Female Tuatara Research Articles

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.

Patterns of Nesting Migrations in the Tuatara (Sphenodon punctatus), A Colonially Nesting Island Reptile

The costs of nesting to female animals can be substantial, and may dictate patterns of nest-site choice. In particular, females that travel longer distances to nest likely incur greater energetic costs and may be at greater risk of losing territories to conspecific competitors. We examined patterns of nesting migrations for a population of Tuatara on Stephens Island, New Zealand, over 5 yr. We found that only about 7% of female Tuatara observed nesting in a rookery also lived in that rookery. Of 140 females for which we had both residence and nesting locations, 42% traveled to a nesting rookery from their residential area. The average distance traveled by these females was 84 m, and travel was directed toward the rookery closest to a female's residence area. Our results demonstrate that female Tuatara on Stephens Island minimize the distance traveled on nesting migrations. We suggest that the patterns in nesting travel we observed in this population may be due to natal philopatry of nesting females, or to females minimizing the costs of nesting-related travel.

Sexual dimorphism, body size, bite force and male mating success in tuatara

287..292 Sexual dimorphisms in body size and head size are common among lizards and are often related to sexual selection on male fighting capacity (organismal performance) and territory defence. However, whether this is generally true or restricted to lizards remains untested. Here we provide data on body and head size, bite performance and indicators of mating success in the tuatara (Sphenodon punctatus), the closest living relative to squamates, to explore the generality of these patterns. First, we test whether male and female tuatara are dimorphic in head dimensions and bite force, independent of body size. Next, we explore which traits best predict bite force capacity in males and females. Finally, we test whether male bite force is correlated with male mating success in a free-ranging population of tuatara (Sphenodon punctatus). Our data confirm that tuatara are indeed dimorphic in head shape, with males having bigger heads and higher bite forces than females. Across all individuals, head length and the jaw closing in-lever are the best predictors of bite force. In addition, our data show that males that are mated have higher absolute but not relative bite forces. Bite force was also significantly correlated to condition in males but not females. Whereas these data suggest that bite force may be under sexual selection in tuatara, they also indicate that body size may be the key trait under selection in contrast to what is observed in squamates that defend territories or resources by biting. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 287‐292.

Nest‐site choice and fidelity in tuatara on Stephens Island, New Zealand

Nest-site microhabitat influences hatching success, hatchling phenotype and offspring sex in reptiles with temperature-dependent sex determination (TSD). How females assess environmental features at potential nest sites, and then use such features in predicting the future incubation regime of the site, is integral to understanding how nest-site choice affects offspring fitness and ultimately female reproductive success. Tuatara Sphenodon punctatus are colonially nesting reptiles with TSD. We examined nest-site fidelity and nest-site choice in tuatara over 5 years on Stephens Island, New Zealand. Female tuatara nested every 2–4 years and showed high fidelity to nesting rookeries. Over 93% of females nested in the same rookery at least twice in 5 years. Approximately 25% of nests contained conspecific cues from previous nesting seasons, indicating that some females choose nest sites based on locations already selected by conspecifics. In experimental plots, female tuatara selected nest sites with loose soil and minimal vegetation, but they showed no preference for shaded compared with unshaded sites. This study provides insight into the development of colonial nesting structures in reptiles in that females are both attracted to nesting areas used by conspecifics, and show strong site fidelity to areas they have used in the past.

Does Nest-Guarding in Female Tuatara (Sphenodon punctatus) Reduce Nest Destruction by Conspecific Females?

Although parental care is generally rare among reptiles, nest-guarding occurs in some species and is usually attributed to defense against nest predation. Nest-guarding also occurs in the Tuatara (Sphenodon punctatus), but nest predation on rodent-free islands does not appear to be a significant threat to nesting success in this species. We studied a population of colonially nesting Tuatara on rodent-free Stephens Island, New Zealand, over four years and tested the hypothesis that female Tuatara guard their nests to defend them from excavation by conspecific females. We located 73 nests for which females could be assigned based on observations during oviposition. Nearly 25% of these nests were subsequently excavated by another female, but only 56% of the nests were guarded by the females that constructed them. We found a trend where guarded nests were less likely to be excavated than unguarded nests. Females were more likely to guard their nests, and guarded nests for longer, as the activity of other females on the date of oviposition increased. Nest-guarding in Tuatara appears to be adaptive in that it tends to reduce the likelihood of nest excavation by other females, but social interactions may affect females' propensity to guard, because guarding behavior was influenced by the activity of conspecifics at the time of oviposition.

Do TSD, sex ratios, and nest characteristics influence the vulnerability of tuatara to global warming?

Tuatara ( Sphenodon punctatus) are threatened New Zealand reptiles with temperature-dependent sex determination (TSD). Higher incubation temperatures produce males, and less than 1 °C separates production of males and females. We investigated variability in nesting ecology to assess whether global warming is likely to result in increasingly male-biased populations. We examined nesting seasons during 1998/1999 and 2002/2003 in New Zealand's largest tuatara population on Stephens Island, and collected hourly temperature recordings and physical descriptions from 70 nests. Nest depths were not significantly different between years, and ranged from 10 to 230 mm from the soil surface to the top egg. Incubation temperatures in successful nests throughout the year-long incubation period ranged from 1.6 to 38.4 °C. Sex ratios of nests were correlated with incubation temperature: 64% males were produced in 1998/1999, a relatively warm season, but we predict an equal sex ratio was produced in 2002/2003. Although temperatures varied over the 2002/2003 season with respect to monthly long-term averages, 2002 was the second warmest year on record. Stephens Island supports a wide range of nesting habitat, a relatively large population of tuatara, and nest characteristics are highly variable. As such, this population is likely to be resilient to global warming in the short term because an equal sex ratio was predicted from a relatively warm season. However, most other islands where tuatara occur are smaller, have smaller populations, and have fewer open areas for nesting and/or shallower soils. These conditions are more likely to produce a male bias in hatchlings because female tuatara do not appear to vary construction of nests with respect to temperature or location. In the extreme, this could lead to the extinction of small populations.

Relationships between Corticosterone Concentration and Season, Time of Day and Confinement in a Wild Reptile (Tuatara,Sphenodon punctatus)

Seasonal and daily variation in basal plasma concentrations of corticosterone and the effect of short-term confinement on plasma corticosterone concentration were examined in wild adult male and female tuatara (Sphenodon punctatus) on Stephens Island, New Zealand. Males and females sampled immediately upon capture at night (active period) showed significant variation in basal plasma corticosterone among four seasons of the year, and females also showed variation in concentration between reproductive states sampled at the same time. Highest basal concentrations in females were seen in November, when the level in gravid females about to nest (4.45±0.66 ng/ml; mean±SE) was almost twice that in nonnesting females at the same time (2.48±0.30 ng/ml). Plasma samples collected from tuatara at dusk, middle of the night, dawn, and middle of the day showed no significant daily variation in corticosterone concentration in either summer or winter. In these samples, significant positive correlations between body temperature and log plasma corticosterone were observed in males in summer and winter, but not in females in either season. Mean corticosterone concentrations in tuatara held in cloth bags for 3 h were significantly higher than in free-roaming controls (P<0.01), with mean concentrations in males about 23 times, and vitellogenic females 9 times, those of their respective controls. Plasma corticosterone concentrations measured in this stress study were compared with previously published concentrations for plasma sex steroids in the same individuals. Corticosterone was positively correlated with progesterone (both sexes), but not with testosterone (both sexes) or estradiol (measured in females only). In summary, wild adult tuatara have low basal levels of plasma corticosterone that vary seasonally, but not diurnally. As in other reptiles, corticosterone concentration shows a significant elevation in response to short-term confinement.

Influence of X‐rays on incubation in tuatara, Sphenodon punctatus

Eggs and hatchlings from female tuatara, Sphenodon punctatus, that were X‐rayed immediately before oviposition were compared with eggs and hatchlings from non‐X‐rayed females. Eggs were incubated at 18, 20 or 22°C, ‐230 or ‐400 kPa water potential, and either half buried or completely buried in the incubation substratum. Incubation times and water fluxes of eggs were recorded. Hatchlings were weighed and measured. Total oxygen consumption of eggs incubating on the surface of substrata at ‐230 kPa and 18, 20 and 22°C was measured. At 20cC and buried at ‐230 kPa, X‐rayed eggs absorbed more water than non‐X‐rayed eggs, although the resulting hatchlings were not significantly different in size and mass. No other egg or hatchling parameter was significantly influenced by X‐rays. The only deformed hatchlings emerged from eggs that had not been X‐rayed. Thus, with the exception of possible germ cell damage, which could not be assessed in this study, we conclude that radiography of females with oviductal eggs had no affect on embryonic development.

Purification, Partial Characterization and Peptide Sequences of Vitellogenin from a Reptile, the Tuatara (Sphenodon punctatus)

Vitellogenin (Vg), a major precursor to egg yolk proteins, was purified from plasma of an estradiol-treated female tuatara (Sphenodon punctatus) by MgCl2–EDTA precipitation and DEAE-cellulose chromatography. The amino acid composition of tuatara Vg is similar to that of other vertebrate Vgs and contains a large proportion of serine (13.7 mol/100 mol of total amino acid). The amino acid sequences of the N-terminus of mature Vg (33 residues) and of several trypsin- and CNBr-generated peptides were determined. Six peptide sequences obtained from tuatara Vg could be aligned with Vg sequences from other vertebrates. Reduced and non-reduced forms of tuatara Vg have the same apparent molecular mass (∼218 kDa) when resolved by SDS-PAGE, indicating that inter-chain disulfide bonds are not a feature of the molecule in this species. Western blot analysis with anti-tuatara Vg antiserum indicated that at least some epitopes are shared among Vgs of turtle, alligator and tuatara.

Eggshell formation during prolonged gravidity of the tuatara Sphenodon punctatus.

Scanning electron microscopy (SEM) was used to examine the process of shell formation in tuatara. Tuatara carry eggs in the oviducts for ∼ 7-8 mo before nesting, a period of gravidity more than three times as long as in any other oviparous reptile. Our aim was to determine whether shell formation occurred rapidly after ovulation, or whether it occurred gradually throughout gravidity. Eggs were obtained from females in early gravidity (May, ∼ 1 mo after ovulation), midgravidity (August and September, 4-5 mo after ovulation), and late gravidity, immediately prior to nesting (December, 8 mo after ovulation). The shell membrane (fibrous layer) was well formed by May, but calcification of the outer surface had only just begun. Vertical columns of calcium carbonate were embedded in the shell membrane and appeared to erupt through the outer surface between early and midgravidity. Changes in the appearance of the outer calcareous layer were evident as gravidity progressed. In all shells, calcium carbonate was present as calcite. The appearance of the inner boundary (innermost layer of eggshell) was variable; some shells had a smooth and amorphous inner boundary as previously reported for tuatara and other reptiles, whereas other shells had an inner boundary composed of small spherical granules on the inner surface of which small calcareous spicules were scattered. A previously published model of the process of shell formation in tuatara eggshells is refined in light of our observations. We interpret the ability of female tuatara to shell their eggs gradually during winter as further evidence of their unusual physiological tolerance of cold conditions. © 1996 Wiley-Liss, Inc.

Plasma Concentrations of Vitellogenin and Sex Steroids in Female Tuatara ( Sphenodon punctatus punctatus) from Northern New Zealand

Vitellogenesis in tuatara ( Sphenodon punctatus) on Stephens Island, New Zealand, at the southern (coolest) end of the geographical range of this species involves a prolonged period of about 3 years of the average 4-year reproductive cycle. These studies used a semiquantitative electrophoretic assay for vitellogenin (Vg). In the present study an ELISA was used to measure plasma levels of Vg in 138 female northern tuatara ( S.p. punctatus) on 11 islands at the warmest end of the tuatara's range. Blood samples were collected in late summer-early autumn, just prior to the expected time of ovulation in those females that would nest the following spring. Plasma Vg levels ranged from nondetectable to 2.9 mg/ml but were not significantly correlated with plasma estradiol, testosterone, or progesterone levels. There was also no significant correlation among the three sex steroids. Plasma Vg and hormone levels were further examined as to whether females were ovulating. Incipient ovulation was inferred using endocrinological criteria derived from studies of tuatara on Stephens Island (i.e., elevated plasma testosterone). Plasma levels of Vg differed significantly between inferred ovulators and nonovulators when data for all islands were combined, although similar ranges were observed in the two groups of females (inferred ovulators: nondetectable to 2382 μg/ml, mean = 505 ± 75; inferred nonovulators: nondetectable to 2864 μg/ml, mean = 460 ± 63). Plasma levels of estradiol, but not progesterone, differed significantly between inferred ovulators and nonovulators when data for all islands were combined. Mean levels in inferred ovulators were: estradiol, 157 ± 16 pg/ml (vs. 34 ± 5 pg/ml in inferred nonovulators); progesterone, 1.1 ± 0.2 ng/ml (vs. 0.7 ± 0.1 ng/m in inferred nonovulators); and testosterone, 5.6 ± 0.5 ng/ml (vs. 0.2 ± 0 ng/ml in inferred nonovulators). Only 37% of the females sampled met the minimum hormonal criteria indicative of ovulation. This suggests that, as in S. punctatus on Stephens Island, female S.p. punctatus on northern islands do not ovulate each year.

Use of annual growth and activity patterns to assess management procedures for captive tuatara(Sphenodon punctatus)

Abstract Surface activity of two captive 4 year old female tuatara were monitored for 1.5 min every 11 min for 24 h (i.e. 115 × 12.5 min cycles), once per week over 13 months (October 1991‐October 1992) using a closed circuit, low light, video surveillance system. Body length and weight measurements were taken monthly. Specific, recognisable cycles of annual and seasonal patterns of activity were recorded. Growth reflected these patterns but was also affected by social interaction. Social interaction and environmental factors are known to affect biological functions and physical development. The close correlation between surface activity and growth in tuatara are significant and characteristic of the species and can be used to gauge the effectiveness of captive management procedures.

Egg production by tuatara on Lady Alice and Stephens Island, New Zealand

Abstract Radiography was used to determine the proportion of female tuatara, Sphenodon punctatus, that carry eggs each year, over 6 years on Stephens Island and 5 years on Lady Alice Island. The smallest female found to be gravid, located on Stephens Island, was 170 mm snout‐vent length. Between 1983 and 1987 the gravidity rate for Lady Alice Island females averaged 28.8% (range = 22.2–45.5%), while between 1982 and 1987 the gravidity rate for Stephens Island females averaged 21.9% (range = 8.2–29.6%). Excluding data from 1986, when only 11 females from Lady Alice Island were sampled, annual gravidity rates did not differ significantly between islands. There was significant annual variation in the proportion of females carrying eggs on Stephens Island, but not on Lady Alice Island. On Stephens Island, gravidity rate was especially low in 1982 (8.2%). There were significant positive, but different, correlations between clutch size and female body size on both islands. Mean clutch size for Lady Alice Island...

Reproductive cycles of male and female tuatara (Sphenodon punctatus) on Stephens Island, New Zealand

Cycles in gonadal activity and plasma sex steroid concentrations were investigated in wild female and male tuatara on Stephens Island, New Zealand. Females nest once every four years on average. Vitellogenesis is spread over the first three years, and mating, ovulation and nesting occur in the fourth. Oviducal eggs are carried for6–8 months before nesting. Although the length of this ovarian cycle is unparalleled among oviparous reptiles, the associated cycles in plasma concentrations of sex steroids are similar to those in other reptiles. Mean plasma concentrations of oestradiol increase during vitellogenesis, peak at mating, fall rapidly before or around ovulation, are low during most of gravidity, and rise slightly during late gravidity‐nesting. Plasma concentrations of testosterone show a similarcycle. Mean plasma concentrations of progesterone are low during vitellogenesis and peak around ovulation. This periovulatory surge falls within1–2 months, and mean concentrations are low during the final 5–6 months of gravidity. Male tuatara show an annual, pre‐nuptial reproductive cycle. Mean plasma concentrations of testosterone are low during winter, rise during spring, peak during midsummer‐early autumn, when pre‐nuptial displays and mating occur, and fall in mid autumn. Limited histological data indicate that spermiogenesis occurs during midsummer‐early autumn, and also support a previous study suggesting that there may be no period of complete testicular and epididymal regression. In comparison with the pre‐nuptial cycles of other temperate‐zone reptiles, the cycle in male tuatara shows a more prolonged duration of testicular activity and of rising or elevated plasma testosterone concentrations. The prolonged reproductive cycles of male and female tuatara may be adaptations to a temperate environment with cool summers and cool, but not freezing, winters.

Identification of female tuatara in ovulatory condition using plasma sex steroid concentrations

Abstract Female tuatara (Sphenodon punctatus) are long-lived reptiles that do not nest each year. Identification of gravid females (those carrying oviducal eggs) has in the past been achieved only with radiography or laparoscopy. Both techniques require considerable equipment and/or operator skill, and both pose an unquantified degree of risk to tuatara. For these reasons, we sought to develop a blood test for identifying females destined to nest in a given year. We observed that on Stephens Island, New Zealand, those females that nested in a given year had significantly higher plasma concentrations of oestradiol, testosterone, and progesterone at the time of mating-ovulation (February-March) than in those females that did not ovulate and nest that year. Used in conjunction, the criteria of a testosterone concentration ≥2.0 ng/ml or a progesterone concentration ≥5.8 ng/ml in plasma, obtained within 3 h of capture in February–March, can be used to distinguish females destined to nest that year from those that do not nest. These criteria may prove useful for comparing nesting frequencies among years on Stephens Island, for comparing nesting frequencies among different islands, and for monitoring reproduction in captivity. To our knowledge, this is the ftrst time that plasma hormone concentrations have been used to identify nesting females in reptiles that do not nest each year.

Slow Estradiol-induced Vitellogenesis in the Tuatara, Sphenodon punctatus

The vitellogenic response to exogenous estradiol-17β in wild male and female tuatara (Sphenodon punctatus) is extremely slow to induce. Tuatara given slowrelease implants containing 4-11 μg estradiol/g body mass showed a marked elevation in plasma estradiol concentrations within 2 d (to levels of up to 2,967 pg/mL) but no marked increases in plasma concentrations of calcium, total protein, or inorganic phosphate within 11 d. A small increase in the amount of electrophoretically detectable vitellogenin in the plasma (expressed as a percentage of total plasma protein) was evident by 11 d. Plasma concentrations of progesterone in individual females and testosterone in individual males were low (<3. 7 ng/mL and <3.8 ng/mL, respectively) during the 11 d. Several individuals captured 4-9 mo after administration of implants showed very high plasma levels of vitellogenin (up to 59% of total plasma protein), calcium (up to 61 mmol/L), total protein (up to 108 g/L), and inorganic phosphate (up to 39 mmol/L); these concentrations are similar to those seen in other estrogen-treated reptiles within much shorter time periods. Estradiol-induced vitellogenesis in tuatara is slower to induce and may occur at a lower maximal rate than in other reptiles examined. These responses are probably explained by the cool body temperatures exhibited by tuatara and are consistent with the slow rate of natural vitellogenesis observed in wild female tuatara.

Variation in plasma constituents during the natural vitellogenic cycle of tuatara, Sphenodon Punctatus

1. 1. The vitellogenic cycle of female tuatara was investigated by monitoring plasma levels of vitellogenin, calcium, total protein, inorganic phosphate (P 1) and cholesterol. 2. 2. Vitellogenin was not detected in females in the non-reproductive condition, but was found perenially in plasma of reproducing females during vitellogenesis, which normally lasts about 3 years out of the 4 year ovarian cycle. 3. 3. No large year-to-year variations were found in the plasma constituents measured and there was no correlation between the oestradiol peak at mating and plasma levels of vitellogenin. 4. 4. The results provide further evidence that tuatara have an extraordinary prolonged and gradual vitellogenic cycle spanning several years for a single clutch of eggs. This type of reproductive cycle is unique among reptiles.

Absence of daily cycles in plasma sex steroids in male and female tuatara ( Sphenodon punctatus), and the effects of acute capture stress on females

The possible existence of daily cycles in plasma concentrations of sex steroids was examined in wild male and female tuatara ( Sphenodon punctatus). Samples were collected from freshly captured animals at dusk, middle of the night, dawn, and middle of the day in January (summer) and July (winter). Males showed daily cycles in mean body temperature ( T b) in both seasons but no daily cycle in mean plasma testosterone concentration in either season. Vitellogenic female tuatara in January and females in mixed reproductive condition in July also showed significant daily variation in T b. However, there were no daily cycles in mean plasma concentrations of estradiol, testosterone, or progesterone in either group of females. Vitellogenic female tuatara subjected to an acute capture stress (3-hr confinement) in January had mean plasma concentrations of estradiol and testosterone that did not differ from those of free-roaming females. However, progesterone and T b were significantly higher in captives than in free-roaming females. The elevation in progesterone may result from physical confinement, the difference in T b, or both. These data suggest that seasonal fluctuations in circulating concentrations of plasma sex steroids in tuatara can be determined using samples collected at different times of the 24-hr cycle. However, the effects of acute capture stress and/or changes in T b on plasma progesterone concentrations need to be considered in future studies on this and possibly other female reptiles.

Ultrastructural observations on the granular leucocytes of the tuatara Sphenodon punctatus (gray)

The granular leucocytes of an active, mature female tuatara, Sphenodon punctatus (Gray) were examined in the electron microscope. Eosinophils contained a lobulated nucleus, homogeneous, dense, irregularly shaped granules, assorted smaller granular inclusions, mitochondria and β-glycogen. Endoplasmic reticulum, Golgi complexes and ribosomes were scanty. Immature neutrophils (myelocytes) were regular in outline and contained a compact nucleus, in the adjacent centrosomal region were paired centrioles with connected microtubules, and Golgi complexes. Ovoid electrondense granules, mitochondria, lipid droplets and numerous microfilaments arranged randomly or in bundles, lay in the cytoplasm. Mature neutrophils were often highly irregular in outline, had a segmented nucleus and contained possibly a second type of granular inclusion. The basophils were regular in outline with a compact nucleus. Numerous ovoid homogeneous, electron-dense granules, mitochondria, β-glycogen particles and some microfilaments were seen in the cytoplasm. The granules in many basophils appeared ‘altered’ or degenerate and most of these contained microtubules The cytology of the granulocytes of the tuatara is compared with that in other vertebrates.

Haematological observations on a hibernating tuatara,Sphenodon punctatus

Abstract Quantitative and biochemical observations were made on the peripheral blood of a hibernating female tuatara. The packed red cell volume (haematocrit) was 30%. There were about 260 000 erythrocytes and 5000 leucocytes per mm3 blood. Lymphocytes and neutrophils were the predominant leucocytes, followed by monocytes, eosinophils, and basophils. Haemoglobin, plasma protein, glucose, sodium, potassium, cholesterol, and triglyceride levels were determined. These haematological data are compared with data from the same animal obtained during the active summer period.