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.

16S rRNA gene-based meta-analysis of the reptile gut microbiota reveals environmental effects, host influences and a limited core microbiota.

An animal's gut microbiota plays an important role in host health, reproduction and digestion. However, many studies focus on only a few individuals or a single species, limiting our ability to recognize emergent patterns across a wider taxonomic grouping. Here, we compiled and reanalysed published 16S rRNA gene sequence data for 745 gut microbiota samples from 91 reptile species using a uniform bioinformatics pipeline to draw broader conclusions about the taxonomy of the reptile gut microbiota and the forces shaping it. Our meta-analysis revealed the significant differences in alpha- and beta-diversity across host order, environment, diet, habitat and conservation status, with host diet and order contributing the most to these differences. We identified the principal bacterial phyla present in the reptile gut microbiota as Bacteroidota, Proteobacteria (mostly Gamma class), and Firmicutes, and detected the bacterial genus Bacteroides in most reptile individuals, thus representing a putative 'core' microbiota. Our study provides novel insights into key drivers of the reptile gut microbiota, highlights existing knowledge gaps and lays the groundwork for future research on these fascinating hosts and their associated microbes.

Characterization of sperm and implications for male fertility in the last of the Rhynchocephalians.

Managing a species of conservation concern can be best achieved when there is information on the reproductive physiology of both sexes available; however, many species lack this critical, baseline information. One such species, the tuatara (Sphenodon punctatus), is the last surviving member of one of the four reptile orders (Rhynchocephalia) and is the only reptile known to lack a male intromittent organ. Culturally and evolutionarily significant, the conservation of this species is a global priority for the maintenance of biodiversity. In light of this, we characterized the morphology, viability and swim speed of mature tuatara sperm for the first time. We found that tuatara sperm are filiform and bear the remarkably conserved three-part sperm structure seen across the animal kingdom. Tuatara sperm are long (mean total length 166 µm), with an approximate head:midpiece:tail ratio of 15:1:17. While tuatara sperm are capable of high levels of within-mating viability (94.53%), the mean viability across all samples was 58.80%. Finally, tuatara sperm had a mean curvilinear velocity swim speed (µ × s - 1) of 82.28. At the population level, there were no differences in viability or mean swim speed between sperm collected from a male's first mating of a season and repeat matings; however, the maximum sperm swim speed increased in observed repeated matings relative to first matings. Interestingly, faster sperm samples had shorter midpieces, but had greater viability and longer head and tail sections. This work expands our understanding of male reproductive characteristics and their variation to a new order, provides wild references for the assessment of captive individuals, lays the groundwork for potential assisted reproductive techniques and highlights variation in male reproductive potential as an important factor for consideration in future conservation programs for this unique species.

Investigating the link between morphological characteristics and diet in an island population of omnivorous reptiles (Sphenodon punctatus).

The 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.

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.

Thermoregulation of a temperate reptile in a forested habitat.

A major focus in zoology is to understand the phenotypic responses of animals to environmental variation. This is particularly important when dealing with ectotherms in a thermally heterogenous environment. We measured body temperatures of a free-ranging, medium sized temperate reptile, the tuatara, Sphenodon punctatus, to investigate its thermal opportunities and the degree to which the animal actively regulates its body temperature. We found high variation in body temperature between individuals, but this variation could not be attributed to sex or body size. However, variation among individuals in timing of burrow use did affect body temperature and in one of the years studied tuatara were found to be more effective in their thermoregulation when sharing a burrow with a seabird (Pachyptila turtur). The strength of this study is that it includes both biotic and behavioural components of the thermal environment of a temperate reptile, areas which are often missing from thermal studies that focus on the abiotic aspects.

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.

Genomic Epidemiology and Management of Salmonella in Island Ecosystems Used for Takahe Conservation.

Translocation and isolation of threatened wildlife in new environments may have unforeseen consequences on pathogen transmission and evolution in host populations. Disease threats associated with intensive conservation management of wildlife remain speculative without gaining an understanding of pathogen dynamics in meta-populations and how location attributes may determine pathogen prevalence. We determined the prevalence and population structure of an opportunistic pathogen, Salmonella, in geographically isolated translocated sub-populations of an endangered New Zealand flightless bird, the takahe (Porphyrio hochstetteri). Out of the nine sub-populations tested, Salmonella was only isolated from takahe living on one private island. The apparent prevalence of Salmonella in takahe on the private island was 32% (95% CI 13-57%), with two serotypes, Salmonella Mississippi and Salmonella houtenae 40:gt-, identified. Epidemiological investigation of reservoirs on the private island and another island occupied by takahe identified environmental and reptile sources of S. Mississippi and S. houtenae 40:gt- on the private island. Single nucleotide polymorphism analysis of core genomes revealed low-level diversity among isolates belonging to the same serotype and little differentiation according to host and environmental source. The pattern observed may be representative of transmission between sympatric hosts and environmental sources, the presence of a common unsampled source, and/or evidence of a recent introduction into the ecosystem. This study highlights how genomic epidemiology can be used to ascertain and understand disease dynamics to inform the management of disease threats in endangered wildlife populations.

Cryptic inbreeding depression in a growing population of a long-lived species.

Genetic effects are often overlooked in endangered species monitoring, and populations showing positive growth are often assumed to be secure. However, the continued reproductive success of a few individuals may mask issues such as inbreeding depression, especially in long-lived species. Here, we test for inbreeding depression in little spotted kiwi (Apteryx owenii) by comparing a population founded with two birds to one founded with 40 birds, both from the same source population and both showing positive population growth. We used a combination of microsatellite genotypes, nest observations and modelling to examine the consequences of assessing population viability exclusively via population growth. We demonstrate (i) significantly lower hatching success despite significantly higher reproductive effort in the population with two founders; (ii) positive growth in the population with two founders is mainly driven by ongoing chick production of the founding pair; and (iii) a substantial genetic load in the population founded with two birds (10-15 diploid lethal equivalents). Our results illustrate that substantial, cryptic inbreeding depression may still be present when a population is growing, especially in long-lived species with overlapping generations.

Microbial Genomics of a Host-Associated Commensal Bacterium in Fragmented Populations of Endangered Takahe.

Isolation of wildlife into fragmented populations as a consequence of anthropogenic-mediated environmental change may alter host-pathogen relationships. Our understanding of some of the epidemiological features of infectious disease in vulnerable populations can be enhanced by the use of commensal bacteria as a proxy for invasive pathogens in natural ecosystems. The distinctive population structure of a well-described meta-population of a New Zealand endangered flightless bird, the takahe (Porphyrio hochstetteri), provided a unique opportunity to investigate the influence of host isolation on enteric microbial diversity. The genomic epidemiology of a prevalent rail-associated endemic commensal bacterium was explored using core genome and ribosomal multilocus sequence typing (rMLST) of 70 Campylobacter sp. nova 1 isolated from one third of the takahe population resident in multiple locations. While there was evidence of recombination between lineages, bacterial divergence appears to have occurred and multivariate analysis of 52 rMLST genes revealed location-associated differentiation of C. sp. nova 1 sequence types. Our results indicate that fragmentation and anthropogenic manipulation of populations can influence host-microbial relationships, with potential implications for niche adaptation and the evolution of micro-organisms in remote environments. This study provides a novel framework in which to explore the complex genomic epidemiology of micro-organisms in wildlife populations.

Presence of antibodies to Salmonella in tuatara (Sphenodon punctatus) sera.

Colonisation of a host by pathogenic microorganisms is a near constant threat to the health of all vertebrates and most species have evolved an efficient adaptive immune response which produces antibodies following exposure to a specific antigen. The strength of this response can be influenced by many factors including sex and season. Tuatara are exposed to Salmonella through contact with infected skinks and soil; however, no gastrointestinal colonisation of tuatara with Salmonella has been found. Using Western blot and flow cytometry we have demonstrated that tuatara possess antibodies which recognise Salmonella antigens, but many of these antibodies are not specific and are cross-reactive with two closely related and ubiquitous bacteria, Escherichia coli and Citrobacter koseri. Our study describes the anti-Salmonella immune responses in tuatara and will help to inform decisions around maintaining wildlife health, as well as providing important insights into the role and development of adaptive immunity in reptilian species.

Using a common commensal bacterium in endangered Takahe as a model to explore pathogen dynamics in isolated wildlife populations.

Predicting and preventing outbreaks of infectious disease in endangered wildlife is problematic without an understanding of the biotic and abiotic factors that influence pathogen transmission and the genetic variation of microorganisms within and between these highly modified host communities. We used a common commensal bacterium, Campylobacter spp., in endangered Takahe (Porphyrio hochstetteri) populations to develop a model with which to study pathogen dynamics in isolated wildlife populations connected through ongoing translocations. Takahe are endemic to New Zealand, where their total population is approximately 230 individuals. Takahe were translocated from a single remnant wild population to multiple offshore and mainland reserves. Several fragmented subpopulations are maintained and connected through regular translocations. We tested 118 Takahe from 8 locations for fecal Campylobacter spp. via culture and DNA extraction and used PCR for species assignment. Factors relating to population connectivity and host life history were explored using multivariate analytical methods to determine associations between host variables and bacterial prevalence. The apparent prevalence of Campylobacter spp. in Takahe was 99%, one of the highest reported in avian populations. Variation in prevalence was evident among Campylobacter species identified. C. sp. nova 1 (90%) colonized the majority of Takahe tested. Prevalence of C. jejuni (38%) and C. coli (24%) was different between Takahe subpopulations, and this difference was associated with factors related to population management, captivity, rearing environment, and the presence of agricultural practices in the location in which birds were sampled. Modeling results of Campylobacter spp. in Takahe metapopulations suggest that anthropogenic management of endangered species within altered environments may have unforeseen effects on microbial exposure, carriage, and disease risk. Translocation of wildlife between locations could have unpredictable consequences including the spread of novel microbes between isolated populations.

Moving house: long-term dynamics of corticosterone secretion are unaltered in translocated populations of a rare reptile (the tuatara, Sphenodon punctatus).

Translocations are an important conservation tool used to restore at-risk species to their historical range. Unavoidable procedures during translocations, such as habitat disturbance, capture, handling, processing, captivity, transport and release to a novel environment, have the potential to be stressful for most species. In this study, we examined acute and chronic stress (through the measurement of the glucocorticoid corticosterone) in a rare reptile (the tuatara, Sphenodon punctatus). We found that: (i) the acute corticosterone response remains elevated during the initial translocation process but is not amplified by cumulative stressors; and (ii) the long-term dynamics of corticosterone secretion are similar in translocated and source populations. Taken together, our results show that translocated tuatara are generally resistant to cumulative acute stressors and show no hormonal sign of chronic stress. Translocation efforts in tuatara afford the potential to reduce extinction risk and restore natural ecosystems.

Reptile reservoirs and seasonal variation in the environmental presence of Salmonella in an island ecosystem, Stephens Island, New Zealand.

A wide variety of Salmonella serotypes occurs within reptilian hosts, but their ecology is poorly understood. We collected cloacal swabs from tuatara (Sphenodon punctatus), fairy prions (Pachyptila turtur), and skinks (Oligosoma spp.) on Stephens Island, New Zealand, to screen for Salmonella. Soil samples were also collected from inside burrows of tuatara and fairy prions and tested for Salmonella. We sampled repeatedly from October 2009 to October 2011. Cloacal swabs were collected from 620 tuatara, and no intestinal shedding of Salmonella was detected. Similarly, no Salmonella was detected in fairy prions. In contrast, we isolated Salmonella from 6.5% of skinks and 8.4% of soil samples. We identified two serovars of Salmonella from 52 isolates, Salmonella Saintpaul and Salmonella Mississippi. Salmonella Mississippi was isolated from skinks only and S. Saintpaul was found in skinks and soil samples. Salmonella persists in this ecosystem with skinks as the main wildlife reservoir, and an environmental reservoir exists in the soil from burrows used by skinks, tuatara, and fairy prions. Salmonella was absent from skinks and the soil in winter, raising the question of bacteria persisting through winter.

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.

Investigating kleptothermy: a reptile-seabird association with thermal benefits.

Studies on interspecific interactions between vertebrates based on thermal benefits are poorly represented in the literature. Ecologists know little about a category of thermoregulation termed kleptothermy. We provide evidence that a close association between a medium-sized reptile (tuatara, Sphenodon punctatus) and a small seabird (fairy prion, Pachyptila turtur) enables the reptile to maintain higher-than-average body temperatures. This is the first multiyear data set to reveal that the presence of an endotherm within a burrow has direct, transferable thermal benefits to an ectotherm. It is possible that such positive interspecific interactions may alleviate thermal stress across the range of a single species. Currently, we know little about the evolutionary consequences of such interactions, and future work should focus on whether these thermal benefits increase fitness through increased growth rates or reproductive output.

De novo sequence assembly and characterisation of a partial transcriptome for an evolutionarily distinct reptile, the tuatara (Sphenodon punctatus).

BACKGROUND: The tuatara (Sphenodon punctatus) is a species of extraordinary zoological interest, being the only surviving member of an entire order of reptiles which diverged early in amniote evolution. In addition to their unique phylogenetic placement, many aspects of tuatara biology, including temperature-dependent sex determination, cold adaptation and extreme longevity have the potential to inform studies of genome evolution and development. Despite increasing interest in the tuatara genome, genomic resources for the species are still very limited. We aimed to address this by assembling a transcriptome for tuatara from an early-stage embryo, which will provide a resource for genome annotation, molecular marker development and studies of development and adaptation in tuatara. RESULTS: We obtained 30 million paired-end 50 bp reads from an Illumina Genome Analyzer and assembled them with Velvet and Oases using a range of kmers. After removing redundancy and filtering out low quality transcripts, our transcriptome dataset contained 32911 transcripts, with an N50 of 675 and a mean length of 451 bp. Almost 50% (15965) of these transcripts could be annotated by comparison with the NCBI non-redundant (NR) protein database or the chicken, green anole and zebrafish UniGene sets. A scan of candidate genes and repetitive elements revealed genes involved in immune function, sex differentiation and temperature-sensitivity, as well as over 200 microsatellite markers. CONCLUSIONS: This dataset represents a major increase in genomic resources for the tuatara, increasing the number of annotated gene sequences from just 60 to almost 16,000. This will facilitate future research in sex determination, genome evolution, local adaptation and population genetics of tuatara, as well as inform studies on amniote evolution.

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.

Ecology and dynamics of the blood parasite, Hepatozoon tuatarae (Apicomplexa), in tuatara (Sphenodon punctatus) on Stephens Island, New Zealand.

We explored infection patterns and temporal dynamics of the protozoan blood parasite Hepatozoon tuatarae (Apicomplexa) infecting the tuatara (Sphenodon punctatus), a protected reptile living on Stephens Island, New Zealand. In March 2006, we surveyed tuatara in five study sites to examine spatial variation in infection prevalence, and four times, from May 2005 to November 2006, we recaptured marked individuals within three study sites to examine the temporal dynamics of infection. We also examined how blood-parasite infection patterns were influenced by host sex, body size, and host infestation with ticks (Amblyomma sphenodonti) and mites (Neotrombicula spp.), which are potential vectors of the blood parasite. Infection prevalence (16.9-24% infected) and intensity (<0.01-0.1% blood cells infected) were low in all samples. Infection intensity varied among the five sampled sites in March 2006, but prevalence did not. Neither infection prevalence nor intensity varied with time, and infections were detected in consecutive samples from recaptured individuals for up to 18 mo. Neither survey showed an influence of host sex on infection, but both surveys showed infection intensity declined with increasing host body size, as did infection prevalence in the spatial survey. In the temporal survey, we found a positive relationship between the tick numbers on hosts and blood-parasite infection intensity, which were stronger in two of the sampling periods and among larger hosts. These data suggest that exposure and susceptibility to infection decreases with host size and that ticks, but not mites, are probably the vectors in this ancient host-parasite association of a long-lived (>50 yr) host.