Hilary C. Miller

Genetic drift outweighs balancing selection in shaping post-bottleneck major histocompatibility complex variation in New Zealand robins (Petroicidae)

The Chatham Island black robin, Petroica traversi, is a highly inbred, endangered passerine with extremely low levels of variation at hypervariable neutral DNA markers. In this study we investigated variation in major histocompatibility complex (MHC) class II genes in both the black robin and its nonendangered relative, the South Island robin Petroica australis australis. Previous studies have shown that Petroica have at least four expressed class II B MHC genes. In this study, the sequences of introns flanking exon 2 of these loci were characterized to design primers for peptide‐binding region (PBR) sequence analysis. Intron sequences were comprised of varying numbers of repeated units, with highly conserved regions immediately flanking exon 2. Polymerase chain reaction primers designed to this region amplified three or four sequences per black robin individual, and eight to 14 sequences per South Island robin individual. MHC genes are fitness‐related genes thought to be under balancing selection, so they may be more likely to retain variation in bottlenecked populations. To test this, we compared MHC variation in the black robin with artificially bottlenecked populations of South Island robin, and with their respective source populations, using restriction fragment length polymorphism analyses and DNA sequencing of the PBR. Our results indicate that the black robin is monomorphic at class II B MHC loci, while both source and bottlenecked populations of South Island robin have retained moderate levels of variation. Comparison of MHC variation with minisatellite DNA variation indicates that genetic drift outweighs balancing selection in determining MHC diversity in the bottlenecked populations. However, balancing selection appears to influence MHC diversity over evolutionary timescales, and the effects of gene conversion are evident.

Changes in Hox genes" structure and function during the evolution of the squamate body plan.

Hox genes are central to the specification of structures along the anterior–posterior body axis, and modifications in their expression have paralleled the emergence of diversity in vertebrate body plans. Here we describe the genomic organization of Hox clusters in different reptiles and show that squamates have accumulated unusually large numbers of transposable elements at these loci, reflecting extensive genomic rearrangements of coding and non-coding regulatory regions. Comparative expression analyses between two species showing different axial skeletons, the corn snake and the whiptail lizard, revealed major alterations in Hox13 and Hox10 expression features during snake somitogenesis, in line with the expansion of both caudal and thoracic regions. Variations in both protein sequences and regulatory modalities of posterior Hox genes suggest how this genetic system has dealt with its intrinsic collinear constraint to accompany the substantial morphological radiation observed in this group.

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.

Influence of major histocompatibility complex genotype on mating success in a free-ranging reptile population

Major histocompatibility complex (MHC) genes are highly polymorphic components of the vertebrate immune system, which play a key role in pathogen resistance. MHC genes may also function as odour-related cues for mate choice, thus ensuring optimal MHC diversity in offspring. MHC-associated mate choice has been demonstrated in some fish, bird and mammal species but it is not known whether this is a general vertebrate phenomenon. We investigated whether MHC-associated mate choice occurs in a wild population of tuatara (Sphenodon punctatus), a territorial and sexually dimorphic reptile. We found weak evidence for MHC-disassortative mating, based on amino acid genotypic distance between pairs, when mated pairs were directly compared with potential pairs in close spatial proximity. No significant association was found between male mating success, number of MHC sequences, microsatellite heterozygosity or MHC lineage. The major determinant of mating success in tuatara was male body size, which was not related to MHC lineage or microsatellite heterozygosity. Our results suggest that male competitive ability is the primary driver of mating success in tuatara. However, MHC-associated preferences also appear to play a role, possibly as a kin avoidance mechanism during territory formation.

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

BackgroundThe 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.ResultsWe 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.ConclusionsThis 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.

Fine-scale genetic structure of a long-lived reptile reflects recent habitat modification.

Anthropogenic habitat fragmentation — ubiquitous in modern ecosystems — has strong impacts on gene flow and genetic population structure. Reptiles may be particularly susceptible to the effects of fragmentation because of their extreme sensitivity to environmental conditions and limited dispersal. We investigate fine‐scale spatial genetic structure, individual relatedness, and sex‐biased dispersal in a large population of a long‐lived reptile (tuatara, Sphenodon punctatus) on a recently fragmented island. We genotyped individuals from remnant forest, regenerating forest, and grassland pasture sites at seven microsatellite loci and found significant genetic structuring (RST = 0.012) across small distances (< 500 m). Isolation by distance was not evident, but rather, genetic distance was weakly correlated with habitat similarity. Only individuals in forest fragments were correctly assignable to their site of origin, and individual pairwise relatedness in one fragment was significantly higher than expected. We did not detect sex‐biased dispersal, but natural dispersal patterns may be confounded by fragmentation. Assignment tests showed that reforestation appears to have provided refuges for tuatara from disturbed areas. Our results suggest that fine‐scale genetic structuring is driven by recent habitat modification and compounded by the sedentary lifestyle of these long‐lived reptiles. Extreme longevity, large population size, simple social structure and random dispersal are not strong enough to counteract the genetic structure caused by a sedentary lifestyle. We suspect that fine‐scale spatial genetic structuring could occur in any sedentary species with limited dispersal, making them more susceptible to the effects of fragmentation.

Minisatellite DNA profiling detects lineages and parentage in the endangered kakapo (Strigops habroptilus) despite low microsatellite DNA variation

An important goal of the conservationmanagement program of the critically endangeredground parrot, the New Zealand kakapo (Strigops habroptilus) is the determination ofparentage and levels of genetic diversitywithin the remaining population. Our previousmicrosatellite DNA studies have shown that allindividuals of this species except one arehomozygous at seven loci examined. Incontrast, we now show that a minisatellite DNAanalysis of kakapo provides sufficientvariation to conduct paternity analyses anddetect heterogeneity within the 86 livingkakapo. The sole remaining Fiordland kakapo,Richard Henry, is shown to be geneticallydivergent from individuals originating from theonly other remaining population on StewartIsland, suggesting that two lineages of kakapoare present in the extant population. This hasparticular significance for the conservationmanagement goal of maintenance of the maximumgenetic diversity in the species as a whole. The example of the kakapo illustrates thatminisatellite DNA markers can be useful incases where microsatellite DNA fails to showsufficient variation.

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.

The First Cytogenetic Map of the Tuatara, Sphenodon punctatus

Tuatara, Sphenodon punctatus, is the last survivor of the distinctive reptilian order Rhynchocephalia and is a species of extraordinary zoological interest, yet only recently have genomic analyses been undertaken. The karyotype consists of 28 macrochromosomes and 8 microchromosomes. A Bacterial Artificial Chromosome (BAC) library constructed for this species has allowed the first characterization of the tuatara genome. Sequence analysis of 11 fully sequenced BAC clones (∼0.03% coverage) increased the estimate of genome wide GC composition to 47.8%, the highest reported for any vertebrate. Our physical mapping data demonstrate discrete accumulation of repetitive elements in large blocks on some chromosomes, particularly the microchromosomes. We suggest that the large size of the genome (5.0 pg/haploid) is due to the accumulation of repetitive sequences. The microchromosomes of tuatara are rich in repetitive sequences, and the observation of one animal that lacked a microchromosome pair suggests that at least this microchromosome is unnecessary for survival. We used BACs bearing orthologues of known genes to construct a low-coverage cytogenetic map containing 21 markers. We identified a region on chromosome 4 of tuatara that shares homology with 7 Mb of chicken chromosome 2, and therefore the orthologous region of the snake Z chromosome. We identified a region on tuatara chromosome 3 that is orthologous to the chicken Z, and a region on chromosome 9 orthologous to the mammalian X. Since the tuatara determines sex by temperature and has no sex chromosomes, this implies that different tuatara autosome regions are homologous with the sex chromosomes of mammals, birds and snakes. We have identified anchor BAC clones that can be used to reliably mark chromosomes 3–7, 10 and 13, some of which are difficult to distinguish based on morphology alone. Fluorescence in situ hybridization mapping of 18S rDNA confirms the presence of a single NOR located on the long arm of chromosome 7, as previously identified by silver staining. Further work to construct a dense physical map will lead to a better understanding of the dynamics of genome evolution and organization in this isolated species.

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.