Identification and partial characterization of α2-macroglobulin from the tuatara (Sphenodon punctatus)

α₂-Macroglobulin (α₂-M), a large molecular mass proteinase-binding protein, was identified in plasma from tuatara (Sphenodon), a rare reptile endemic to New Zealand. In this genus, α₂-M constitutes 11–13% of total plasma protein (∼2.2–3.9 mg/ml). Analysis of blood samples collected at approximately monthly intervals from individual tuatara indicated that the plasma level of α₂-M remains fairly constant. The subunits of tuatara α₂-M have an apparent molecular mass of ∼160 kDa as determined by SDS-polyacrylamide gel electrophoresis and the intact protein is an oligomer that contains inter-chain disulfide bonds. N-terminal sequence analyses of tuatara α₂-M revealed a distinct similarity to α-macroglobulins of other vertebrates and that at least two types of α₂-M subunits are present in plasma of tuatara.     

Identification of a 130-kDa albumin in tuatara (Sphenodon) and detection of a novel albumin polymorphism

Electrophoretic, immunochemical, and protein sequence analyses were performed on plasma albumin of the tuatara (Sphenodon), a rare reptile endemic to New Zealand. The analyses revealed that, unlike other terrestrial vertebrates, tuatara do not seem to possess a 60- to 75-kDa plasma albumin. The common form of plasma albumin in this genus has an apparent molecular mass of 130 kDa, making it by far the largest albumin reported for any terrestrial vertebrate. Starch gel electrophoresis of samples from tuatara on 24 of the 30 islands inhabited by this genus resolved two forms of the 130-kDa albumin (albumins A and C). A third albumin of approximately 170 kDa (albumin B), reflecting a novel alloalbuminemia, was found in tuatara in three geographically isolated populations. Albumin A appears to be restricted to populations at the southern extremity of the tuatara's distribution, while albumin C was found in all but four (southern) populations. Possible explanations for the origin and distribution of these albumins are discussed.     

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.     

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.     

Circadian emergence and movement of captive juvenile tuatara (Sphenodon spp.)

Abstract

Knowledge of the circadian behaviour of young tuatara (Sphenodon spp.) is relatively scarce because tuatara are difficult to observe in the wild. We document diurnal, nocturnal and crepuscular emergence and movements (half‐body movement, walking and running) of three groups of captive juvenile tuatara (2‐ and 3‐year‐old Sphenodon guntheri, and 5‐year‐old S. punctatus). Juvenile tuatara emerge predominantly at night, but move around above ground, mainly during the day and around sunset. Differences in emergence andmove‐ment scores between the three study groups were evident, probably linked with age, species or housing conditions, which were inevitably coupled in our study. We found that 2‐year‐old tuatara in captive conditions emerged less frequently than, but once above ground, moved more than 3‐ and 5‐year‐olds in semi‐captive conditions. Activities in semi‐captive conditions were not correlated with temperature, light or humidity. We conclude that young tuatara may be primarily adapted to nocturnal activity, but thermal restrictions and possible hardwired adaptations to avoid predators and conspecifics may make day‐time movements safer.     

Mark—Recapture Accurately Estimates Census for Tuatara,a Burrowing Reptile

Abstract: Estimates of population size are necessary for effective management of threatened and endangered species, but accurate estimation is often difficult when species are cryptic. We evaluated effectiveness of mark–recapture techniques using the Lincoln–Peterson estimator for predicting true census size of a population of tuatara (Sphenodon punctatus), a burrowing reptile that is a conservation priority in New Zealand. We found that Lincoln–Peterson estimates (NŇ = 85) were accurate for predicting the census size (N = 87) after only a 3-day mark–recapture survey. We recommend this method as a cost-effective way to accurately estimate population size for isolated, inaccessible tuatara populations, because it requires limited personnel, expertise, and time, and has low environmental impact on fragile sites.     

Eradications as reverse invasions: lessons from Pacific rat (Rattus exulans) removals on New Zealand islands

Eradications of kiore or Pacific rats (Rattus exulans) from islands around New Zealand have been followed by responses from resident species of coastal plants, invertebrates, reptiles and seabirds. These responses are compared with an invasion by ship rats (Rattus rattus), which devastated populations of invertebrates, birds and bats. Post-eradication responses only approximate the effects of invasions because recovery is limited to the residual pool of native species. Greater effects of kiore are indicated by adding incompatible species confined to rat-free locations. The extended list includes at least 15 species of invertebrates, two species of frogs, tuatara (Sphenodon punctatus), 11 species of lizards and 9 species of seabirds. The analyses indicate direct and indirect effects of kiore similar to those reported after ship rat invasions. This is despite indications from the literature that kiore are the least damaging of the three commensal rat species.     

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

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.     

Distribution and phylogenetic analyses of an endangered tick,Amblyomma sphenodonti

Abstract

In this study we investigate the geographic distribution, genetic diversity, and phylogenetic relationships of an endangered tick, Amblyomma sphenodonti (Family Ixodidae). Amblyomma sphenodonti and its host, the tuatara (Sphenodon), are found only on small offshore islands around New Zealand. Our results show that Amblyomma sphenodonti has a more severely restricted geographic distribution than its host, as it was found on only eight of 28 islands (four out of 12 island groups) where tuatara still live. The prevalence of A. sphenodonti is likely to have been affected by low host density and fluctuations in host population size as tuatara populations became isolated on offshore islands. Analysis of A. sphenodonti cytochrome oxidase 1 (CO1) sequences indicated a lack of gene flow between islands, with fixed differences in CO1 sequences between islands, but almost no genetic diversity within island populations. A similar phylogenetic pattern to that observed in tuatara mtDNA was observed, indicating co‐evolution of two species, at least since the Pleistocene. Phylogenetic analysis using 18S rRNA sequences suggest that A. sphenodonti is not closely related to other Amblyomma species, and that a separate genus for this species may be warranted. However, data from other ixodid ticks are required before the distinctiveness of A. sphenodonti can be confirmed and the phylogenetic relationships among ixodid ticks fully understood.     

Social network structure and parasite infection patterns in a territorial reptile, the tuatara (Sphenodon punctatus)

We investigated whether the parasite load of an individual could be predicted by its position in a social network. Specifically, we derived social networks in a solitary, territorial reptile (the tuatara, Sphenodon punctatus), with links based on the sharing of space, not necessarily synchronously, in overlapping territories. Tuatara are infected by ectoparasitic ticks (Amblyomma sphenodonti), mites (Neotrombicula spp.) and a blood parasite (Hepatozoon tuatarae) which is transmitted by the tick. We recorded the location of individual tuatara in two study plots twice daily during the mating season (March) in 2 years (2006 and 2007) on Stephens Island, New Zealand. We constructed weighted, directed networks to represent pathways for parasite transmission, where nodes represented individual tuatara and edges connecting the nodes represented the extent of territory overlap among each pair of individuals. We considered a network-based hypothesis which predicted that the in-strength of individuals (the sum of edge weights directed towards a node) in the derived network would be positively related to their parasite load. Alternatively, if the derived social network did not reflect actual parasite transmission, we predicted other factors such as host sex, size or territory size may better explain variation in parasite infection patterns. We found clear positive relationships between the in-strength of tuatara and their tick loads, and infection patterns with tick-borne blood parasites. In particular, the extent that individuals were connected to males in the network consistently predicted tick loads of tuatara. However, mite loads of tuatara were significantly related to host sex, body size and territory size, and showed little association with network measures. The results suggest that the pathway of transmission of parasites through a population will depend on the transmission mechanism of the parasite, but that social networks provide a powerful predictive tool for some parasites.     

Differences in dietary and plasma fatty acids between wild and captive populations of a rare reptile, the tuatara (Sphenodon punctatus)

Tuatara (Sphenodon) are rare reptiles endemic to New Zealand. Wild tuatara on Stephens Island (study population) prey on insects as well as the eggs and chicks of a small nesting seabird, the fairy prion (Pachyptila turtur). Tuatara in captivity (zoos) are fed diets containing different insects and lacking seabirds. We compared the fatty acid composition of major dietary items and plasma of wild and captive tuatara. Fairy prions (eaten by tuatara in the wild) were rich in C20 and C22 polyunsaturated fatty acids (PUFA), especially the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In contrast, items from the diet of captive tuatara contained no C20 and C22 PUFA and were higher in medium-chain and less unsaturated fatty acids. Plasma from wild tuatara was higher in n-3 PUFA [including alpha-linoleic acid (C18:3n-3), EPA and DHA], and generally lower in oleic acid (C18:1) and palmitic acid (C16:0), than plasma from captive tuatara in the various fractions (phospholipid, triacylglycerol, cholesterol ester and free fatty acids). Plasma from wild adult tuatara showed strong seasonal variation in fatty acid composition, reflecting seasonal consumption of fairy prions. Differences in the composition of diets and plasma between wild and captive tuatara may have consequences for growth and reproduction in captivity. Accepted: 3 August 1998     

Seasonal and spatial dynamics of ectoparasite infestation of a threatened reptile, the tuatara (Sphenodon punctatus)

Abstract The conservation of threatened vertebrate species and their threatened parasites requires an understanding of the factors influencing their distribution and dynamics. This is particularly important for species maintained in conservation reserves at high densities, where increased contact among hosts could lead to increased rates of parasitism. The tuatara (Sphenodon punctatus) (Reptilia: Sphenodontia) is a threatened reptile that persists at high densities in forests (～ 2700 tuatara/ha) and lower densities in pastures and shrubland (< 200 tuatara/ha) on Stephens Island, New Zealand. We investigated the lifecycles and seasonal dynamics of infestation of two ectoparasites (the tuatara tick, Amblyomma sphenodonti, and trombiculid mites, Neotrombicula sp.) in a mark‐recapture study in three forest study plots from November 2004 to March 2007, and compared infestation levels among habitat types in March 2006. Tick loads were lowest over summer and peaked from late autumn (May) until early spring (September). Mating and engorgement of female ticks was highest over spring, and larval tick loads subsequently increased in early autumn (March). Nymphal tick loads increased in September, and adult tick loads increased in May. Our findings suggest the tuatara tick has a 2‐ or 3‐year lifecycle. Mite loads were highest over summer and autumn, and peaked in March. Prevalences (proportion of hosts infected) and densities (estimated number of parasites per hectare) of ticks were similar among habitats, but tick loads (parasites per host) were higher in pastures than in forests and shrub. The prevalence and density of mites was higher in forests than in pasture or shrub, but mite loads were similar among habitats. We suggest that a higher density of tuatara in forests may reduce the ectoparasite loads of individuals through a dilution effect. Understanding host–parasite dynamics will help in the conservation management of both the host and its parasites.     

A Genomic Comparison of Faster-Sex, Faster-X, and Faster-Male Evolution Between Drosophila melanogaster and Drosophila pseudoobscura

A genomic comparison of Drosophila melanogaster and Drosophila pseudoobscura provides a unique opportunity to investigate factors involved in sequence divergence. The chromosomal arrangements of these species include an autosomal segment in D. melanogaster which is homologous to part of the X chromosome in D. pseudoobscura. Using orthologues to calculate rates of nonsynonymous (d_N) substitutions, we found genes on the X chromosome to be significantly more diverged than those on the autosomes, but it is not true for segment 3L-XR which is autosomal in D. melanogaster (3L) and X-linked in D. pseudoobscura (XR). We also found that the median d_N values for genes having reproductive functions in either the male, the female, or both sexes are higher than those for sequences without reproductive function and even higher for sequences involved in male-specific function. These estimates of divergence for male sex-related sequences are most likely underestimates, as the very rapidly evolving reproductive genes would tend to lose homology sooner and thus not be included in the comparison of orthologues. We also noticed a high proportion of male reproductive genes among the othologous genes with the highest rates of d_N. Reproductive genes with and without an orthologue in D. pseudoobscura were compared among D. melanogaster, D. simulans, and D. yakuba and it was found that there were in fact higher rates of divergence in the group without a D. pseudoobscura orthologue. These results, from widely separated taxa, bolster the thesis that sexual system genes experience accelerated rates of change in comparison to nonsexual genes in evolution and speciation. [Reviewing Editor: Dr. Willie J. Swanson]     

First evidence of methylation in the genome of <Emphasis Type="Italic">Drosophila willistoni</Emphasis>

DNA methylation has been studied abundantly in vertebrates and recent evidence confirms that this phenomenon could be disseminated among some invertebrates groups, including Drosophila species. In this paper, we used the Methylation-Sensitive Restriction Endonuclease (MSRE) technique and Southern blot with specific probes, to detect methylation in the Drosophila willistoni species. We found differential cleavage patterns between males and females that cannot be explained by Mendelian inheritance, pointing to a DNA methylation phenomenon different from the Drosophila melanogaster one. The sequencing of some of these bands showed that these fragments were formed by different DNA elements, among which rDNA. We also characterized the D. willitoni dDnmt2 sequence, through a Mega Blast search against the D. willistoni Trace Archive Database using the D. melanogaster dDnmt2 nucleotide sequence as query. The complete analysis of D. willistoni dDnmt2 sequence showed that its promoter region is larger, its dDnmt2 nucleotide sequence is 33% divergent from the D. melanogaster one, Inverted Terminal Repeats (ITRs) are absent and only the B isoform of the enzyme is produced. In contrast, ORF2 is more conserved. Comparing the D. willistoni and D. melanogaster dDnmt2 protein sequences, we found higher conservation in motifs from the large domain, responsible for the catalysis of methyl transfer, and great variability in the region that carries out the recognition of specific DNA sequences (TRD). Globally, our results reveal that methylation of the D. willistoni genome could be involved in a singular process of species-specific dosage compensation and that the DNA methylation in the Drosophila genus can have diverse functions. This could be related to the evolutionary history of each species and also to the acquisition time of the dDnmt2 gene.     

Genetic identification of units for conservation in tomato frogs, genus Dyscophus

Dyscophus antongilii and D. guineti are two morphologically very similar microhylid frogs from Madagascar of uncertain taxonomy. D. antongilii is currently included in Appendix I of the Convention on the International Trade in Endangered Species (CITES) and its exportation is banned completely. In contrast, D. guineti does not receive any legal protection and it is regularly exported. Field data on ecology and behaviour are to a large extent lacking. Here we report on a genetic survey of D. antongilii and D. guineti using nuclear and mitochondrial DNA markers. Sequences of a fragment of 501 bp of the mitochondrial cytochrome b gene from one population of D. antongilii and two populations of D. guineti resulted in a single haplotype network, without haplotype sharing among the populations. However, haplotypes of D.␣guineti were only 1–4 mutational steps from those of D. antongilii, and did not form a clade. The analysis of eight microsatellites newly developed and standardized for D. antongilii revealed an excess of homozygotes and the absence of Hardy–Weinberg equilibrium. The microsatellite data clearly distinguished between D. antongilii and D. guineti, and fixed differences were observed at one locus. Although confirmation of the status of Dyscophus antongilii and D. guineti as separate species requires further data, our study supports the definition of these two taxa as different evolutionary significant units under the adaptive evolutionary conservation concept.     

Status of the tuatara,Sphenodon punctatus,on Hongiora and Ruamahua-iti Islands,Aldermen Group,New Zealand

Abstract

Results from a survey of the tuatara populations on Hongiora and Ruamahua-iti Islands, conducted in March 1986, do not support an earlier report that the Hongiora population is declining (Crook 1973). On Hongiora, 49 captures were made of 43 tuatara over 12 man-hours, and on Ruamahua-iti, 67 captures of 60 tuatara were made over 12.5 man-hours. The mean body lengths of Hongiora tuatara (male=250.4 mm, female=220.8 mm) were significantly larger than those of Ruamahua-iti animals (male= 187.6mm, female= 179.3 mm). We question the interpretation that the smaller average size of tuatara on Ruamahua-iti Island indicates that they are younger than those on Hongiora Island.     

Experimental interspecific hybridization in Daphnia

Hybridization is a common phenomenon in Daphnia (Cladocera; Anomopoda); interspecific hybrids have been found between several species and hybrids are found in many European lakes. Although much information on the morphology, ecology and genetics of hybrids is available, little is known about the level of reproductive isolation among species or about the relative fitness of hybrids and parental species. In order to facilitate studies on differentiation and speciation processes and comparative experimental studies on hybrids and recombinant genotypes, we present the first successful laboratory crossing experiments of two different Daphnia species, D. galeata and D. cucullata. Males and sexual females from two D. galeata and two D. cucullata clones were reciprocally crossed, juveniles hatched from resting eggs and reared until maturity. Hatching and juvenile survival rates of hybrids were relatively low (12.1% and 24%, respectively). D. galeata and D. cucullata clones vary in their level of successful interspecific matings and in the number of subsequent offspring. In general, hybrid crosses between D. cucullata females and D. galeata males were more successful than reciprocal crosses.     

F supergroup <Emphasis Type="Italic">Wolbachia</Emphasis> in bush crickets: what do patterns of sequence variation reveal about this supergroup and horizontal transfer between nematodes and arthropods?

Wolbachia pipientis, an intracellular, α-proteobacterium, is commonly found in arthropods and filarial nematodes. Most infected insects are known to harbor strains of Wolbachia from supergroups A or B, whereas supergroups C and D occur only in filarial nematodes. Here, we present molecular evidence from two genes (ftsZ and 16S rDNA) that 2 Orthopterans (the bush cricket species Orocharis saltator and Hapithus agitator; Gryllidae: Eneopterinae) are infected with Wolbachia from the F supergroup. Additionally, a series of PCR tests revealed that these bush cricket specimens did not harbor nematodes, thus indicating that our positive results were not a by-product of nematodes being present in these cricket samples. Patterns of molecular variation suggest that (1) strains of F supergroup Wolbachia exhibit less genetic variation than the nematode-specific C and D supergroups but more than the A and B supergroups found in arthropods and (2) that there is no evidence of recombination within F supergroup strains. The above data support previous findings that F supergroup Wolbachia is not only harbored in both nematodes and arthropods, but that horizontal transfer has likely occurred recently between these diverse taxonomic groups (although the exact details of such horizontal transmissions remain unclear). Moreover, the limited genetic variation and lack of recombination in the F supergroup suggest that this clade of Wolbachia has radiated relatively rapidly with either (1) little time for recombination to occur or (2) selection against recombination as occurs in the mutualistic C and D strains of Wolbachia – both of which remain to be explored further.     

Development of tolerance against toxic cyanobacteria in Daphnia

We tested whether previous exposure to a toxic strain of cyanobacteria (Microcystis) affects survival, growth, and reproduction of a common herbivore, Daphnia magna. Samples from three natural populations of D. magna were each divided into two parts; one part was fed a mixture of toxic Microcystis and the non-toxic green alga Scenedesmus whereas the other part was fed only Scenedesmus. After four weeks, we compared the ability of these two populations to withstand the toxic Microcystis by assessing survivorship, growth, and reproduction. We found that the ability of D. magna to cope successfully with toxic Microcystis is improved if the animals have experienced previous exposure to toxic Microcystis. This suggests that the toxin may less affect the D. magna populations that are repeatedly exposed to toxic cyanobacteria in their natural habitat than populations lacking prior exposure. Since the ability to tolerate toxins is manifested in both improved survival and larger size of the animals, it may have considerable impact on zooplankton community composition in fresh-waters.