Gut microbiome of the sole surviving member of reptile order Rhynchocephalia reveals biogeographic variation, influence of host body condition and a substantial core microbiota in tuatara across New Zealand.

Tuatara are the sole extant species in the reptile order Rhynchocephalia. They are ecologically and evolutionarily unique, having been isolated geographically for ~84 million years and evolutionarily from their closest living relatives for ~250 million years. Here we report the tuatara gut bacterial community for the first time. We sampled the gut microbiota of translocated tuatara at five sanctuaries spanning a latitudinal range of ~1000 km within Aotearoa New Zealand, as well as individuals from the source population on Takapourewa (Stephens Island). This represents a first look at the bacterial community of the order Rhynchocephalia and provides the opportunity to address several key hypotheses, namely that the tuatara gut microbiota: (1) differs from those of other reptile orders; (2) varies among geographic locations but is more similar at sites with more similar temperatures and (3) is shaped by tuatara body condition, parasitism and ambient temperature. We found significant drivers of the microbiota in sampling site, tuatara body condition, parasitism and ambient temperature, suggesting the importance of these factors when considering tuatara conservation. We also derived a 'core' community of shared bacteria across tuatara at many sites, despite their geographic range and isolation. Remarkably, >70% of amplicon sequence variants could not be assigned to known genera, suggesting a largely undescribed gut bacterial community for this ancient host species.

Initial collection, characterization, and storage of tuatara (Sphenodon punctatus) sperm offers insight into their unique reproductive system.

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.

Behavioral variation in nesting phenology may offset sex-ratio bias in tuatara.

The nest environment for eggs of reptiles has lifelong implications for offspring performance and success, and, ultimately, for population viability and species distributions. However, understanding the various abiotic and biotic drivers of nesting is complex, particularly regarding variation in nesting behavior of females and consequences for sex ratios in species with temperature-dependent sex determination (TSD). We investigated how nest construction and nesting phenology affect the incubation environment of a reptile with TSD, the tuatara (Sphenodon punctatus), a species that is at risk from climate-mediated male bias in population sex ratios. Using longitudinal behavioral data, we addressed the following questions. (1) Does nesting behavior vary with seasonal or location cues? (2) Does variation in nesting phenology or nest construction affect the incubation environment? We aimed to investigate whether female tuatara could modify nesting behavior to respond to novel environments, including a warming climate, allowing for successful incubation and balanced population sex ratios, maintaining population viability throughout their historic range. We predicted that earlier nesting after warm winters increased the likelihood that females will be produced, despite the sex determining system where males are produced from warmer temperatures. Further research is needed to understand the extent to which nesting behavior varies by individual through time, and across the range of tuatara, and the importance of habitat variability in maintaining production of females under future climate warming.

Temperature selection by juvenile tuatara (Sphenodon punctatus) is not influenced by temperatures experienced as embryos.

Most reptiles thermoregulate to achieve body temperatures needed for biological processes, such as digestion and growth. Temperatures experienced during embryogenesis may also influence post-hatching growth rate, potentially through influencing post-hatching choice of temperatures. We investigated in laboratory settings whether embryonic temperatures (constant 18°C, 21°C and 22°C) influence selected body temperatures (Tsel) of juvenile tuatara (Sphenodon punctatus), providing a possible mechanism for differences in growth rates. We found that incubation temperature does not influence Tsel. Although the average daily mean Tsel was 21.6 ± 0.3°C, we recorded individual Tsel values up to 33.5°C in juvenile tuatara, which is higher than expected and above the panting threshold of 31-33°C reported for adults. We found diel patterns of Tsel of juvenile tuatara, observing a general pattern of two apparent peaks and troughs per day, with Tsel being significantly lower around dawn and at 1500h than any other time. When comparing our results with other studies on tuatara there is a remarkable consistency in mean Tsel of ~ 21°C across tuatara of different ages, sizes and acclimatization histories. The ability of juvenile tuatara to withstand a wide range of temperatures supports their former widespread distribution throughout New Zealand and warrants further investigation into their plasticity to withstand climate warming, particularly where they have choices of habitat and the ability to thermoregulate.

Sex ratio bias and extinction risk in an isolated population of Tuatara (Sphenodon punctatus).

Understanding the mechanisms underlying population declines is critical for preventing the extinction of endangered populations. Positive feedbacks can hasten the process of collapse and create an 'extinction vortex,' particularly in small, isolated populations. We provide a case study of a male-biased sex ratio creating the conditions for extinction in a natural population of tuatara (Sphenodon punctatus) on North Brother Island in the Cook Strait of New Zealand. We combine data from long term mark-recapture surveys, updated model estimates of hatchling sex ratio, and population viability modeling to measure the impacts of sex ratio skew. Results from the mark-recapture surveys show an increasing decline in the percentage of females in the adult tuatara population. Our monitoring reveals compounding impacts on female fitness through reductions in female body condition, fecundity, and survival as the male-bias in the population has increased. Additionally, we find that current nest temperatures are likely to result in more male than female hatchlings, owing to the pattern of temperature-dependent sex determination in tuatara where males hatch at warmer temperatures. Anthropogenic climate change worsens the situation for this isolated population, as projected temperature increases for New Zealand are expected to further skew the hatchling sex ratio towards males. Population viability models predict that without management intervention or an evolutionary response, the population will ultimately become entirely comprised of males and functionally extinct. Our study demonstrates that sex ratio bias can be an underappreciated threat to population viability, particularly in populations of long-lived organisms that appear numerically stable.

Securing the demographic and genetic future of tuatara through assisted colonization.

Climate change poses a particular threat to species with fragmented distributions and little or no capacity to migrate. Assisted colonization, moving species into regions where they have not previously occurred, aims to establish populations where they are expected to survive as climatic envelopes shift. However, adaptation to the source environment may affect whether species successfully establish in new regions. Assisted colonization has spurred debate among conservation biologists and ecologists over whether the potential benefits to the threatened species outweigh the potential disruption to recipient communities. In our opinion, the debate has been distracted by controversial examples, rather than cases where assisted colonization may be a viable strategy. We present a strategic plan for the assisted migration of tuatara (Sphenodon punctatus), an endemic New Zealand reptile. The plan includes use of extant populations as reference points for comparisons with assisted-colonization populations with respect to demography, phenotypic plasticity, and phenology; optimization of genetic variation; research to fill knowledge gaps; consideration of host and recipient communities; and inclusion of stakeholders in the planning stage. When strategically planned and monitored, assisted colonization could meet conservation and research goals and ultimately result in the establishment of long-term sustainable populations capable of persisting during rapid changes in climate.

Support for a rare pattern of temperature-dependent sex determination in archaic reptiles: evidence from two species of tuatara (Sphenodon).

BACKGROUND: The sex of many reptiles is determined by the temperature an embryo experiences during its development. Three patterns of temperature-dependent sex determination (TSD) have been defined, but one pattern where only males are produced above an upper temperature threshold (Type IB) is controversial. Here we report new data on the relationship between constant temperature incubation and sexual phenotype in two species of tuatara (Sphenodon), archaic reptiles of enormous zoological significance as the sole representatives of a once widespread reptilian order. RESULTS: In both species, the pattern observed with constant incubation temperatures from 18 to 23 degrees C (or 24 degrees C) supported a female-->male (FM or Type IB) pattern of TSD: in Sphenodon guntheri males were produced above a pivotal temperature of 21.6 degrees C, and in S. punctatus (unnamed subspecies on Stephens Island, Cook Strait), males were produced above a pivotal temperature of 22.0 degrees C. The pivotal temperatures and scaling parameters differed between species (p < 0.001). The thermosensitive period (TSP), where temperature influences gonad morphogenesis, occurs between 0.25 and 0.55 of embryonic development. While it is possible that the more common female-->male-->female (FMF or Type II) pattern exists, with a second pivotal temperature above 23-24 degrees C, we review several lines of evidence to the contrary. Most notably, we show that in S. punctatus, the warmest natural nests during the TSP produce predominantly males. CONCLUSION: An FM pattern of TSD could be currently adaptive in promoting sexual size dimorphism in tuatara. However, an FM pattern has particularly serious consequences for S. guntheri because current patterns of global warming could exacerbate the male bias already present in the relic population.

T cell function in tuatara (Sphenodon punctatus).

Tuatara are the sole survivors of an entire order of reptiles that thrived during the age of the dinosaurs. Therefore, knowledge of their physiology is critical to understanding the phylogeny of reptiles. Previous studies of the immune system of the tuatara did not assess T cell function. We analyzed T cell function among six captive tuatara by assessing concanavalin A (Con A), phytohemagglutinin (PHA) and mixed lymphocyte reaction (MLR) induced T cell proliferation. Peripheral blood mononuclear cells from six out of six and four out of four tuatara tested exhibited significant proliferative responses to Con A and PHA, respectively, as measured by an MTT reduction assay. A lower level of proliferation was detected in an MLR. However, Con A activated lymphocytes were not cytotoxic for a xenogeneic murine mastocytoma cell line (P815).

Burrowing seabirds and reptiles: impacts on seeds, seedlings and soils in an island forest in New Zealand.

Stephens Island (Cook Strait, New Zealand) is home to large populations of fairy prions (Pachyptila turtur) and tuatara (Sphenodon punctatus, an ancient reptile), which share burrows in the ground. It has been assumed that guano deposition by seabirds increases nutrient availability to plants, and that large populations of the carnivorous tuatara are the result of flow-through effects from plants to invertebrate herbivores. We examined the within-island scale effects of seabirds and tuatara on forest soils and vegetation along a gradient of burrow densities. High burrow densities were correlated with extremely low soil pH (down to 3.4), very high soil P (up to 3.9 mg P g-1), and high litter deposition but low ground litter. Seedling numbers declined marginally as burrow numbers increased. Seedling emergence rates from field soils, and germination and growth of a phytometer species also decreased in soils from areas with high burrow densities. In contrast, soil available N (NO3- and NH4+) was high everywhere and did not change with burrow density. Furthermore, of three common shrub species examined (Macropiper excelsum, Coprosma repens and Melicytus ramiflorus), only one (Macropiper excelsum) showed an increase in leaf N concentration with increased burrow density, and only one (C. repens) showed increased herbivory (number of holes per leaf and area per leaf removed) with increased burrow density. Birds descending through trees may increase litter loss. There is little evidence for changes in herbivory or nutrient availability to herbivores along burrow density gradients. We suggest that, although overall nutrient availability may be increased by the presence of these animals, very high densities have negative effects on seedling populations.