Low genetic divergence obscures phylogeny among populations of Sphenodon,remnant of an ancient reptile lineage

Tuatara (two species of Sphenodon) are the last representatives of a branch of an ancient reptilian lineage, Sphenodontia, that have been isolated on the New Zealand landmass for 82 million years. We present analyses of geographic variation in allozymes, mitochondrial DNA, nuclear DNA sequences, and one-way albumin immunological comparisons. These all confirm a surprisingly low level of genetic diversity within Sphenodon for such an ancient lineage. We hypothesise a recent extended population bottleneck, probably during the Pliocene/Pleistocene glaciation cycles, to explain the current paucity of variation. All data sets reveal clear genetic differentiation between the northern populations and those in Cook Strait, but offer conflicting views of the history and taxonomic relationships of the Cook Strait population on North Brother Island, currently recognised as Sphenodon guntheri. Allozymes show this population to be the most divergent of all tuatara populations, but preliminary mitochondrial DNA data indicate few differences between S. guntheri and Cook Strait Sphenodon punctatus. Interpretation of the trees is confounded by the lack of a suitable outgroup. As in other cases of conflicting nuclear and mitochondrial data sets, the different data sets likely reveal different aspects of the animals' evolutionary history, and introgression is not uncommon between species pairs.     

Two patterns of variation among MHC class I loci in Tuatara (Sphenodon punctatus)

The genes of the major histocompatibility complex (MHC) are a central component of the immune system in vertebrates and have become important markers of functional, fitness-related genetic variation. We have investigated the evolutionary processes that generate diversity at MHC class I genes in a large population of an archaic reptile species, the tuatara (Sphenodon punctatus), found on Stephens Island, Cook Strait, New Zealand. We identified at least 2 highly polymorphic (UA type) loci and one locus (UZ) exhibiting low polymorphism. The UZ locus is characterized by low nucleotide diversity and weak balancing selection and may be either a nonclassical class I gene or a pseudogene. In contrast, the UA-type alleles have high nucleotide diversity and show evidence of balancing selection at putative peptide-binding sites. Twenty-one different UA-type genotypes were identified among 26 individuals, suggesting that the Stephens Island population has high levels of MHC class I variation. UA-type allelic diversity is generated by a mixture of point mutation and gene conversion. As has been found in birds and fish, gene conversion obscures the genealogical relationships among alleles and prevents the assignment of alleles to loci. Our results suggest that the molecular mechanisms that underpin MHC evolution in nonmammals make locus-specific amplification impossible in some species.     

Nuclear mitochondrial pseudogenes as molecular outgroups for phylogenetically isolated taxa: a case study in Sphenodon

'Living fossil' taxa, by definition, have no close relatives, and therefore no outgroup to provide a root to phylogenetic trees. We identify and use a molecular outgroup in the sole extant lineage of sphenodontid reptiles, which separated from other reptiles 230 million years ago. We isolated and sequenced a partial nuclear copy of the mitochondrial cytochrome b gene. We confirm the copy is indeed not mitochondrial, is older than all extant mitochondrial copies in Sphenodon (tuatara), and is therefore useful as a molecular outgroup. Under phylogenetic analysis, the nuclear copy places the root of the tuatara mitochondrial gene tree between the northern and the southern (Cook Strait) groups of islands of New Zealand that are the last refugia for Sphenodon. This analysis supports a previous mid-point rooted mitochondrial gene tree. The mitochondrial DNA tree conflicts with allozyme analyses which place a Cook Strait population equidistant to all northern and other Cook Strait populations. This population on North Brother Island is the only natural population of extant S. guntheri; thus, we suggest that the current species designations of tuatara require further investigation.     

Genetic diversity and differentiation at MHC genes in island populations of tuatara (Sphenodon spp.)

Neutral genetic markers are commonly used to understand the effects of fragmentation and population bottlenecks on genetic variation in threatened species. Although neutral markers are useful for inferring population history, the analysis of functional genes is required to determine the significance of any observed geographical differences in variation. The genes of the major histocompatibility complex (MHC) are well‐known examples of genes of adaptive significance and are particularly relevant to conservation because of their role in pathogen resistance. In this study, we survey diversity at MHC class I loci across a range of tuatara populations. We compare the levels of MHC variation with that observed at neutral microsatellite markers to determine the relative roles of balancing selection, diversifying selection and genetic drift in shaping patterns of MHC variation in isolated populations. In general, levels of MHC variation within tuatara populations are concordant with microsatellite variation. Tuatara populations are highly differentiated at MHC genes, particularly between the northern and Cook Strait regions, and a trend towards diversifying selection across populations was observed. However, overall our results indicate that population bottlenecks and isolation have a larger influence on patterns of MHC variation in tuatara populations than selection.     

Chromosomes of tuatara, Sphenodon, a chromosome heteromorphism and an archaic reptilian karyotype

We examined karyotypes of the endemic New Zealand reptile genus Sphenodon (tuatara) from five populations, finding a karyotype unchanged for at least one million years. Animals karyotyped were from five geographically distinct populations, representing three groups, namely S. guntheri, S. punctatus (Cook Strait group), and S. punctatus (northeastern North Island group). All five populations have a diploid chromosome number of 2n = 36, consisting of 14 pairs of macrochromosomes and four pairs of microchromosomes. Chromosomal differences were not found between the five populations nor between female and male animals, except for one animal with a structural heteromorphism. Similarity between Sphenodon and Testudine karyotypes suggests an ancestral karyotype with a macrochromosome complement of 14 pairs and the ability to accumulate variable numbers of microchromosome pairs. Our research supports molecular phylogenies of the Reptilia.     

Genetic variation in island populations of tuatara (<Emphasis Type="Italic">Sphenodon</Emphasis> spp) inferred from microsatellite markers

Tuatara (Sphenodon spp) populations are restricted to 35 offshore islands in the Hauraki Gulf, Bay of Plenty and Cook Strait of New Zealand. Low levels of genetic variation have previously been revealed by allozyme and mtDNA analyses. In this new study, we show that six polymorphic microsatellite loci display high levels of genetic variation in 14 populations across the geographic range of tuatara. These populations are characterised by disjunct allele frequency spectra with high numbers of private alleles. High F _ST (0.26) values indicate marked population structure and assignment tests allocate 96% of all individuals to their source populations. These genetic data confirm that islands support genetically distinct populations. Principal component analysis and allelic sequence data supplied information about genetic relationships between populations. Low numbers of rare alleles and low allelic richness identified populations with reduced genetic diversity. Little Barrier Island has very low numbers of old tuatara which have retained some relictual diversity. North Brother Island’s tuatara population is inbred with fixed alleles at 5 of the 6 loci.     

Demographic effects of temperature‐dependent sex determination: will tuatara survive global warming?

Global climate change is of particular concern for small and isolated populations of reptiles with temperature-dependent sex determination because low genetic variation can limit adaptive response in pivotal temperatures, leading to skewed sex ratios. We explore the demographic consequences of skewed sex ratios on the viability of a tuatara population characterized by low genetic diversity. We studied the rare species of tuatara ( Sphenodon guntheri ) on the 4 ha North Brother Island in New Zealand over two nesting seasons and captured 477 individuals, with a 60% male bias in the adult population. Females first breed at 15 years and have extremely low rates of gravidity, producing clutches of three to eight eggs every 9 years. Simulations of the population using population viability analysis showed that the current population is expected to persist for at least 2000 years at hatchling sex ratios of up to 75% male, but populations with 85% male hatchlings are expected to become extinct within approximately 300 years (some eight generations). Incorporation of inbreeding depression increased the probability of extinction under male biased sex ratios, with no simulated populations surviving at hatchling sex ratios >75% male. Because recent models have predicted that climate change could lead to the production of all male S. guntheri hatchlings by 2085, we examined whether periodic intervention to produce mixed or female biased sex ratios would allow the population to survive if only males were produced in natural nests. We show that intervention every 2–3 years could buffer the effects of climate change on population sex ratios, but translocation to cooler environs might be more cost-effective. Climate change threatens tuatara populations because neither modified nesting behaviour nor adaptive response of the pivotal temperature can modify hatchling sex ratios fast enough in species with long generation intervals.     

On the relative roles of selection and genetic drift in shaping MHC variation

Genes of the major histocompatibility complex (MHC) have provided some of the clearest examples of how natural selection generates discordances between adaptive and neutral variation in natural populations. The type and intensity of selection as well as the strength of genetic drift are believed to be important in shaping the resulting pattern of MHC diversity. However, evaluating the relative contribution of multiple microevolutionary forces is challenging, and empirical studies have reported contrasting results. For instance, balancing selection has been invoked to explain high levels of MHC diversity and low population differentiation in comparison with other nuclear markers. Other studies have shown that genetic drift can sometimes overcome selection and then patterns of genetic variation at adaptive loci cannot be discerned from those occurring at neutral markers. Both empirical and simulated data also indicate that loss of genetic diversity at adaptive loci can occur faster than at neutral loci when selection and population bottlenecks act simultaneously. Diversifying selection, on the other hand, explains accelerated MHC divergence as the result of spatial variation in pathogen‐mediated selective regimes. Because of all these possible scenarios and outcomes, collecting information from as many study systems as possible, is crucial to enhance our understanding about the evolutionary forces driving MHC polymorphism. In this issue, Miller and co‐workers present an illuminating contribution by combining neutral markers (microsatellites) and adaptive MHC class I loci during the investigation of genetic differentiation across island populations of tuatara Sphenodon punctatus. Their study of geographical variation reveals a major role of genetic drift in shaping MHC variation, yet they also discuss some support for diversifying selection.     

Microsatellite DNA loci identify individuals and provide no evidence for multiple paternity in wild tuatara (Sphenodon: Reptilia)

Six new microsatellite DNA loci are isolated from a genomic library of the sphenodontid reptile tuatara (Sphenodon) and presented here as a tool for identifying individuals for future paternity and kinship studies. These loci, in combination with four previously published loci, are sufficient to discriminate between clutch-mate siblings from Stephens Island and Brothers Island populations. These populations represent high and low levels of genetic diversity in tuatara populations respectively. An estimate of minimum number of fathers of each clutch found no evidence for multiple paternity in any clutch. These newly isolated loci complete the development of an array of genetic tools for use in tuatara to enhance ongoing conservation and management of wild, translocated and captive populations.     

Conservation implications of a long-term decline in body condition of the Brothers Island tuatara (Sphenodon guntheri)

Understanding the factors that drive the dynamics of remnant populations of long-lived species presents a unique challenge for conservation management. The long-lived Brothers Island tuatara Sphenodon guntheri is represented by one natural, self-sustaining population on 4-ha North Brother Island, New Zealand, and two small, translocated populations. The North Brother Island population was almost driven to extinction by extreme habitat modification and collecting in the late 19th century. Analysis of a long-term (1957–2001) dataset, following the population's recovery, reveals a significant decline in tuatara body condition over time, which is more pronounced in females. Declining body condition, coupled with very low reproductive output, may be symptomatic of a density-dependent response to elevated population size exacerbated by resource limitation. Sex-specific effects that disadvantage females could compromise this small population, particularly as it exhibits a male-biased sex ratio. We recommend removal of infrequently used structures and habitat restoration to alleviate intense resource competition. Population-level manipulation should be considered if future monitoring indicates an increasingly male-biased sex ratio and continued decline of female body condition.     

Selection maintains MHC diversity through a natural population bottleneck

A perceived consequence of a population bottleneck is the erosion of genetic diversity and concomitant reduction in individual fitness and evolutionary potential. Although reduced genetic variation associated with demographic perturbation has been amply demonstrated for neutral molecular markers, the effective management of genetic resources in natural populations is hindered by a lack of understanding of how adaptive genetic variation will respond to population fluctuations, given these are affected by selection as well as drift. Here, we demonstrate that selection counters drift to maintain polymorphism at a major histocompatibility complex (MHC) locus through a population bottleneck in an inbred island population of water voles. Before and after the bottleneck, MHC allele frequencies were close to balancing selection equilibrium but became skewed by drift when the population size was critically low. MHC heterozygosity generally conformed to Hardy-Weinberg expectations except in one generation during the population recovery where there was a significant excess of heterozygous genotypes, which simulations ascribed to strong differential MHC-dependent survival. Low allelic diversity and highly skewed frequency distributions at microsatellite loci indicated potent genetic drift due to a strong founder affect and/or previous population bottlenecks. This study is a real-time examination of the predictions of fundamental evolutionary theory in low genetic diversity situations. The findings highlight that conservation efforts to maintain the genetic health and evolutionary potential of natural populations should consider the genetic basis for fitness-related traits, and how such adaptive genetic diversity will vary in response to both the demographic fluctuations and the effects of selection.     

Implications of social dominance and multiple paternity for the genetic diversity of a captive-bred reptile population (tuatara)

Captive breeding is an integral part of many species recovery plans. Knowledge of the genetic mating system is essential for effective management of captive stocks and release groups, and can help to predict patterns of genetic diversity in reintroduced populations. Here we investigate the poorly understood mating system of a threatened, ancient reptile (tuatara) on Little Barrier Island, New Zealand and discuss its impact on the genetic diversity. This biologically significant population was thought to be extinct, due to introduced predators, until 8 adults (4 males, 4 females) were rediscovered in 1991/92. We genotyped these adults and their 121 captively-bred offspring, hatched between 1994 to 2005, at five microsatellite loci. Multiple paternity was found in 18.8% of clutches. Male variance in reproductive success was high with one male dominating mating (77.5% of offspring sired) and one male completely restricted from mating. Little Barrier Island tuatara, although clearly having undergone a demographic bottleneck, are retaining relatively high levels of remnant genetic diversity which may be complemented by the presence of multiple paternity. High variance in reproductive success has decreased the effective size of this population to approximately 4 individuals. Manipulation to equalize founder representation was not successful, and the mating system has thus had a large impact on the genetic diversity of this recovering population. Although population growth has been successful, in the absence of migrants this population is likely at risk of future inbreeding and genetic bottleneck.     

Diet of tuatara (Sphenodon punctatus) translocated to Ōrokonui Ecosanctuary in southern New Zealand

ABSTRACT

Scats from tuatara (Sphenodon punctatus) were investigated in autumn at ōrokonui Ecosanctuary on South Island, New Zealand. Eighty-seven tuatara had been translocated there 5–7 months previously, either directly from Stephens Island/Takapourewa or via captivity. Tuatara at ōrokonui fed on diverse invertebrates, including beetles, millipedes, spiders, dipteran flies and cave wētā. Prey occurrence in large scats (presumed to come from adults) was similar in frequency to that in small scats (presumed to be from medium–large juveniles), apart from a higher incidence of spiders in those from adults. Tuatara scats contained scarabaeid and large carabid beetles more frequently, and tenebrionid beetles less frequently, than reported on Stephens Island. Unlike tuatara on Stephens Island, those at ōrokonui do not have access to seabirds or tree wētā, and showed no certain predation on passerines or reptiles. Some differences in diet composition may reflect differences in prey availability resulting from the past presence of rodents at ōrokonui.     

Diversity at the major histocompatibility complex Class II in the platypus, Ornithorhynchus anatinus

The platypus (Ornithorhynchus anatinus) is the sole survivor of a previously widely distributed and diverse lineage of ornithorhynchid monotremes. Its dependence on healthy water systems imposes an inherent sensitivity to habitat degradation and climate change. Here, we compare genetic diversity at the major histocompatibility complex (MHC) Class II-DZB gene and 3 MHC-associated microsatellite markers with diversity at 6 neutral microsatellite markers in 70 platypuses from across their range, including the mainland of Australia and the isolated populations of Tasmania, King Island, and Kangaroo Island. Overall, high DZB diversity was observed in the platypus, with 57 DZB β1 alleles characterized. Significant positive selection was detected within the DZB peptide-binding region, promoting variation in this domain. Low levels of genetic diversity were detected at all markers in the 2 island populations, King Island (endemic) and Kangaroo Island (introduced), with the King Island platypuses monomorphic at the DZB locus. Loss of MHC diversity on King Island is of concern, as the population may have compromised immunological fitness and reduced ability to resist changing environmental conditions.     

How do reproductive skew and founder group size affect genetic diversity in reintroduced populations?

Reduced genetic diversity can result in short-term decreases in fitness and reduced adaptive potential, which may lead to an increased extinction risk. Therefore, maintaining genetic variation is important for the short- and long-term success of reintroduced populations. Here, we evaluate how founder group size and variance in male reproductive success influence the long-term maintenance of genetic diversity after reintroduction. We used microsatellite data to quantify the loss of heterozygosity and allelic diversity in the founder groups from three reintroductions of tuatara ( Sphenodon ), the sole living representatives of the reptilian order Rhynchocephalia. We then estimated the maintenance of genetic diversity over 400 years (∼10 generations) using population viability analyses. Reproduction of tuatara is highly skewed, with as few as 30% of males mating across years. Predicted losses of heterozygosity over 10 generations were low (1–14%), and populations founded with more animals retained a greater proportion of the heterozygosity and allelic diversity of their source populations and founder groups. Greater male reproductive skew led to greater predicted losses of genetic diversity over 10 generations, but only accelerated the loss of genetic diversity at small population size (<250 animals). A reduction in reproductive skew at low density may facilitate the maintenance of genetic diversity in small reintroduced populations. If reproductive skew is high and density-independent, larger founder groups could be released to achieve genetic goals for management.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. MHC Class I genes in the Tuatara (Sphenodon spp.): evolution of the MHC in an ancient reptilian order

The major histocompatibility complex (MHC) is an extremely dynamic region of the genome, characterized by high polymorphism and frequent gene duplications and rearrangements. This has resulted in considerable differences in MHC organization and evolution among vertebrate lineages, particularly between birds and mammals. As nonavian reptiles are ancestral to both mammals and birds, they occupy an important phylogenetic position for understanding these differences. However, little is known about reptile MHC genes. To address this, we have characterized MHC class I sequences from the tuatara (Sphenodon spp.), the last survivor of an ancient order of reptiles, Sphenodontia. We isolated two different class I cDNA sequences, which share 93% sequence similarity with each other but are highly divergent from other vertebrate MHC genes. Southern blotting and polymerase chain reaction amplification of class I sequences from seven adult tuatara plus a family group indicate that these sequences represent at least two to three loci. Preliminary analysis of variation among individuals from an island population of tuatara indicates that these loci are highly polymorphic. Maximum likelihood analysis of reptile MHC class I sequences indicates that gene duplication has occurred within reptilian orders. However, the evolutionary relationships among sequences from different reptilian orders cannot be resolved, reflecting the antiquity of the major reptile lineages.     

Plasma corticosterone concentrations in wild and captive juvenile tuatara (Sphenodon punctatus)

Abstract

High mortality and abnormal growth patterns commonly found in captive juvenile tuatara were hypothesised to be due in part to the effects of long‐term chronic stress of captivity. This study compared plasma concentrations of the reptilian adrenal steroid, corticosterone, in wild juvenile tuatara on Stephens Island, Cook Strait, and in captive juveniles of Stephens Island origin, held in New Zealand institutions, in February and August 1992. Seasonal variation in plasma concentration of corticosterone in wild juveniles in four seasons of the year was also examined. This is the first study of seasonal cycles in plasma corticosterone in a wild juvenile reptile. Plasma corticosterone concentrations were significantly higher in captive juvenile females (4.21 ± 0.27 ng/ml; mean ± SE) compared with wild juvenile females (2.44 ± 0.42 ng/ml) in February (P < 0.05), but not in August, and there was no difference in concentration between captive and wild juvenile males in either month. There was significant seasonal variation in plasma corticosterone in wild juvenile females (P < 0.05). However, there was no seasonal variation observed in wild juvenile males, and the magnitude of the variation in plasma corticosterone was low in both sexes (1.28 ± 0 ng/ml ‐4.65 ±3.41 ng/ml). Although mean plasma corticosterone was higher in captive juvenile females compared with wild juvenile females in February 1992, the value in captive females was within the range of mean plasma corticosterone concentrations observed in the seasonal study, and may be therefore due to asynchronicity of seasonal cycles, rather than stress. Further research is required; however, lack of correlation between plasma corticosterone concentrations and either growth rate or density indicate that captive juvenile tuatara in New Zealand are not suffering from pronounced chronic stress.     

Is MHC diversity a better marker for conservation than neutral genetic diversity? A case study of two contrasting dolphin populations

Genetic diversity is essential for populations to adapt to changing environments. Measures of genetic diversity are often based on selectively neutral markers, such as microsatellites. Genetic diversity to guide conservation management, however, is better reflected by adaptive markers, including genes of the major histocompatibility complex (MHC). Our aim was to assess MHC and neutral genetic diversity in two contrasting bottlenose dolphin (Tursiops aduncus) populations in Western Australia—one apparently viable population with high reproductive output (Shark Bay) and one with lower reproductive output that was forecast to decline (Bunbury). We assessed genetic variation in the two populations by sequencing the MHC class II DQB, which encompasses the functionally important peptide binding regions (PBR). Neutral genetic diversity was assessed by genotyping twenty‐three microsatellite loci. We confirmed that MHC is an adaptive marker in both populations. Overall, the Shark Bay population exhibited greater MHC diversity than the Bunbury population—for example, it displayed greater MHC nucleotide diversity. In contrast, the difference in microsatellite diversity between the two populations was comparatively low. Our findings are consistent with the hypothesis that viable populations typically display greater genetic diversity than less viable populations. The results also suggest that MHC variation is more closely associated with population viability than neutral genetic variation. Although the inferences from our findings are limited, because we only compared two populations, our results add to a growing number of studies that highlight the usefulness of MHC as a potentially suitable genetic marker for animal conservation. The Shark Bay population, which carries greater adaptive genetic diversity than the Bunbury population, is thus likely more robust to natural or human‐induced changes to the coastal ecosystem it inhabits.     

Variation in MHC genotypes in two populations of house sparrow (Passer domesticus) with different population histories

Small populations are likely to have a low genetic ability for disease resistance due to loss of genetic variation through inbreeding and genetic drift. In vertebrates, the highest genetic diversity of the immune system is located at genes within the major histocompatibility complex (MHC). Interestingly, parasite‐mediated selection is thought to potentially maintain variation at MHC loci even in populations that are monomorphic at other loci. Therefore, general loss of genetic variation in the genome may not necessarily be associated with low variation at MHC loci. We evaluated inter‐ and intrapopulation variation in MHC genotypes between an inbred (Aldra) and a relatively outbred population (Hestmannøy) of house sparrows (Passer domesticus) in a metapopulation at Helgeland, Norway. Genomic (gDNA) and transcribed (cDNA) alleles of functional MHC class I and IIB loci, along with neutral noncoding microsatellite markers, were analyzed to obtain relevant estimates of genetic variation. We found lower allelic richness in microsatellites in the inbred population, but high genetic variation in MHC class I and IIB loci in both populations. This suggests that also the inbred population could be under balancing selection to maintain genetic variation for pathogen resistance.