzees [82], but in some bats the VNO may be rudimentary or even absent [83]. 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 [76, 84]. 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 primordial 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 non-sensory epithelium covers the mushroom body [2, 14, 85]. 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, 37, 76]. It was suggested that the anole VNO is: “present but less well-differentiated than in most lizards” [2]. We noted that in late stages of the brown anole (18–19) embryos the VNO is characterized by medial localization of the dorsal dome in relation to its duct, which is associated with the anterior tip of the lacrimal duct (Fig. 15F). Moreover, the VNO possesses a weakly developed ventral channel, poorly developed mushroom body and concave dorsal and dorsomedial wall.
Results of histological study indicated that the vomeronasal sensory epithelium is ciliated and has well-visible pigment aggregated within its central layer (Figs. 15E and F). 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. Figure 15D). The ultrastructural studies indicated that this epithelium contains different populations of cells [15, 85, 86]. 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, 85, 86]. The columns are located basally 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 [87]. The penetration of vomeronasal sensory epithelium by connective tissue, without formation of columns, was found in some mammals: rodents and rabbit [79]. 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 (17–19) (Figs. 15E and F), but such explanation seems to be unlikely (see latter). 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 [41]. Similar cells were found in the subepithelial tissue of the brown anole (see Fig. 15F). 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, 79]. 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 [88, 89]. 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 [89] Interestingly, we did not observe similar pigmentation in the sensory olfactory epithelium of the brown anole (see Fig. 11D and Figs. 12D and E) and the pigment granules have not been noted in vomeronasal sensory epithelium in other vertebrates [89].
The mushroom body in squamates [14, 15, 85, 86], the floor of the VNO in Sphenodon [16], and the lateral or dorsolateral wall of the organ in mammals [82, 90, 91] 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. However, 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 cilia (Fig. 15F’’). The presence of the cilia on the apical surface of the vomeronasal sensory epithelium was reported in some papers in the nineteenth century, but such findings were later denied by studies concerning mammals and reptiles (see [21]). Recently their presence was noted in embryonic VNO of Iguana (Squamata: Iguania) in a study using light microscopy [46], but similarly as in the present investigation on the brown anole it is impossible to evaluate from which type of cell of the vomeronasal sensory epithelium they emerge. Interestingly, there is no information about the cilia on the sensory part of the VNO epithelium in adult anoles in other studies using light microscopy (e.g. [2]). The presence of ciliated dendrites in the mammalian VNO was found in some ultrastructural studies in dog [92], bat [93], and rabbit [94]. The presence of cilia on the luminal surface of the vomeronasal sensory epithelium emerging from supporting cells was confirmed by ultrastructural study in bat [93] and newborn/suckling rats [90, 95]. Moreover, ciliated cells as a separate type of cells in vomeronasal sensory epithelium were found in anurans and salamanders [96]. Ciliated epithelium is also present in the VNO vestige of humans and chimpanzees [82]. The centrioles and potential precursors of cilia have been found in many ultrastructural studies in squamates and mammals [87, 92, 93, 97]. Thus it was suggested that the processes of cilia formation may be blocked in the neurons of vomeronasal sensory epithelium [97]. Moreover, it seems that cilia are not necessary for vomerolfaction and may be lost in adult ontogenesis. Thus the presence of temporary cilia in the sensory epithelium of the VNO in embryos or newly born/hatched animals may mark newly formed cells [93]. The presence of cilia in the adult and embryonic vomeronasal sensory epithelium and identification of their origin require future investigations using ultrastructural or immunohistological studies.
Lacrimal duct
Most tetrapods, except some plethodontid salamanders [98] 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, 99–103]. In squamates the lacrimal duct is usually directly associated with the VNO. Moreover, in most non-ophidian squamates it is connected with the choanal groove and, in some cases, with the nasal cavity (see later). Close association of the VNO and the lacrimal duct was also found in the Gymnophiona [20, 104]. 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, 40, 102]), except for crocodilians and birds, in which the VNO is absent. It means that the secretion of the Harderian gland is involved in vomeronasal chemoreception, rather than eye lubrication [23, 24, 40, 105]. In fact the flow of the Harderian secretion gland or orbital fluid to the VNO was experimentally tested in snakes [106], frogs [99, 103] and Gymnophiona [104]. 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, 75, 107, 108]. In the brown anole, the primodium of the lacrimal duct was first 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 Additional file 1). 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 covers the posterior extent of the lateral nasal prominence and reduces the length of the nasolacrimal groove. This hypothesis needs to be confirmed 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 [51], Fig. 4, stage 32 in [109], Fig. 2h in [110], and Fig. 2, stage 4 in [111].
The orbital end of the lacrimal duct of tetrapods usually forms two canaliculi or sometimes one [23, 101, 112]. 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. [100–102]). Thus, still little is known about development of the lacrimal canaliculi. In this study, these structures were first distinguishable at stage 9 of the brown anole, just after the formation of the lacrimal duct primordium. Two canaliculi developed as a two branches emerging from the lacrimal duct, and growing toward the inner surface of the lower eyelid (Fig. 8C). At stage 9 the lacrimal duct closely approaches the choanal diverticulum of the primitive outer choanal tube. However, the fusion between these structures does not occur until the beginning of the late developmental phase of the naso-palatal complex (stages 11–12), when the latter forms a relatively well-developed choanal groove (Figs. 9B and E). The lacrimal duct of Sphenodon discharges exclusively into the nasal cavity [20]. In contrast to that condition, the lacrimal duct of most non-ophidian squamates establishes a relatively extensive connection with the choanal groove. Anteriorly, it is more or less associated with the VNO duct or enters the VNO lumen directly in some forms [6]. 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]. 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, 106]. 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, 40].
Despite the fact that Bellairs & Boyd [23] provided the most comprehensive description concerning the relationships of the choanal groove and the lacrimal duct, they did not make a clear distinction between the choanal groove and the lateral choanal fissure, which is here considered to be a part of the outer choanal tube. Nevertheless, according to their drawing, it can be stated that the lacrimal duct connects the lateral choanal fissure in Anguis fragilis (anguid lizard), Chamaeleo bitaeniatus (now Trioceros bitaeniatus [77]), Tupinambis teguixin (teiids) and Mabuya megalura (representative of skinks; now Trachylepis megalura [77]). 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 part of the lacrimal duct extension runs backward toward the outer choana (Fig. 14D). This extension is lined with the epithelium characteristic for the choanal groove (with the presence of cilia and goblet cells) (Fig. 13B); thus it could be considered as a part of this structure. However, the progress of development suggests that the posterior part of the lacrimal duct extension gradually grows posteriorly starting from the beginning of the late developmental phase (see Figs. 14A–D). Moreover, despite the fact that Bellairs & Boyd [6] suggested that “transition from stratified to ciliated epithelium” may approximate the border between the fused lacrimal duct and choanal groove and the fact that the squamate lacrimal duct is usually considered as a structure delivering the secretion of the Harderian gland to the VNO, rather than a structure involved in secretion by itself [13, 23, 40], some exceptions in tetrapods can be noted. “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 [100]. Moreover, goblet cells in addition to cilia were also found in the human lacrimal duct [112]. If our interpretation is correct, then it could be assumed that the posterior part of the lacrimal duct extension (white arrows in Fig. 14D) 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.
According to data obtained in this study, it can be hypothesized that the connection of the lacrimal duct and the outer choanal tube in the taxa listed above is secondary and that it is caused by the posterior elongation of the lacrimal duct extension. It cannot be excluded that such secondary connection of the lacrimal duct and the outer choanal tube in the brown anole may be achieved in further ontogenesis. Interestingly, in monitor lizards two lacrimal ducts are present on each side of the snout [22]. In this peculiar condition the longer one connects to the VNO duct, as mentioned above, while the shorter one discharges into the inner choanal tube (defined there as the “ventral conchal zone”) “above the inner choana” [22]. It could be assumed that the condition represented by the shorter lacrimal duct of monitor lizards may represent the “primary” connection of this structure with the nasal cavity. Thus, it seems that such a primary connection of the lacrimal duct and the nasal cavity does not occur in the other squamates, because the anterior part of the outer choanal tube in this group develops into the choanal groove, which is usually not considered to be a part of the adult nasal cavity.
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 [113] which serve an adhesive function [114]. However, many extant forms are not strictly associated with a fully arboreal lifestyle [29–32]. 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 [77] and represents a trunk-crown ecomorph [115, 116]. In fact, behavioral evidence, 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 [117]. Increases in the tongue flick 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 [118]. The tongue extrusions were also observed in the field for A. trinitatis [119]. 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) [120]. However, some findings suggest that lingual gustation in squamates cannot replace vomerolfaction and thus be responsible for chemical discrimination of prey or conspecifics [12].
Morphological studies also showed that the VNO is functional at least in A. garmani, A. grahami and A. lineatopus [2], which are classified as crown-giant, trunk-crown and trunk-ground ecomorphs respectively [32, 115, 116]. Some features of anole adult-like morphology may constitute adaptations to vomeronasal chemoreception. For instance, the closure of the choanal groove may efficiently 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 crown-giants 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 falsified by the fact that well-developed olfactory systems in arboreal geckoes are present [34, 121].
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 welldeveloped chemosensory abilities, is ancestral [34]. In fact, our studies may indirectly confirm this notion at least for Pleurodonta, since the morphology of the adult brown anole probably does not represent the ancestral condition. The loss/significant reduction of the concha, characteristic for Anolis and the “Uma-type”, but not for “Dipsosaurus-type” including Iguanidae, is secondary, since at the beginning of the middle developmental phase the concha and extraconchal space were easily distinguishable. Nevertheless, members of the Iguania in contrast to the rest of the squamates (Scleroglossa) are characterized by a less developed VNO [34], lower tongue-flick rate [122] and lower abundance of vomeronasal and olfactory receptor cells [122]. The weakly or moderately developed chemosensory abilities in Iguania are difficult to explain, since the phylogenetic position of this group is still a matter of debate [47]. 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. [70]), or reversal according to molecular phylogeny, which suggests the nested position of Iguania (e.g. [28, 69]).