Reptile venom evolution in general is an area of controversy and lizard venom is particularly contentious. This is despite the growing pile of robust genetic evidence and integrated morphological/molecular analyses, in which a well-supported clade is consistently recovered containing Anguimorpha, Iguania and Serpentes. This clade was given the name  by us of “Toxicofera” to reflect the shared presence of venom systems (however rudimentary or incipient) within the group. Toxinological studies have further corroborated close evolutionary relationships between snakes and anguimorph lizards, showing that Anguimorpha lizard venom glands express proteins homologous to toxins found in the venom of front-fanged snakes, including those of the iconic komodo dragon as well as other anguimorph lizards such heladermatid lizards (beaded lizards and gila monsters) and anguid lizards.

Our original publication concerning this “early origin” hypothesis prefers the term “venom system” over “venom”—thus the original “Toxicofera hypothesis (of venom evolution) concerns the single early origin of the venom system, not “venom” per se. In that all lineages possessed uniquely derived mandibular and maxillary glands distinguished by having segregated protein and mucus secreting regions, with the enlargement of the protein-secreting region relative to that of the mucus-secreting region.

Some of the controversy surrounding the “Toxicofera hypothesis” (sensu HEA) of venom evolution has been blamed on inconsistencies in definitions of the word “venom” and the designation of certain species as “venomous”. In reality, the various definitions of “venom” differ little—the consensus is that venom is an actively delivered (e.g., via a bite or a string) secretion that functions (i.e., has been selected for the purpose of) in the subjugation of prey or the deterrence of predators/competitors.

Confusion has thus been fueled less by differing definitions of “venom”, than by different applications of the term, and particularly its conflation with the term “venom system”. It must be stressed that taxa possessing a “venom system” are not necessarily “venomous”—in particular we have frequently referred to the venom system of iguanian lizards as “incipient” (e.g.,see here and here for a discussion of the use of the term incipient in evolutionary contexts). So, the “Toxicofera hypothesis of venom evolution”, more properly considered, concerns the early evolution of the venom system—i.e., the synapomorphy of the Toxicofera clade, which (as above) is the presence of protein-secreting oral glands (i.e., an incipient venom system) which may be considered exapted for the subsequent development of sophisticated venom delivery systems.

In making a selectable contribution to the subjugation of prey, it is not necessary for a venom to be capable of rapidly killing or incapacitating the prey item. Venoms do not necessarily function as stand-alone prey subjugation mechanisms—they may be used in concert with physical means of subjugation. Thus, a venom system need only marginally increase a predator’s chances of successfully securing a meal, e.g., by slightly weakening a potential prey animal, making it easier to subdue physically [38].

Criticisms of (some variants of) the Toxicofera hypothesis of venom evolution notwithstanding, it is clear that a core set of toxins are present in the venoms of all anguimorph lizards studied to date including CRiSP, kallikrein, B-type natriuretic peptides and type III phospholipase A2 (PLA2). Some of the previously listed toxins are responsible for the hypotensive effect of intravenous injection of crude venom in rats, such as aortic smooth muscle relaxation by natriuretic peptides equipotent to forms recovered from venomous snakes . In addition, kinin release from kininogen by kallikrein enzymes is another source of hypotensive effects resulting from lizard venoms. Further, it has also been previously noted that injection of rodents and birds with V. griseus venoms resulted in paralysis.

Reports of human envenomation by monitor lizards have been noted both in the literature and anecdotally. A bite report documented effects for a V. griseus bite to a zoo keeper that included symptoms similar to that of the bird and rodent studies: dizziness, muscular weakness and soreness in the extremities, facial and eye muscle pain, respiratory distress and pain (local and systemic). Additional reports of V. griseus bites to zoo keepers recorded similar symptoms including dysphagia, chest tightness, muscle soreness in the extremities, facial pain, dizziness, and difficulty walking. In all the above V. griseus cases, the bite victims were experienced zoo keepers who did not view the bites with concern upon their occurrence. Vikrant and Verma (2014) reported a lethal bite by the related V. bengalensis that induced local pain, blood loss, as well as nausea, diaphoresis, dizziness, and breathlessness in the victim and eventually led to an acute kidney injury and cardiac arrest. Thus, while this case is extraordinary, the possibility of dangerous bites from varanid lizards to humans, under exceptional circumstances, should not be discounted. Another case report described a bite by a juvenile V. komodoensis that led to faintness, prolonged bleeding and transient hypotension.

Anecdotally, a great many varanid lizard bites to biologists, zookeepers and amateur reptile enthusiasts have resulted in little that could be attributed to the action of toxins; however, some bite victims do report burning sensations, prolonged bleeding and inflammation disproportionate to the mechanical damage inflicted. In addition to published cases, we have been contacted by keepers of varanid species who had symptomatic cases, sometimes with taxon-specific effects: 3 cases of V. albigularis reporting extreme muscle soreness and weakness lasting days, 4 cases of V. kordensis producing significant local swelling of the bitten finger and adjacent fingers, 5 cases of V. varius with pronounced bleeding lasting 3–4 h, a V. salvadorii bite with similar symptoms to that of V. varius and a V. komodoensis bite also producing apparent anticoagulant effects. These effects are consistent with the characterised venom chemistry of anguimorph lizards by us.

When compared with helodermatid lizard bites, which have received far more research attention than those of their varanoid cousins, a key point becomes evident: duration of contact. Typically, a helodermatid lizard will stay attached, chewing more venom into the bite site, while a monitor lizard is less likely to hold onto something that is not a food item but in some cases may hold on for up to a half hour when defensively biting. This likely leads to differences in the amount of oral fluids inoculated to the victim, with symptomatic human bites typically being those in which the varanid lizard chewed for a prolonged period of time. Interestingly, while feeding on large prey items, varanid lizards seem to have a tendency to shake it violently and chew vigorously until it is subdued, which may facilitate venom delivery as well as potentiating mechanical damage.

Varanoid lizards are characterised by a refined mandibular venom gland that is homologous with that of the helodermatid lizards. Most anguimorph lizards have simple-structured mandibular venom glands, however, Heloderma and Lanthanothus/Varanus have independently evolved complex glands. Both lineages derived their compartmentalised venom glands from the ancestral anguimorph lizard condition of an enlarged, mixed sero-mucous gland in which the protein- and mucous-secreting regions are not segregated into distinct glandular structures. In both cases, sophisticated structures with separated protein and mucous regions, a structured lumen for storing liquid venom, and a thick membranous cover, have evolved. Further to this, morphological as well as molecular evolutionary studies have indicated that these glands are homologous with the venom glands of snakes. The fact that varanid lizards possess highly developed dental glands suggests that those glands in one way or another play an important role in their life. By way of comparison, it has been shown by us that with venomous snakes which switch to eggs or constriction as an alternate form of prey capture rapidly lose the functionality of their venom glands, with atrophying occurring in short periods of (evolutionary) time [25,29,52,53].

The evolution of a complex venom system is likely only possible under certain contingent circumstances—i.e., when both environmental conditions select for it and a species’ overall evolutionary trajectory facilitates it. For example, in Iguania the incipient venom glands never developed any significant complexity, probably due to the mainly insectivorous/herbivorous nature of these lizards. In addition, when a species evolves an alternative method of subduing prey that renders the venom system redundant, or switches to a diet with no need for subjugation (e.g., plants), the venom system often degrades. The cost of venom production is presumably high enough to justify the presence of active secretory and delivery apparatuses only when it confers an evolutionary advantage. It is notable, however, that in iguanian species which include a large quantity of vertebrates in their diet, the glands are larger and the protein-secreting region more developed (though we stress that this is not equivalent to them being ‘venomous’ per se but it is strongly suggestive of a functional role in predation).

Work on helodermatid lizards by us has demonstrated a striking level of proteomic conservation within the venoms of this clade, with the same basic toxin groups present in similar quantities despite the most recent common ancestor (MRCA) of the five Heloderma species existing ~15–20 million years ago. In contrast, we have shown that the venoms of varanid lizards are much more diverse, which is strongly suggestive of accelerated evolution under selection pressure.

The venoms of varanoid lizards remain understudied in evolutionary toxinology; however, multiple sources of evidence point to the adaptive evolution of venom in varanoid lizards. We have studied the differential complexity of anguimorph lizard venoms across a wide taxonomical range and considered its evolutionary context as part of a combined predatory arsenal. According to our results, varanoid lizard venom is largely based on kallikrein toxins that previous studies have shown to be homologous with those present in the venom of advanced snakes. Additional components are present in various species with profile complexity seemingly being a function of size and habitat where larger monitors possess the most complex venom and smaller or aquatic species the least. The high level of variability of varanid venoms relative to the high levels of conservation in helodermatid lizards, points to active evolution under selection pressure.

Kallikrein enzymes have undergone significant structural and functional diversification within the clade. There was evidence of gene duplication and diversification of the molecular surface biochemistry which is paralleled by the diverse activities of the kallikrein enzymes in varanid venoms. However, the ancestral and still dominant action is cleavage of the alpha and beta chains of fibrinogen in a destructive anticoagulant manner that did not form clots. Predators likely benefit from inducing blood loss or altering the blood pressure of their prey, as this will increase the chance of successful subjugation by weakening the prey. Previous work has shown that the Type III PLA2 in V. varius venom block platelet aggregation, thus interfering with blood clotting via the same pathway as do homologous toxins in the venom of Heloderma species. In the case of some monitor lizards, in particular the larger species such as V. varius or V. komodoensis, this type of toxic action might be beneficial even if a prey manages to escape the initial attack but succumbs to blood loss or shock in the aftermath. Field studies on V. komodoensis by us and other have indeed shown such post-bite mortality in a significant percentage (~20%) of prey animals. Thus supporting a predator role of venom in varanid and other anguimorph lizards.

Anatomical variations in the anguimorph lizard venom systems . (A) Varanus komodoensis, magnetic resonance imaging: (A1) showing the six compartments (pink/red) of the mandibular venom gland and the infralabial mucus gland (yellow); (A2) longitudinal MRI section showing large ducts emerging separately from each mandibular venom gland compartment; and (A3) transverse MRI section showing the mandibular venom gland large central lumen (red) and labial gland individual lobes (yellow). Transverse histology of Masson’s Trichrome stained sections: (A4) the intratubular lumina of the mandibular venom gland that feed into the large central lumen; (A5) a mucus infralabial lobule (note that the six large dark folds are histology artifacts). (B) Lanthanotus borneensis: (B1) dissection of the mandibular venom gland (computationally highlighted red) and the mucus gland (computationally highlighted yellow); (B2) transverse histology section of the mandibular venom gland compartments (Kochva 1974); (B3) longitudinal MRI of the mandibular venom gland and the infralabial mucus gland. (C) Heloderma horridum: (C1) MRI of the mandibular venom gland; (C2) dissection of the mandibular venom gland (computationally highlighted red) and the mucus gland (computationally highlighted yellow); and (C3) histology of the venom gland intracellular storage granules (computationally highlighted red) and the nucleus (computationally highlighted black). (D) Gerrhonotus infernalis: (D1) longitudinal MRI of the multiple compartments (one per tooth); and (D2) Masson’s Trichrome stained longitudinal histology revealing the seromucous mixed gland arrangement with the protein-secreting regions (dark reddish-purple) ventral to the mucus-secreting region (grayish-green). Mandibular gland histology sections: (E) the Iguania species Anolis equestris stained with Masson’s Trichrome histology showing the mixed seromucous gland; or (F) the Xantusiidae species Lepidophyma flavimaculatum stained with Periodic Acid Schiff’s showing the purely mucus-secreting gland. 

Anatomical variations in the anguimorph lizard venom systems . (A) Varanus komodoensis, magnetic resonance imaging: (A1) showing the six compartments (pink/red) of the mandibular venom gland and the infralabial mucus gland (yellow); (A2) longitudinal MRI section showing large ducts emerging separately from each mandibular venom gland compartment; and (A3) transverse MRI section showing the mandibular venom gland large central lumen (red) and labial gland individual lobes (yellow). Transverse histology of Masson’s Trichrome stained sections: (A4) the intratubular lumina of the mandibular venom gland that feed into the large central lumen; (A5) a mucus infralabial lobule (note that the six large dark folds are histology artifacts). (B) Lanthanotus borneensis: (B1) dissection of the mandibular venom gland (computationally highlighted red) and the mucus gland (computationally highlighted yellow); (B2) transverse histology section of the mandibular venom gland compartments (Kochva 1974); (B3) longitudinal MRI of the mandibular venom gland and the infralabial mucus gland. (C) Heloderma horridum: (C1) MRI of the mandibular venom gland; (C2) dissection of the mandibular venom gland (computationally highlighted red) and the mucus gland (computationally highlighted yellow); and (C3) histology of the venom gland intracellular storage granules (computationally highlighted red) and the nucleus (computationally highlighted black). (D) Gerrhonotus infernalis: (D1) longitudinal MRI of the multiple compartments (one per tooth); and (D2) Masson’s Trichrome stained longitudinal histology revealing the seromucous mixed gland arrangement with the protein-secreting regions (dark reddish-purple) ventral to the mucus-secreting region (grayish-green). Mandibular gland histology sections: (E) the Iguania species Anolis equestris stained with Masson’s Trichrome histology showing the mixed seromucous gland; or (F) the Xantusiidae species Lepidophyma flavimaculatum stained with Periodic Acid Schiff’s showing the purely mucus-secreting gland.