ABSTRACTTranslocations are increasingly used to restore populations, yet seldom are simultaneous over large climate gradients into different latitudes, and rarely consider both a host and its parasites. Tuatara (Sphenodon punctatus) is a long‐lived reptile endemic to Aotearoa New Zealand (NZ). Once found throughout NZ, tuatara populations are now sustained on offshore islands and increased through translocation, including to pest‐free sanctuaries. Here, we study the simultaneous translocations of adult tuatara to four mainland sanctuaries a decade following release, investigating populations established both north and south of the founding population that span nearly 1000 km in latitude. We compared changes in body condition and snout‐vent length (SVL) of tuatara, and abundance of a host‐specific, ectoparasitic tick for tuatara among sites, plus evidence for survival and emergence of the next generation of tuatara. We found a general increase in SVL and maintenance of body condition between release in 2012 and 2023 at all sites, with some differences between males and females. However, tuatara at some sites showed more growth and/or higher body condition by 2023, particularly correlating with site temperature. Although ticks persisted on founding tuatara, there were fewer ticks on the next generation and those at the southern site. Evidence for a second generation of tuatara was also weakest at the southern site. This study shows that long‐distance translocations north and south of a source population can yield promising outcomes for survival and growth of a long‐lived reptile in current climates, though with differences in outcomes among sites.

Squamate reptiles are amongst the most successful terrestrial vertebrate lineages, with over 10,000 species across a broad range of ecosystems. Despite their success, squamates are also amongst the least studied lineages immunologically. Recently, a universal lack of γδ T cells in squamates due to deletions of the genes encoding the T cell receptor (TCR) γ and δ chains was discovered. Here, we begin to address how the loss of γδ T cells may have impacted the evolution of the squamate immune system. Using the skink Tiliqua rugosa, we found that squamates have not significantly increased the complexity of conventional T cell receptor beta (TCRβ or TRB) chain V regions compared to that of the nearest living squamate relative, the tuatara, Sphenodon punctatus or other amniotes. Our analyses include a putative new TCR locus. This novel locus contains V, D, and J gene segments that undergo V(D)J recombination, albeit with a limited number of gene segments in most squamate species. Based on conserved residues, the predicted protein chain would be expected to form a heterodimer with TCRα. This new TCR locus appears to be derived from an ancient duplication of the TRB locus and is homologous to the recently described T cell receptor epsilon (TRE). TRE is absent from the genomes of the tuatara and all Archosaurs examined and appears squamate specific.

The Lepidosauria is the most species-rich group of land-dwelling vertebrates. The group includes around 12,000 species of lizards and snakes (Squamata) and one species of Rhynchocephalia, the tuatara Sphenodon punctatus from New Zealand1. Squamates owe their success to their generally small size, but also to their highly mobile skull that enables them to manipulate large prey. These key features of lizard and snake skulls are not seen in Sphenodon, which makes it important to understand the nature of their common ancestor. Lepidosaurs originated in the Triassic 252–201 million years ago, but confusion has arisen because of incomplete fossils, many of which are generalized lepidosauromorphs, neither squamates nor rhynchocephalians2–5. Here we report a reasonably complete skull and skeleton of a definitive rhynchocephalian from the Middle Triassic (Anisian) Helsby Sandstone Formation of Devon, UK that is around 3–7 million years older than the oldest currently known lepidosaur. The new species shows, as predicted, a non-mobile skull but an open lower temporal bar and no large palatine teeth, and it seems to have been a specialized feeder on insects. This specimen helps us understand the initial diversification of Lepidosauria as part of the Triassic Revolution, when modern-style terrestrial ecosystems emerged.

Limited gut bacterial response of tuatara (Sphenodon punctatus) to dietary manipulation and captivity.

The theoretical trade-off between immune and endocrine investment in mating animals has received mixed empirical support, particularly in reptiles. We investigated the relationship between male sexual characteristics, diet, and immune response to stress in an island population of tuatara (Sphenodon punctatus) across two mating seasons. Tuatara are promiscuous, with a highly skewed mating system where males face significant competition for access to mates and postcopulatory competition for fertilization success. We found that tuatara sperm viability and swim speed were negatively associated with male body condition and the ratio of heterophils to lymphocytes. Additionally, sperm swim speed was negatively associated with spine area, mite load, and the total number of circulating white blood cells, but was positively associated with tick number. This is likely a function of social dynamics in this system where larger male size predicts greater spatial overlap with potential rivals and increased tick load. Because the production of sexual characteristics may be costly, we also investigated the effect of diet on sperm quality. We did not identify an association between diet and sperm viability. However, sperm swim speed was negatively associated with carbon-13 and positively associated with nitrogen-15. We suspect that these results reflect the influence of seabird-based nutrients in this island ecosystem, particularly polyunsaturated fatty acid, and antioxidant damage on tuatara sperm. In total, these results provide evidence of a trade-off between pre- and post-copulatory sexual characteristics and the immune and endocrine systems in male tuatara.

Abstract Observations of reptile courting and mating behaviour are relatively scant in the literature. Here, using the largest sample of observed courting attempts and matings in tuatara (Sphenodon punctatus) ever published, we describe novel behaviours in this rare species: mirrored head bobbing between courting pairs, purring vocalizations by displaying males, and several potential instances of anejaculation in otherwise normal matings. These results highlight the complexity of reptile mating displays and have implications for how we manage this vulnerable species.

Leukocyte profiles are broadly used to assess the health status of many species. Reference intervals, and an understanding of the factors that may influence these intervals, are necessary for adequate interpretation of leukograms. Using a data set that spans over three decades, we investigated variation in leukocyte profile in several populations of the evolutionarily unique reptile, the tuatara (Sphenodon punctatus). To do this, we first established reference intervals for each leukocyte type according to best practices. Next, we determined that source population and sampling date were the two most important predictors of leukocyte makeup. We found significant differences in the ratio of heterophils: lymphocytes (H:L) between populations, with tuatara on the more resource-stressed sampling island having a significantly higher ratio of H:L. Finally, we found that sampling location, sex, and life stage did not explain variation in the responses of tuatara to stimulation with Concanavalin A and lipopolysaccharide in both 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide and Griess assay experiments. Our results offer important insight into the function of leukocytes in reptiles.

Some species have held fast for millions of years as constants in a changing world. Often called “living fossils,” these species capture scientific and public interest by showing us the vestiges of an earlier world. If living fossils are defined by a holistic pattern of low evolutionary rates or stasis, however, then classifying a species as a living fossil involves the application of sophisticated norms of scientific evidence. Using examples from Crocodilia and the tuatara (Sphenodon punctatus), I show how scientists’ evidential criteria for classifying living fossils are contentious and underspecified in many cases, threatening the concept’s explanatory interest and its adequacy for sustaining a collective problem agenda as proposed by Scott Lidgard and Alan Love. While debates over the definition of the living fossil concept may appear fruitless, I suggest they can be productive insofar as the debate leads to clarified and improved evidential standards for classification. To this end, I formulate a view of the living fossil concept as an investigative kind, and compare two theoretical frameworks as a basis for shared evidential norms: the Zero Force Evolutionary Law framework, introduced by Daniel McShea and Robert Brandon, and the statistical model selection framework first developed by Gene Hunt in the 2000s.

Background: DNA barcoding allows for species identification and description of genetic diversity. However, in the Middle East, information on genetic diversity is accumulating at a slower pace compared to that of other regions. Methods: The COI sequence of 24 lizard and snake species in Qatar that represent major families within the order Squamata were sampled and amplified via PCR using RepCOI primers (apart from one species). Purified amplicons were then aligned, and high- quality sequences were uploaded to BOLD. Using Sphenodon punctatus as the outgroup, the phylogenetic analysis was conducted using raxmlGUI software following the maximum likelihood method. Results: The COI sequence from each of the species was obtained and the consensus sequences were submitted to GenBank. In the phylogenetic analysis, a close relationship between members of the Agamidae and Serpentes was confirmed. While members of the same genus often showed sister-taxa relationships, and species in the same family were clustered with reasonably high bootstrap supports, the COI-based phylogeny was not able to resolve the relationships among genera within the families or identify relationships with high resolution at deeper lineages. Conclusion: Although ideal for species identification, COI gene sequencing is limited in phylogenetic inference due to high mutation rates that restrict its effectiveness for resolving relationships at deep phylogenetic levels. However, COI gene sequencing can be combined with nuclear markers for a more in-depth analysis.

Biodiversity is the most emphasized term in ecology that keeps the ecosystem wonderful and stable. However, invasion and endangerment are two phenomena of disturbance of biodiversity. The features of these kinds of species should be determined for further animal protection. Through four main case studies, the article describes how such species proliferate or nearly distinct. Python Bivattatus, Rhinella Marina, Sphenodon punctatus, and Ambystoma mexicanum would be focused, and typical features of reproduction, niche, and feeding behavior indicate the phenomena. It is concluded that invasion is related to climate differentiation, energy cost, and adaptation. The specialty of poikilothermal animals in the ecosystem is important to discover the mystery of their invasion and endangerment. Furthermore, the relationship between invasion and endangerment is revealed, and several government solutions are mentioned.

Understanding the origins of the vertebrate brain is fundamental for uncovering evolutionary patterns in neuroanatomy. Regarding extinct species, the anatomy of the brain and other soft tissues housed in endocranial spaces can be approximated by casts of these cavities (endocasts). The neuroanatomical knowledge of Rhynchocephalia, a reptilian clade exceptionally diverse in the early Mesozoic, is restricted to the brain of its only living relative, Sphenodon punctatus, and unknown for fossil species. Here, we describe the endocast and the reptilian encephalization quotient (REQ) of the Triassic rhynchocephalian Clevosaurus brasiliensis and compare it with an ontogenetic series of S. punctatus. To better understand the informative potential of endocasts in Rhynchocephalia, we also examine the brain-endocast relationship in S. punctatus. We found that the brain occupies 30% of its cavity, but the latter recovers the general shape and length of the brain. The REQ of C. brasiliensis (0.27) is much lower than S. punctatus (0.84-1.16), with the tuatara being close to the mean for non-avian reptiles. The endocast of S. punctatus is dorsoventrally flexed and becomes more elongated throughout ontogeny. The endocast of C. brasiliensis is mostly unflexed and tubular, possibly representing a more plesiomorphic anatomy in relation to S. punctatus. Given the small size of C. brasiliensis, the main differences may result from allometric and heterochronic phenomena, consistent with suggestions that S. punctatus shows peramorphic anatomy compared to Mesozoic rhynchocephalians. Our results highlight a previously undocumented anatomical diversity among rhynchocephalians and provide a framework for future neuroanatomical comparisons among lepidosaurs.

ABSTRACTThe morphological characteristics that impact feeding ecology in ectotherms, particularly reptiles, are poorly understood. We used morphometric measures and stable isotope analysis (carbon-13 and nitrogen-15) to assess the link between diet and functional morphology in an island population of an evolutionarily unique reptile, the tuatara (Sphenodon punctatus). First, we established a significant positive correlation between overall body size, gape size, and fat store in tuatara (n=56). Next, we describe the relationship between stable isotope profiles created from whole blood and nail trim samples and demonstrate that nail trims offer a low-impact method of creating a long-term dietary profile in ectotherms. We used nitrogen-15 values to assess trophic level in the population and found that tuatara on Takapourewa forage across multiple trophic levels. Finally, we found a significant relationship between gape size and carbon-13 (linear regression: P<0.001), with tuatara with large gapes showing dietary profiles that suggest a higher intake of marine (seabird) prey. However, whether body size or gape size is the primary adaptive characteristic allowing for more optimal foraging is yet unknown.This article has an associated First Person interview with the first author of the paper.

Cloacal virome of an ancient host lineage – The tuatara (Sphenodon punctatus) – Reveals abundant and diverse diet-related viruses

During development, the embryonic cartilaginous skull in most vertebrates is partially replaced by bones with endochondral and perichondral ossifications. Muscle attachments are thought to influence the patterns of ossification and, hence, the differentiation of the skull. To investigate the association between muscle attachments and early ossifications of reptilian embryos, we conducted digital 3D reconstructions of the cranium, the head, and the neck musculature from a histological section series of a late term embryonic tuatara, Sphenodon punctatus, with a total body length of 52 mm. As the sole living rhynchocephalian species, it is an important outgroup in comparative studies of squamate evolution. We found that head and neck muscles are largely associated with early ossification of the basal plate and the palatoquadrate, and with three other ossifications in an older specimen with a total body length of 72 mm. These results suggest that tensile forces resulting from embryonic muscle contraction are largely, but not exclusively, correlated with the area of endochondral ossification in the chondrocranium and palatoquadrate in tuatara. Beyond little-known genetic factors, the complexity of chondrocranial architecture, the progress of its development, and the effect of multiple muscle transmitting forces in the chondrocranium must be considered to provide a more comprehensive discussion of the mechanical properties of the embryonic skull.

Sphenodontian reptiles are an extremely old evolutionary lineage forming the closest relatives to squamates (lizards and snakes) and were globally distributed and more diverse than squamates during the first half of their evolutionary history. However, the majority of their fossils are highly fragmentary, especially within sphenodontines—the group including its single surviving species, Sphenodon punctatus (the tuatara of New Zealand)—thus severely hampering our understanding on the origins of the tuatara. Here, we present a new sphenodontian species from the Early Jurassic of North America (Arizona, USA) represented by a nearly complete articulated skeleton and dozens of upper and lower jaws forming the most complete ontogenetic series in the sphenodontian fossil record. CT-scanning provides plentitude of data that unambiguously place this new taxon as one of the earliest evolving and oldest known sphenodontines. Comparisons with Sphenodon reveal that fundamental patterns of mandibular ontogeny and skeletal architecture in Sphenodon may have originated at least ~190Mya. In combination with recent findings, our results suggest strong morphological stability and an ancient origin of the modern tuatara morphotype.

Keratin intermediate filament chains in the European common wall lizard (Podarcis muralis) and a potential keratin filament crosslinker

Successful reproduction is critical to the persistence of at-risk species; however, reproductive characteristics are understudied in many wild species. New Zealand’s endemic tuatara (Sphenodon punctatus), the sole surviving member of the reptile order Rhynchocephalia, is restricted to 10% of its historic range. To complement ongoing conservation efforts, we collected and characterized mature sperm from male tuatara for the first time. Semen collected both during mating and from urine after courting contained motile sperm and had the potential for a very high percentage of viable sperm cells (98%). Scanning electron microscopy revealed a filiform sperm cell with distinct divisions: head, midpiece, tail, and reduced end piece. Finally, our initial curvilinear velocity estimates for tuatara sperm are 2–4 times faster than any previously studied reptile. Further work is needed to examine these trends at a larger scale; however, this research provides valuable information regarding reproduction in this basal reptile.

In response to anthropogenic threats, conservation translocations are increasingly used to combat species' population and range declines. However, moving animals outside of their current distribution can mean introducing them to novel conditions, even in the case of reintroductions to formerly inhabited areas due to ecosystem changes following extirpation. This exposure to novel conditions introduces uncertainty that can undermine decision making for species conservation. Here we propose two strategies, which we define as conservative and extrapolative, for approaching and managing novelty and the resulting uncertainty in conservation translocations. Conservative strategies are characterised by the avoidance and removal of novel conditions as much as possible, whereas extrapolative strategies are more experimental, allowing exposure to novel conditions and monitoring outcomes to increase understanding of a species' ecology. As each strategy carries specific risks and opportunities, they will be applicable in different scenarios. Extrapolative strategies suit species in recovery which can afford some experimental management, or species facing novel and emerging threats which require less traditional translocations, such as assisted colonisations. We provide examples, applying our framework to two endemic New Zealand species with long histories of translocation management: tuatara (Sphenodon punctatus), a reptile and takahē (Porphyrio hochstetteri), a flightless bird.

Population bottlenecks can reduce genetic diversity and may lead to inbreeding depression. However, some studies have provided evidence that long lifespans buffer negative genetic effects of bottlenecks. Others have cautioned that longevity might merely mask the effects of genetic drift, which will still affect long-term population viability. We used microsatellite data from actual populations of tuatara (Sphenodon punctatus) and eastern massasaugas (Sistrurus catenatus) as a starting point for simulated population declines to evaluate the performance of bottleneck tests under a range of scenarios. We quantified losses in genetic diversity for each scenario and assessed the power of commonly used tests (i.e., M-ratio, heterozygosity excess, and mode-shift) to detect known bottlenecks in these moderate- to long-lived species. Declines in genetic diversity were greater in bottlenecks simulated for eastern massasaugas, the shorter-lived species, and mode-shift and heterozygosity excess tests were more sensitive to population declines in this species. Conversely, M-ratio tests were more sensitive to bottlenecks simulated in tuatara. Despite dramatic simulated population declines, heterozygosity excess and mode-shift tests often failed to detect bottlenecks in both species, even when large losses in genetic diversity had occurred (both allelic diversity and heterozygosity). While not eliminating type II error, M-ratio tests generally performed best and were most reliable when a critical value (Mc) of 0.68 was used. However, in tuatara simulations, M-ratio tests had high rates of type I error when Mc was calculated assuming θ = 10. Our results suggest that reliance on these tests could lead to misguided species management decisions.

Abstract AimCorrelative species distribution models (SDMs) are typically trained using only the contemporary distribution of species; however, recent records might reflect an incomplete description of a species' niche, limiting the reliability of predictions. SDMs linking fossil records have the potential to improve conservation decisions under human‐induced climate change. Here, we built SDMs using presence records from contemporary and Holocene records to enable estimations of climatically suitable area under current and future climate scenarios.LocationAotearoa New ZealandTaxonTuatara, Sphenodon punctatusMethodsFor an evolutionary relict found in Aotearoa New Zealand, the tuatara (Sphenodon punctatus), we built SDMs using presence records from contemporary and Holocene records to estimate climatically suitable area under current and future climate scenarios. We also use our detailed knowledge of the Holocene distribution and remnant populations to examine niche shifts following the arrival of humans and associated introduction of mammalian predators. To build SDMs, we use presence records from four sources: (a) remnant populations, (b) radiocarbon‐dated fossil deposits from the Holocene, (c) other fossil deposits containing tuatara bones of Holocene age and iv) islands from which tuatara are known or highly likely to have become extinct.ResultsWe found shifts in the niche of tuatara due to niche unfilling. Incorporating locations of Holocene deposits and/or all past locations in SDMs led to larger areas of climatically suitable area being identified compared to SDMs derived from remnant populations only. Using all presence records, under climate change projections for 2090, climatically suitable area increased slightly. However, many areas retain potential as translocation sites (e.g. northern South Island), some areas become unsuitable (e.g. inland Canterbury) and/or involve extrapolation into novel climates (e.g. Northland).Main ConclusionSDMs incorporating locations of Holocene deposits and/or all past locations identified areas of critical habitat for tuatara under current and future climate scenarios, that would not have been identified using contemporary occurrences only. Our results highlight the need to consider past locations when assessing habitat suitability for conservation translocations, both for tuatara and other relict species.