Molecular evidence for a terrestrial origin of snakes

Biologists have debated the origin of snakes since the nineteenth century. One hypothesis suggests that snakes are most closely related to terrestrial lizards, and reduced their limbs on land. An alternative hypothesis proposes that snakes are most closely related to Cretaceous marine lizards, such as mosasaurs, and reduced their limbs in water. A presumed close relationship between living monitor lizards, believed to be close relatives of the extinct mosasaurs, and snakes has bolstered the marine origin hypothesis. Here, we show that DNA sequence evidence does not support a close relationship between snakes and monitor lizards, and thus supports a terrestrial origin of snakes.     

Homology and synapomorphy‐symplesiomorphy—neither synonymous nor equivalent but different perspectives on the same phenomenon

In a recent debate, either synapomorphy and symplesiomorphy or only synapomorphy have been claimed to be synonymous or equivalent to homology. In my view, exactly the same relationship exists between homology supported by a congruence test on the one hand and synapomorphy as well as symplesiomorphy on the other hand. Both conditions become established at the same time with the process of rooting of an unrooted topology. I, however, do not consider the concept of homology equal or synonymous to that of synapomorphy and symplesiomorphy. In my view, they represent different perspectives on the same phenomenon, i.e. correspondence by common origin. Homology has no implication on the direction of transformation, whereas symplesiomorphy as “primitive” condition and synapomorphy as “derived” condition refer directly to phylogenesis, the real historical evolutionary process of speciation and transformation. In addition, synapomorphy and symplesiomorphy might also refer to a character state that refers to the absence of a structure/organ, which creates problems with traditional homology concepts. Hennig's terms synapomorphy and symplesiomorphy are necessary and sufficient for the evolutionary interpretation of character states. For what is corroborated in an unrooted topology as the result of a congruence test, I suggest as a new term “synmorphy” because it can well be applied also to those characters where one state represents the absence of a structure/organ. The place for homology in morphological cladistics, however, is restricted to the characterization of the relationship between different character states of one transformation series (i.e. character).     

Molecular evidence and marine snake origins

A molecular phylogeny was used to refute the marine scenario for snake origins. Nuclear gene sequences suggested that snakes are not closely related to living varanid lizards, thus also apparently contradicting proposed relationships between snakes and marine mosasaurs (usually considered to be varanoids). However, mosasaurs share derived similarities with both snakes and living varanids. A reanalysis of the morphological data suggests that, if the relationships between living taxa are constrained to the proposed molecular tree, with fossil forms allowed to insert in their optimal positions within this framework, mosasaurs cluster with snakes rather than with varanids. Combined morphological and molecular analyses also still unite marine lizards with snakes. Thus, the molecular data do not refute the phylogenetic evidence for a marine origin of snakes.     

Homology and misdirection

Hennig (1966) recognized symplesiomorphies as homologies, and that view is logically correct under the concept of homology (homogeny) prevalent among evolutionists since 1870. Nelson and Platnick (1981) instead wanted homology to exclude symplesiomorphies for reasons that they never made clear but which certainly included opposition to Hennig. They and some of their followers, most recently Platnick (2013) and Brower and de Pinna (2013), have continued to advocate that anti‐Hennigian position, often under the slogan “homology equals synapomorphy,” while ironically passing themselves off as cladists and often using ambiguous or falsified citations to pretend that legitimate phylogeneticists think likewise. Such authors have seldom shown much concern for accuracy or logic, with the result that a great deal of print has been wasted. Those problems can be avoided simply by maintaining a Hennigian view and so discarding the purported equivalence of homology and synapomorphy.     

Primary homologies of the circumorbital bones of snakes

Some snakes have two circumorbital ossifications that in the current literature are usually referred to as the postorbital and supraorbital. We review the arguments that have been proposed to justify this interpretation and provide counter‐arguments that reject those conjectures of primary homology based on the observation of 32 species of lizards and 81 species of snakes (both extant and fossil). We present similarity arguments, both topological and structural, for reinterpretation of the primary homologies of the dorsal and posterior orbital ossifications of snakes. Applying the test of similarity, we conclude that the posterior orbital ossification of snakes is topologically consistent as the homolog of the lacertilian jugal, and that the dorsal orbital ossification present in some snakes (e.g., pythons, Loxocemus, and Calabaria) is the homolog of the lacertilian postfrontal. We therefore propose that the terms postorbital and supraorbital should be abandoned as reference language for the circumorbital bones of snakes, and be replaced with the terms jugal and postfrontal, respectively. The primary homology claim for the snake “postorbital” fails the test of similarity, while the term “supraorbital” is an unnecessary and inaccurate application of the concept of a neomorphic ossification, for an element that passes the test of similarity as a postfrontal. This reinterpretation of the circumorbital bones of snakes is bound to have important repercussions for future phylogenetic analyses and consequently for our understanding of the origin and evolution of snakes. J. Morphol. 274:973–986, 2013. © 2013 Wiley Periodicals, Inc.     

CONCEPTS AND TESTS OF HOMOLOGY IN THE CLADISTIC PARADIGM

Abstract— Logical equivalence between the notions of homology and synapomorphy is reviewed and supported. So-called transformational homology embodies two distinct logical components, one related to comparisons among different organisms and the other restricted to comparisons within the same organism. The former is essentially hierarchical in nature, thus being in fact a less obvious form of taxic homology. The latter is logically equivalent to so-called serial homology in a broad sense (including homonomy, mass homology or iterative homology). Of three tests of homology proposed to date (similarity, conjunction and congruence) only congruence serves as a test in the strict sense. Similarity stands at a basic level in homology propositions, being the source of the homology conjecture in the first place. Conjunction is unquestionably an indicator of non-homology, but it is not specific about the pairwise comparison where non-homology is present, and depends on a specific scheme of relationship in order to refute a hypothesis of homology. The congruence test has been previously seen as an application of compatibility analysis. However, congruence is more appropriately seen as an expression of strict parsimony analysis. A general theoretical solution is proposed to determine evolution of characters with ambiguous distributions, based on the notion of maximization of homology propositions. According to that notion, ambiguous character-state distributions should be resolved by an optimization that maximizes reversals relative to parallelisms. Notions of homology in morphology and molecular biology are essentially the same. The present tendency to adopt different terminologies for the two sources of data should be avoided, in order not to obscure the fundamental uniformity of the concept of homology in comparative biology. “A similar hierarchy is found both in ‘structures’ and in ‘functions’. In the last resort, structure (i.e. order of parts) and function (order of processes) may be the very same thing […].” L. von Bertalanlfy “[…] it is the fact that certain criteria enable us to match parts of things consistently which suggests that mechanisms of certain kinds must have been involved in their origin.” N. Jardine and C. Jardine     

The bony labyrinth of Platecarpus (Squamata: Mosasauria) and aquatic adaptations in squamate reptiles

Mosasaurs were among the last marine reptiles that lived before the Cretacesous–Paleogene extinction. Little is known about the sensory evolution of mosasaurs in relation to their aquatic lifestyle. In this study, the braincase of Platecarpus was CT-scanned and virtual models were constructed showing the bony labyrinth — or the inner ear — a sensory apparatus for balance and hearing. The virtual inner ear consists of the semicircular canals, vestibule, and cochlea. Compared with extant squamates, Platecarpus resembles sea snakes in having a small vestibule with a flat dorsal surface, but it differs from non-mosasaurian squamates in having rounded semicircular canals. Phylogenetic linear regression analysis supports a linear relationship, independent from phylogeny, between the length of the three semicircular canals and the length of the skull. The semicircular canals of Platecarpus are shorter than predicted, but the fossil data fell within the 95% prediction interval calculated from the extant data and the skull length of Platecarpus. Although size reduction of the bony labyrinth has been associated with aquatic adaptions in mammals, our results suggest that in squamates, semicircular canal size is related to skull size rather than habitat preference.     

Mitochondrial genome of the Komodo dragon: efficient sequencing method with reptile-oriented primers and novel gene rearrangements.

The mitochondrial genome of the Komodo dragon (Varanus komodoensis) was nearly completely sequenced, except for two highly repetitive noncoding regions. An efficient sequencing method for squamate mitochondrial genomes was established by combining the long polymerase chain reaction (PCR) technology and a set of reptile-oriented primers designed for nested PCR amplifications. It was found that the mitochondrial genome had novel gene arrangements in which genes from NADH dehydrogenase subunit 6 to proline tRNA were extensively shuffled with duplicate control regions. These control regions had 99% sequence similarity over 700 bp. Although snake mitochondrial genomes are also known to possess duplicate control regions with nearly identical sequences, the location of the second control region suggested independent occurrence of the duplication on lineages leading to snakes and the Komodo dragon. Another feature of the mitochondrial genome of the Komodo dragon was the considerable number of tandem repeats, including sequences with a strong secondary structure, as a possible site for the slipped-strand mispairing in replication. These observations are consistent with hypotheses that tandem duplications via the slipped-strand mispairing may induce mitochondrial gene rearrangements and may serve to maintain similar copies of the control region.     

Unusual histology and morphology of the ribs of mosasaurs (Squamata)

We report the presence of two previously unrecognized features in the dorsal ribs of mosasaurs: first, the presence of extremely dense, pervasive extrinsic fibres (anchoring soft tissue to bone, sometimes called Sharpey's fibres); and second, high intraspecific variation in costal bone compactness. Extensive extrinsic fibres are developed in the dorsal ribs of the mosasaurs Tylosaurus proriger and Eonatator sternbergi. The dorsal ribs of these mosasaurs are also characterized by a longitudinally ridged texture that almost completely covers the bone. Pervasive extrinsic fibres and ridged textures are absent in the mosasaur Selmasaurus russelli as well as the dorsal ribs of extant semi‐aquatic reptiles (e.g. crocodyliforms) and mosasaurs' close extant relatives and analogues (e.g. snakes and varanids). Similar ridged textures characterize the dorsal ribs of several other mosasaur taxa but are developed to a lesser extent (e.g. Mosasaurus, Clidastes, Platecarpus and Ectenosaurus), but in no other taxa have pervasive extrinsic fibres been reported. We interpret these osteohistological features in T. proriger and E. sternbergi as evidence of tendinous attachment of extensive and highly differentiated axial musculature capable of producing great stresses, most likely related to stabilization of the trunk relative to contralateral movements of the tail during carangiform locomotion. We also report the compactness indices (percentage of space occupied by bone rather than cavities) for these large mosasaur ribs, which are much higher than previously reported. This suggests high intraspecific variation in bone compactness that complicates its use in reconstructing mosasaur palaeoecology.     

Homology in classical and molecular biology

Hypotheses of homology are the basis of comparative morphology and comparative molecular biology. The kinds of homologous and nonhomologous relations in classical and molecular biology are explored through the three tests that may be applied to a hypothesis of homology: congruence, conjunction, and similarity. The same three tests apply in molecular comparisons and in morphology, and in each field they differentiate eight kinds of relation. These various relations are discussed and compared. The unit or standard of comparison differs in morphology and in molecular biology; in morphology it is the adult or life cycle, but with molecules it is the haploid genome. In morphology the congruence test is decisive in separating homology and nonhomology, whereas with molecular sequence data similarity is the decisive test. Consequences of this difference are that the boundary between homology and nonhomology is not the same in molecular biology as in morphology, that homology and synapomorphy can be equated in morphology but not in all molecular comparisons, and that there is no detected molecular equivalent of convergence. Since molecular homology may reflect either species phylogeny or gene phylogeny, there are more kinds of homologous relation between molecular sequences than in morphology. The terms paraxenology and plerology are proposed for two of these kinds--respectively, the consequence of multiple xenology and of gene conversion.     

Homology in coding and non-coding DNA sequences: a parsimony perspective

Putative synapomorphy assessment (primary homology assessment) is distinct for DNA strings having a codon structure (hereafter, coding DNA) versus those lacking it (hereafter, non-coding DNA). The first requires the identification of a reading frame and of usually few in-frame insertions and deletions. In non-coding DNA, where length variation is much more common, putative synapomorphy assessment is considerably less straightforward and highly depends on the alignment method. Appreciating the existence of evolutionary constraints, alignments that consider patterns associated with specific putative evolutionary events are favored. Once the sequences have been aligned, the postulated putative evolutionary events need to be coded as an additional step. In order for the alignments and the alignment coding to be falsifiable, they should be carried out using justified and explicitly formulated criteria. Alternative coding methods for the most common patterns present in alignments of non-coding DNA are discussed here. Simpler putative synapomorphy assessment will not always correlate to more reliable phylogenetic information because simplicity does not necessarily correlate to the degree of homoplasy. The use of non-coding DNA can result in more laborious coding, but at the same time in more corroborated hypotheses, mirroring their accuracy for phylogenetic inference.     

Anatomy and relationships of Pachyrhachis problematicus,a primitive snake with hindlimbs

The anatomy of Pachyrhachis problematicus, an elongate, limb-reduced squamate from the Upper Cretaceous of Israel, is described and evaluated in detail. Previously considered a snake-like ''lizard'' of uncertain affinities, it is here shown to be the most primitive snake, and the sister-group to all other snakes. Pachyrhachis exhibits numerous derived characters uniting it with modern snakes (scolecophidians and alethinophidians): e.g. mobile premaxilla-maxilla articulation, braincase enclosed by frontals and parietals, sagittal parietal crest, absence of tympanic recess, single postdentary bone, over 140 presacral vertebrae, and complete loss of shoulder girdle and forelimb. However, it is more primitive than all modern snakes in retaining some strikingly primitive (lizard-like) features: presence of a jugal, squamosal, normal sacral attachment, and well-developed hindlimb composed of femur, tibia, fibula, and tarsals. Pachyrhachis provides additional support for the hypothesis that snakes are most closely related to Cretaceous marine lizards (mosasauroids). Almost all of the derived characters proposed to unite snakes and mosasauroids are highly developed in Pachyrhachis: the mobile mandibular symphysis, intramandibular joint, long and recurved pterygoid teeth, quadrate suspended by the supratemporal, loosely united pelvic elements (ilium, ischium, and pubis), and separate astragalus and calcaneum.     

Similarity,parsimony and conjectures of homology: The chelonian shoulder girdle revisited

Much has been written about the definition and recognition of biological homology. Homology is usually defined as similarity inherited from a common ancestor (e.g., papers in Hall, 1994). It is recognised through cladistic analysis: Patterson (1982) and de Pinna (1991) have cogently argued that homology can be equated with synapomorphy (a shared evolutionary novelty uniting a monophyletic group). Such identification involves two stages: first, a possible homology is proposed on the basis of morphological similarity. This similarity might be structural, topological, developmental, or any combination thereof. Next, a cladistic analysis is performed, involving the trait in question and all other informative traits identified. If the trait is congruent with the resultant phylogeny, it is accepted as homologous in all taxa which possess it. If the trait is incongruent with the phylogeny, it is interpreted as homoplasious in certain taxa. This has been termed the test of congruence (Patterson, 1982; de Pinna, 1991). Rieppel (1996) has recently suggested that the test of congruence might be circular, and that as a result certain inferences about the evolution of the chelonian shoulder girdle (Lee, 1996) are poorly substantiated. Here I argue that the test of congruence is not circular, and that the disputed conclusions about the evolution of chelonian shoulder girdle can be defended on the basis of parsimony. More generally, I suggest how considerations of parsimony can and should be used to arbitrate between conflicting conjectures of homology that are both congruent with an accepted phylogeny.     

On homology

Homology in cladistics is reviewed. The definition of important terms is explicated in historical context. Homology is not synonymous with synapomorphy: it includes symplesiomorphy, and Hennig clearly included both plesiomorphy and synapomorphy as types of homology. Homoplasy is error, in coding, and is analogous to residual error in simple regression. If parallelism and convergence are to be distinguished, homoplasy would be evidence of the former and analogy evidence of the latter. We discuss whether there is a difference between molecular homology and morphological homology, character state homology, nested homology (additive characters), and serial homology. We conclude by proposing a global definition of homology. ©The Will Henning Society 2011.     

Poikilotherms as reservoirs of Q-fever (Coxiella burnetii) in Uttar Pradesh.

Water snakes (Natrix natrix), rat snakes (Ptyas korros), cobras (Naja naja), pythons (Python molurus), tortoises (Kachuga sp.), plankton fish (Cirrhina mrigala), frogs (Rana tigrina), toads (Bufo sp.) and monitors (Varanus indicus) were screened for evidence of Q-fever infection by the capillary agglutination test on sera to detect antibodies and/or by attempts to demonstrate Coxiella burnetii in spleen and liver samples. Sero-reactors were observed among water and rat snakes, pythons and tortoises. The organism was isolated from the spleen and liver of the monitor, tortoise and python.     

Feeding behaviour in Cylindrophis and its bearing on the evolution of alethinophidian snakes

Cylindrophis ruffus ingests prey using two distinct mechanisms. During initial phases of prey transport, lateral movements of the rear of the braincase combine with small unilateral movements of the toothed bones of each side; prey is usually constricted during this phase to permit the snake to push its head over the prey. Once transport has carried the leading part of the prey into the anterior oesophagus, Cylindrophis begins to use bilaterally synchronized movements of the jaw apparatus combined with low-amplitude, short wave-length flexions of the anterior vertebral column. Transport of prey is many times faster during the bilateral phase than during the unilateral phase.
Radiographic and cinematographic evidence indicates that the mandibular tips of Cylindrophis do not separate more than 1.5–2.0 times the resting distance between the dentary tips. Although this limits potential gape size, the intramandibular joint is highly mobile, allowing the mandibles to conform to a variety of prey shapes. Manipulations of anaesthetized and fresh, dead specimens revealed that the palatomaxillary arches are tightly attached to the ventral bones of the snout, movements of each arch being reflected in equivalent movements of the ipsilateral elements of the snout.
Cylindrophis represents a functional stage intermediate between most lizards with limited palatomaxillary kinesis and advanced snakes with considerable palatomaxillary mobility. Contrary to previous hypotheses, however, upper jaw liberation in Cylindrophis is due to liberation of the ventral snout, not to reduction of attachments to the braincase and snout. This suggests that the nose played a crucial role in the evolution of the feeding apparatus in alethinophidian snakes.     

The evolution of the basicranium in the Henophidia (Reptilia: Serpentes)

In lizards, a short Vidian canal pierces the base of the basipterygoid process. In snakes, the anterior opening of the primitively short Vidian canal lies on the dorsal surface of the basisphenoid, trapped in an intracranial position by downgrowths of the frontal and parietal which meet the lateral edges of the basisphenoid-parasphenoid complex. This condition is observed in anilioid snakes which retain other primitive features in the braincase: the paroccipital process and the spheno-occipital tubercle (Aniliidae only) and participation of the basioccipital in the apertura lateralis of the recessus scalae tympani.
Subsequent evolution within booid snakes shows a shift of the anterior opening of the Vidian canal around the anterior edge of the basisphenoid, thus acquiring a secondary extracranial position. This occurs in parallel within boine and pythonine snakes. Dinilysia shows a parallel development of die condition observed in advanced booid snakes. Pseudoboa , with its short Vidian canal opening in-tracranially, demonstrates that caenophidians originated from a basal henophidian or pre-henophidian stock. The Acrochordidae show a basicranium that can be interpreted as either primitive henophidian or primitive caenophidian.     

The naso-frontal joint in snakes as revealed by high-resolution X-ray computed tomography of intact and complete skulls

The naso-frontal joint of snakes is described on the basis of high-resolution X-ray computed tomography scans of single individuals of spirit-preserved snake specimens. The suspension of the snout unit from the braincase at the naso-frontal joint shows some broad evolutionary trends among snakes with potential phylogenetic implications, such as sutured or fused medial frontal flanges formed by the medial frontal pillars and the frontal subolfactory processes (in alethinophidians), the restriction of the usually extended dorsoventral contact of the medial nasal flange with the medial frontal flanges to a dorsal or ventral contact (in macrostomatans), and the transfer of the main element of snout suspension from the nasal to the septomaxilla (in colubroids). Some phylogenetic implications of the morphological characters identified in this study are outlined and discussed.     

Hybridization between mtDNA-defined phylogeographic lineages of black ratsnakes (Pantherophis sp.)

Phylogeographic analyses using mitochondrial DNA (mtDNA) have revealed many examples of apparently deep historical subdivisions ('phylogroups') within many vertebrates. It remains unclear whether these phylogroups represent independently evolving, adaptively differentiated lineages or groups that show little functional differentiation and, hence, will merge on contact. Here, we use mtDNA sequence data to evaluate the phylogeographic relationships between two of the northernmost populations of black ratsnakes (Pantherophis obsoletus complex) in Ontario, Canada and previously analysed populations in the United States. We then use population-level analyses to evaluate the level of adaptive divergence between previously established mtDNA phylogroups. Phylogenetic analyses show that southern Ontario snakes have mtDNA haplotypes that fall within the Central mtDNA phylogroup, as designated by Burbrink et al. (2000). In contrast, snakes in eastern Ontario carry either Central or Eastern-specific haplotypes. Within the hybrid region, we found highly variable frequencies of mtDNA haplotypes among isolated sub-populations, no association between variation in cytonuclear (mtDNA) and nuclear (microsatellite DNA) markers, no difference in survival or reproductive success among snakes with different mtDNA haplotypes, and no effect of mate similarity in mtDNA on female clutch size. These results argue that the Eastern and Central phylogroups have merged in this region, likely due to a lack of adaptive differentiation between individuals in each lineage. Hence, in these snakes, phylogeographic structure in mtDNA is more a reflection of historical isolation rather than adaptive divergence. The observed reticulation between lineages and lack of evidence for hybrid disgenesis also bears on the classification of these lineages as distinct species.