A novel method of caenophidian snake sex identification using molecular markers based on two gametologous genes

Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female‐specific 250‐bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.     

On the vomer in Acrochordidae (Reptilia: Serpentes), and its cladistic significance

It is reported that in certain features the form of the vomer is significantly different in Caenophidia than in Henophidia (except acrochordids). In Henophidia the vomer typically has one or a few apertures for the exit of the vomeronasal nerve from the bony surround of the vomeronasal organ, well- or moderately-developed vertical and horizontal (palatal) posterior laminae, and only a partially-developed cup-like enclosure for the vomeronasal organ. In Caenophidia the vomer typically has very many tiny foramina for the passage of the vomeronasal nerve, the horizontal posterior lamina in particular is much reduced or absent, and the vomer forms a globular enclosure for the vomeronasal organ. A comparison with the vomer in lizards suggests that the henophidian type of vomer is primitive within snakes and the caenophidian type is derived. Scolecophidia are not discussed. The vomer in acrochordids closely resembles that of Caenophidia, and this form of vomerine morphology is proposed as a synapomorphy indicating the strict monophyly of the group acrochordids-Caenophidia. The acrochordids have been treated very differently by various snake taxonomists and their phyletic position has always been highly problematical. The synapomorphy proposed herein contributes to a solution of this problem.     

ZW,XY, and yet ZW: Sex chromosome evolution in snakes even more complicated

Snakes are historically important in the formulation of several central concepts on the evolution of sex chromosomes. For over 50 years, it was believed that all snakes shared the same ZZ/ZW sex chromosomes, which are homomorphic and poorly differentiated in “basal” snakes such as pythons and boas, while heteromorphic and well differentiated in “advanced” (caenophidian) snakes. Recent molecular studies revealed that differentiated sex chromosomes are indeed shared among all families of caenophidian snakes, but that boas and pythons evolved likely independently male heterogamety (XX/XY sex chromosomes). The historical report of heteromorphic ZZ/ZW sex chromosomes in a boid snake was previously regarded as ambiguous. In the current study, we document heteromorphic ZZ/ZW sex chromosomes in a boid snake. A comparative approach suggests that these heteromorphic sex chromosomes evolved very recently and that they are poorly differentiated at the sequence level. Interestingly, two snake lineages with confirmed male heterogamety possess homomorphic sex chromosomes, but heteromorphic sex chromosomes are present in both snake lineages with female heterogamety. We point out that this phenomenon is more common across squamates. The presence of female heterogamety in non‐caenophidian snakes indicates that the evolution of sex chromosomes in this lineage is much more complex than previously thought, making snakes an even better model system for the evolution of sex chromosomes.     

Behaviour of the ZW sex bivalent in the snake Bothrops jararaca

The behavior of the ZW sex bivalent was investigated in female meiosis of the poisonous snake Bothrops jararaca. The Z is euchromatic and synapses end to end with the W. The W chromosome shows a heterochromatic segment distally in the short arm. Pairing occurs between the long arm of the W and the slightly longer arm of the mediocentric Z. A sex vesicle, similar to the one found in the XY placental mammals, does not occur in snakes. The Z and W chromosomes segregate reductionally in the first meiotic division and equationally in the second.This work is dedicated to the memory of my father Lino Pires de Camargo     

Evolutionary stability of sex chromosomes in snakes

Amniote vertebrates possess various mechanisms of sex determination, but their variability is not equally distributed. The large evolutionary stability of sex chromosomes in viviparous mammals and birds was believed to be connected with their endothermy. However, some ectotherm lineages seem to be comparably conserved in sex determination, but previously there was a lack of molecular evidence to confirm this. Here, we document a stability of sex chromosomes in advanced snakes based on the testing of Z-specificity of genes using quantitative PCR (qPCR) across 37 snake species (our qPCR technique is suitable for molecular sexing in potentially all advanced snakes). We discovered that at least part of sex chromosomes is homologous across all families of caenophidian snakes (Acrochordidae, Xenodermatidae, Pareatidae, Viperidae, Homalopsidae, Colubridae, Elapidae and Lamprophiidae). The emergence of differentiated sex chromosomes can be dated back to about 60 Ma and preceded the extensive diversification of advanced snakes, the group with more than 3000 species. The Z-specific genes of caenophidian snakes are (pseudo)autosomal in the members of the snake families Pythonidae, Xenopeltidae, Boidae, Erycidae and Sanziniidae, as well as in outgroups with differentiated sex chromosomes such as monitor lizards, iguanas and chameleons. Along with iguanas, advanced snakes are therefore another example of ectothermic amniotes with a long-term stability of sex chromosomes comparable with endotherms.     

The emerging phylogenetic pattern of parthenogenesis in snakes

Parthenogenesis occurs across a variety of vertebrate taxa. Within squamate reptiles (lizards and snakes), a group for which the largest number of cases has been documented, both obligate and facultative types of parthenogenesis exists, although the obligate form in snakes appears to be restricted to a single basal species of blind snake, Indotyphlops braminus. By contrast, a number of snake species that otherwise reproduce sexually have been found capable of facultative parthenogenesis. Because the original documentation of this phenomenon was restricted to subjects held in captivity and isolated from males, facultative parthenogenesis was attributed as a captive syndrome. However, its recent discovery in nature shifts the paradigm and identifies this form of reproduction as a potentially important feature of vertebrate evolution. In light of the growing number of documented cases of parthenogenesis, it is now possible to review the phylogenetic distribution in snakes and thus identify subtle variations and commonalities that may exist through the characterization of its emerging properties. Based on our findings, we propose partitioning facultative parthenogenesis in snakes into two categories, type A and type B, based on the sex of the progeny produced, their viability, sex chromosome morphology, and ploidy, as well as their phylogenetic position. Furthermore, we introduce a hypothesis (directionality of heterogamety hypothesis) to explain the production of female‐only parthenogens in basal alethinophidian snakes and male‐only parthenogens in caenophidian (advanced) snakes.     

Postovipositional development of the sand snake Psammophis sibilans (Serpentes:Lamprophiidae) in comparison with other snake species

Characterizing and comparing developmental progress across different species helps to interpret how different or similar body forms evolved. We present an embryonic table for the oviparous African Sand Snake Psammophis sibilans from the Lamprophidae family, describing its postovipositional in ovo development. Psammophis is a good model of a genus that is widely distributed in Africa and Asia and includes 22 species. We describe ten embryonic stages based on the development of externally visible morphological characteristics such as; pharyngeal arches, facial processes, eyes, scales, body pigmentation and body colour pattern development. This study discusses the development of this snake and compares it with that of the closely related brown house snake Lamprophis fulliginosus (Lamprophidae) and the medically important venomous cobras Naja haje haje and Naja kaouthia from the sister lineage Elapidae. The distantly related basal snake Python sebae, which displays different morphology and behaviour, was chosen for deeper insight into the evolution of body structures within the snake clade. We found interspecific differences in the relative stage of development of embryonic structures at the time of oviposition and during postovipositional embryonic development. One of the outcomes of this study is that embryonic structures such as the pharyngeal processes, eye pigmentation and scales are interspecifically conservative in regard to timing of morphodifferentiation, while body pigmentation, colour and colour pattern are interspecifically plastic in their temporospatial development.     

The phylogeny and classification of caenophidian snakes inferred from seven nuclear protein-coding genes

More than 80% of the approximately 3000 living species of snakes are placed in the taxon Caenophidia (advanced snakes), a group that includes the families Acrochordidae, Viperidae, Elapidae, Atractaspididae, and the paraphyletic 'Colubridae'. Previous studies using DNA sequences have involved few nuclear genes (one or two). Several nodes have therefore proven difficult to resolve with statistical significance. Here, we investigated the higher-level relationships of caenophidian snakes with seven nuclear protein-coding genes and obtained a well-supported topology. Accordingly, some adjustments to the current classification of Caenophidia are made to better reflect the relationships of the groups. The phylogeny also indicates that, ancestrally, caenophidian snakes are Asian and nocturnal in origin, although living species occur on nearly all continents and are ecologically diverse.     

Satellite DNA and evolution of sex chromosomes

The satellite DNA (satellite III) which is mainly represented in the female of Elaphe radiata (Ophidia, Colubridae) has been isolated and its buoyant density has been determined (=1.700 g cm^–3). In situ hybridisation of radioactive complementary RNA of this satellite DNA with the chromosomes of different species has revealed that it is mainly concentrated on the W sex chromosome and its sequences are conserved throughout the sub-order Ophidia. From hybridisation studies these sequences are absent from the primitive family Boidae which represents a primitive state of differentiation of sex chromosomes. Chromosome analysis and C-banding have also revealed the absence of heteromorphism and of an entirely heterochromatic chromosome in the species belonging to the primitive family and their presence in the species of highly evolved families. It is suggested that the origin of satellite DNA (satellite III) in the W chromosome is the first step in differentiation of W from the Z in snakes by generating asynchrony in the DNA replication pattern of Z and W chromosomes and thus conceivably reducing the frequency of crossing-over between them which is the prerequisite of differentiation of sex chromosomes. Presence of similar sex chromosome associated satellite DNA in domestic chicken suggests its existence in a wider range of vertebrates than just the snakes.     

Phylogeny, biogeography and classification of the snake superfamily Elapoidea: a rapid radiation in the late Eocene

The snake superfamily Elapoidea presents one of the most intransigent problems in systematics of the Caenophidia. Its monophyly is undisputed and several cohesive constituent lineages have been identified (including the diverse and clinically important family Elapidae), but its basal phylogenetic structure is obscure. We investigate phylogenetic relationships and spatial and temporal history of the Elapoidea using 94 caenophidian species and approximately 2300–4300 bases of DNA sequence from one nuclear and four mitochondrial genes. Phylogenetic reconstruction was conducted in a parametric framework using complex models of sequence evolution. We employed Bayesian relaxed clocks and Penalized Likelihood with rate smoothing to date the phylogeny, in conjunction with seven fossil calibration constraints. Elapoid biogeography was investigated using maximum likelihood and maximum parsimony methods. Resolution was poor for early relationships in the Elapoidea and in Elapidae and our results imply rapid basal diversification in both clades, in the late Eocene of Africa (Elapoidea) and the mid-Oligocene of the Oriental region (Elapidae). We identify the major elapoid and elapid lineages, present a phylogenetic classification system for the superfamily (excluding Elapidae), and combine our phylogenetic, temporal and biogeographic results to provide an account of elapoid evolution in light of current palaeontological data and palaeogeographic models.
© The Willi Hennig Society 2009.     

Sex chromosome associated satellite DNA: Evolution and conservation

Satellites visible in female but not in male DNA were isolated from the snakesElaphe radiata (satellite IV, p = 1.708 g · cm^–3) andBungarus fasciatus (BK¹ minor, p=1.709 g · cm^–3). The satellites cross hybridize. Hybridization of³H labelled nick translated BK minor satellite DNA with the total male and female DNA and/or chromosomes in situ of different species of snakes revealed that its sequences are conserved throughout the snake group and are mainly concentrated on the W chromosome. Snakes lacking sex chromosomes do possess related sequences but there is no sex difference and visible related satellites are absent. The following conclusions have been reached on the basis of these results. 1. The W chromosome associated satellite DNA is related to similar sequences scattered in the genome. 2. The origin and increment in the number of the W satellite DNA sequence on the W chromosome is associated with the heterochromatinization of the W. 3. Satellite sequences have become distributed along the length of the W and resulted in morphological differentiation of sex chromosomes. 4. Evolutionary conservation of W satellite DNA strongly suggests that functional constraints may have limited sequence divergence.     

Conserved repeated DNA sequences in vertebrate sex chromosomes

Summary Satellite DNA isolated from female Elapid snakes contains nucleotide sequences which are quantitatively derived from the W sex-determining chromosome. Certain of these sequences are highly conserved in vertebrates, including mammals, where they are arranged in a sex-specific pattern in Southern blots. Sex reversed mice (Sxr) show a DNA arrangement of these sequences in conformity with their phenotypic sex, suggesting that this DNA is closely connected with the determination of sex. In situ hybridization of the snake sequences with mouse chromosomes reveals a concentration of related DNA at the proximal tip of the mouse Y chromosome. The possible nature and significance of these observations is discussed.     

A mitogenomic study on the phylogenetic position of snakes

Phylogenetic relationships of squamates (lizards, amphisbaenians and snakes) have received considerable attention, although no consensus has been reached concerning some basal divergences. This paper focuses on the Serpentes (snakes), whose phylogenetic position within the Squamata remains uncertain despite a number of morphological and molecular studies. Some mitogenomic studies have suggested a sister-group relationship between snakes and varanid lizards, while other studies have identified snakes and lizards as sister groups. However, recent studies using nuclear data have presented a different scenario, with snakes being more closely related to anguimorph and iguanian lizards. In this mitogenomic study we have examined the above hypotheses with the inclusion of amphisbaenians, one gekkotan and one acrodont lizard, taxa not represented in previous mitogenomic studies. To this end we have also extended the representation of snakes by sequencing five additional snake genomes: two scolecophidians ( Ramphotyphlops australis and Typhlops mirus ) two henophidians ( Eunectes notaeus and Boa constrictor ) and one caenophidian ( Elaphe guttata ). The phylogenetic analysis recovered snakes and amphisbaenians as sister groups, thereby differing from previous hypotheses. In addition to a discussion on previous morphological and molecular studies in light of the results presented here, the current study also provides some details regarding features of the new snake mitochondrial genomes described.     

Chromosomal evolution in serpentes; a comparison of G and C chromosome banding patterns of some colubrid and boid genera

G and C-chromosome banding techniques have been used to compare the structure of the karyotype in a variety of colubrid and boid snakes. The comparison of G-band patterns indicates that while some band sequences have been conserved, either as whole chromosomes or entire arms, there is also evidence of considerable rearrangement especially in the smaller chromosomes. In the colubrid Elaphe subocularis there is also evidence that there has been a relocation of the centromere on chromosome 2 without any accompanying inversion in the sequence of G-bands. Finally, G-banding has facilitated the demonstration of a simple pericentric inversion distinguishing the Z and W chromosomes in Acrantophis dumereli. This represents the first report of differentiated sex chromosomes in a boid snake. The combined banding data thus indicates that snake chromosomes are certainly not lacking in variability. The use of C-banding to detect constitutive heterochromatin has confirmed that in some boids and colubrids macrochromosomes have been derived from microchromosomes by the additions of heterochromatin.     

Sexual divergence in diets and morphology in Fijian sea snakes Laticauda colubrina (Laticaudinae)

In the Fiji Islands, female yellow‐lipped sea kraits (Laticauda colubrina) grow much larger than males, and have longer and wider heads than do conspecific males of the same body length. This morphological divergence is accompanied by (and may be adaptive to) a marked sex divergence in dietary habits. Adult female sea kraits feed primarily on large conger eels, and take only a single prey item per foraging bout. In contrast, adult males feed upon smaller moray eels, and frequently take multiple prey items. Prey size increases with snake body size in both males and females, but the sexes follow different trajectories in this respect. Female sea kraits consume larger eels relative to predator head size and body length than do males. Thus, the larger relative head size of female sea kraits is interpreted as an adaptation to consuming larger prey items. Our results are similar to those of previous studies on American water snakes (natricines) and Australian file snakes (acrochordids), indicating that similar patterns of sex divergence in dietary habits and feeding structures have evolved convergently in at least three separate lineages of aquatic snakes.     

The anatomy of the upper cretaceous snake Najash rionegrina Apesteguía & Zaher, 2006, and the evolution of limblessness in snakes

Najash rionegrina Apesteguía & Zaher, 2006 , a terrestrial fossil snake from the Upper Cretaceous of Argentina, represents the first known snake with a sacrum associated with robust, well‐developed hind limbs. Najash rionegrina documents an important gap in the evolutionary development towards limblessness, because its phylogenetic affinities suggest that it is the sister group of all modern snakes, including the limbed Tethyan snakes Pachyrhachis, Haasiophis, and Eupodophis. The latter three limbed marine fossil snakes are shown to be more derived morphologically, because they lack a sacrum, but have articulated lymphapophyses, and their appendicular skeleton is enclosed by the rib cage, as in modern snakes.     

Behaviour of sex chromosome associated satellite DNAs in somatic and germ cells in snakes

Sex chromosome associated satellite DNAs isolated from the snakes Elaphe radiata (sat III) (Singh et al., 1976) and Bungarus fasciatus (Elapidae) (minor satellite) are evolutionarily conserved throughout the suborder Ophidia. An autosome limited satellite DNA (B. fasciatus major satellite) is not similarly conserved. Both types of satellites have been studied by in situ hybridisation in various somatic tissues and germ cells where it has been observed that the W sex chromosome remains condensed in interphase nuclei. In growing oocytes however, the W chromosome satellite rich heterochromatin decondenses completely whilst the autosomal satellite rich regions remain condensed. Later, the cycle is reversed and the W chromosome condenses whilst the autosomal satellite regions decondense. In a primitive snake (Eryx johni johni) where the sex chromosomes are not differentiated and where there is no satellite DNA specific to them, these phenomena are absent. — The differential behaviour of autosomal and sex chromosome associated satellite DNAs is discussed in the light of gene regulation.     

Radiation within the advanced snakes (Caenophidia) with special emphasis on African opistoglyph colubrids, based on mitochondrial sequence data

A parsimony analysis of parts of the mitochondrial genes 12S and 16S (722 base pairs) from 43 species of the advanced snakes (Caenophidia) resulted in two most parsimonious topologies. Based on a strict consensus of these the following objectives were addressed: (1) Which groupings of caenophidian taxa can be recognized? (2) Is Acrochordus sister group to, or included in, Caenophidia? (3) Are boigine snakes (sensu stricto) a monophyletic grouping and where do these taxa belong in a broader caenophidian context? (4) What are the systematic affinities of the African egg-eating genusDasypeltis ? The phylogeny was then used to discuss: (5) the evolution of the posterior maxillary dentition and the composition of the retinal visual cells. The results reveal that, when using Boa constrictor as outgroup,Acrochordus is the sistergroup to the remainder of the ingroup, and a further eight clades are defined. Five genera of elapids did not appear to be monophyletic and a number of colubrids (sensu lato) such as Mehelya,Lycodonomorphus , Lamprophis, Atractaspis, and Buhoma (formerly Geodipsas), which have traditionally been problematic to place systematically, did not group with any of the larger clades. These taxa are together with the elapids representatives of very early radiations in the evolution of the Advanced snakes. The homalopsine Enhydris enhydris appears as a sistergroup to the viperine clade (Clade 1). When plotted onto the topology the posterior maxillary dentition appear to express three, maybe four, independent origins of the Opistoglyph State. The retinal evolution also appeared very complex. The suggested very primitive placing of the Boigine snakes (sensu stricto) due to their lack of double cones in the retina of the eye was not supported here; instead the sequence data suggests this observation to be the result of a secondary loss.     

A New Snake Skull from the Paleocene of Bolivia Sheds Light on the Evolution of Macrostomatans

Macrostomatan snakes, one of the most diverse extant clades of squamates, display an impressive arsenal of cranial features to consume a vast array of preys. In the absence of indisputable fossil representatives of this clade with well-preserved skulls, the mode and timing of these extraordinary morphological novelties remain obscure. Here, we report the discovery of Kataria anisodonta n. gen. n. sp., a macrostomatan snake recovered in the Early Palaeocene locality of Tiupampa, Bolivia. The holotype consists of a partial, minute skull that exhibits a combination of booid and caenophidian characters, being the presence of an anisodont dentition and diastema in the maxilla the most distinctive trait. Phylogenetic analysis places Kataria basal to the Caenophidia+Tropidophiidae, and represents along with bolyeriids a distinctive clade of derived macrostomatans. The discovery of Kataria highlights the morphological diversity in the maxilla among derived macrostomatans, demonstrating the relevance of maxillary transformations in the evolution of this clade. Kataria represents the oldest macrostomatan skull recovered, revealing that the diversification of macrostomatans was well under way in early Tertiary times. This record also reinforces the importance of Gondwanan territories in the history of snakes, not only in the origin of the entire group but also in the evolution of ingroup clades.