Dianne Gleeson

Ecdysozoan Mitogenomics: Evidence for a Common Origin of the Legged Invertebrates, the Panarthropoda

Ecdysozoa is the recently recognized clade of molting animals that comprises the vast majority of extant animal species and the most important invertebrate model organisms—the fruit fly and the nematode worm. Evolutionary relationships within the ecdysozoans remain, however, unresolved, impairing the correct interpretation of comparative genomic studies. In particular, the affinities of the three Panarthropoda phyla (Arthropoda, Onychophora, and Tardigrada) and the position of Myriapoda within Arthropoda (Mandibulata vs. Myriochelata hypothesis) are among the most contentious issues in animal phylogenetics. To elucidate these relationships, we have determined and analyzed complete or nearly complete mitochondrial genome sequences of two Tardigrada, Hypsibius dujardini and Thulinia sp. (the first genomes to date for this phylum); one Priapulida, Halicryptus spinulosus; and two Onychophora, Peripatoides sp. and Epiperipatus biolleyi; and a partial mitochondrial genome sequence of the Onychophora Euperipatoides kanagrensis. Tardigrada mitochondrial genomes resemble those of the arthropods in term of the gene order and strand asymmetry, whereas Onychophora genomes are characterized by numerous gene order rearrangements and strand asymmetry variations. In addition, Onychophora genomes are extremely enriched in A and T nucleotides, whereas Priapulida and Tardigrada are more balanced. Phylogenetic analyses based on concatenated amino acid coding sequences support a monophyletic origin of the Ecdysozoa and the position of Priapulida as the sister group of a monophyletic Panarthropoda (Tardigrada plus Onychophora plus Arthropoda). The position of Tardigrada is more problematic, most likely because of long branch attraction (LBA). However, experiments designed to reduce LBA suggest that the most likely placement of Tardigrada is as a sister group of Onychophora. The same analyses also recover monophyly of traditionally recognized arthropod lineages such as Arachnida and of the highly debated clade Mandibulata.

Relationship between avian malaria distribution and an exotic invasive mosquito in New Zealand

Abstract Although the mosquito vector responsible for the epizootic outbreaks of avian malaria in Hawaiian avifauna, Culex quinquefasciatus, has spread rapidly in New Zealand over the past three decades, no survey for malarial parasites has been conducted for more than 50 years. Avian malaria often causes extreme morbidity and mortality in novel hosts, and much of New Zealands native avifauna has likely had no prior exposure, so the impact of this spread on native biodiversity is potentially serious. Wild non‐native birds were surveyed for malarial parasites at multiple locations, to test the hypothesis that the prevalence of malarial infection is associated positively with the known distribution of C. quinquefasciatus. Blood samples collected were analysed using a Polymerase Chain Reaction (PCR) assay. There was a strong pattern of decreasing percentage of samples positive for the blood parasite PCR marker from north to south, closely matching the known distribution of C. quinquefasciatus. A subsample of 10 PCR products were sequenced, and all identified as the malarial parasite Plasmodium relictum. Since historical surveys found no malarial parasites in the New Zealand avifauna, with a few minor exceptions, this suggests that this invasive exotic vector may be one factor driving the emergence of avian malaria. Sampling of wild non‐native birds at Orana Park, Christchurch, where a disease outbreak occurred recently in a captive native bird population, suggested that such outbreaks may spill‐back into local wild bird populations. The high prevalence of malarial infection observed in certain non‐native species, particularly blackbirds, indicates that they may act as reservoirs of infection to native species.

Incorporating Genotype Uncertainty into Mark–Recapture-Type Models For Estimating Abundance Using DNA Samples

Sampling DNA noninvasively has advantages for identifying animals for uses such as mark-recapture modeling that require unique identification of animals in samples. Although it is possible to generate large amounts of data from noninvasive sources of DNA, a challenge is overcoming genotyping errors that can lead to incorrect identification of individuals. A major source of error is allelic dropout, which is failure of DNA amplification at one or more loci. This has the effect of heterozygous individuals being scored as homozygotes at those loci as only one allele is detected. If errors go undetected and the genotypes are naively used in mark-recapture models, significant overestimates of population size can occur. To avoid this it is common to reject low-quality samples but this may lead to the elimination of large amounts of data. It is preferable to retain these low-quality samples as they still contain usable information in the form of partial genotypes. Rather than trying to minimize error or discarding error-prone samples we model dropout in our analysis. We describe a method based on data augmentation that allows us to model data from samples that include uncertain genotypes. Application is illustrated using data from the European badger (Meles meles).

Multiple mutations and gene duplications conferring organophosphorus insecticide resistance have been selected at the rop-1 locus of the sheep blowfly, Lucilia cuprina

Sequences of the esterase gene αE7 were compared across 41 isogenic (IV) strains of the sheep blowfly, Lucilia cuprina, and one strain of the sibling species, L. sericata. The 1.2-kb region sequenced includes sites of two insecticide resistance mutations. Gly137Asp confers resistance to organophosphorus insecticides (OPs), particularly preferring diethyl OPs such as diazinon, while Trp251Leu prefers dimethyl OPs, and particularly malathion, with the additional presence of carboxylester moieties. We found that there are just eight haplotypes among the 41 chromosomes studied: two Gly137Asp containing haplotypes, two Trp251Leu containing haplotypes, and four susceptible haplotypes, including the L. sericata sequence. While phylogenetic analysis of these haplotypes suggests that the Asp137 and Leu251 mutations each arose at least twice, evidence for recombination was detected across the region, therefore single origins for these resistance mutations cannot be ruled out. Levels of linkage disequilibrium in the data are high and significant hitchhiking is indicated by Fay and Wu’ s H test but not the Tajima test. A test of haplotype diversity indicates a paucity of diversity compared with neutral expectations. Both these results are consistent with a very recent selective sweep at the LcαE7 locus. Interestingly, gene duplications of three different combinations of OP resistant haplotypes were identified in seven of the isogenic (IV) strains. All three types of duplication involve an Asp137 and a Trp251 haplotype. To examine whether more haplotypes existed before the hypothesised selective sweep, fragments of αE7 surrounding the resistance mutations were amplified from pinned material dating back to before OPs were used. Four new sequence haplotypes, not sampled in the survey of extant haplotypes, were obtained that are all associated with susceptibility. This is suggestive of a higher historical level of susceptible allelic diversity at this locus.

A framework for estimating the sensitivity of eDNA surveys.

Imperfect sensitivity, or imperfect detection, is a feature of all survey methods that needs to be accounted for when interpreting survey results. Detection of environmental DNA (eDNA) is increasingly being used to infer species distributions, yet the sensitivity of the technique has not been fully evaluated. Sensitivity, or the probability of detecting target DNA given it is present at a site, will depend on both the survey method and the concentration and dispersion of target DNA molecules at a site. We present a model to estimate target DNA concentration and dispersion at survey sites and to estimate the sensitivity of an eDNA survey method. We fitted this model to data from a species‐specific eDNA survey for Oriental weatherloach, Misgurnus anguillicaudatus, at three sites sampled in both autumn and spring. The concentration of target DNA molecules was similar at all three sites in autumn but much higher at two sites in spring. Our analysis showed the survey method had ≥95% sensitivity at sites where target DNA concentrations were ≥11 molecules per litre. We show how these data can be used to compare sampling schemes that differ in the number of field samples collected per site and number of PCR replicates per sample to achieve ≥95% sensitivity at a given target DNA concentration. These models allow researchers to quantify the sensitivity of eDNA survey methods to optimize the probability of detecting target species, and to compare DNA concentrations spatially and temporarily.

Pheromone evolution within the genera Ctenopseustis and Planotortrix (Lepidoptera: Tortricidae) inferred from a phylogeny based on cytochrome oxidase I gene variation

Abstract Members of the genera Ctenopseustis and Planotortrix (Lepidoptera: Tortricidae) comprise 12 species of leafroller moths that are native to New Zealand. Females of this species complex produce a range of sex pheromones, typically blends of ( Z )-5-tetradecenyl acetate, ( Z )-7-tetradecenyl acetate, and ( Z )-8-tetradecenyl acetate, that are unusual within the Tortricidae. To order the events in the evolution of this chemical communication system, 523 bp of the cytochrome oxidase I gene were sequenced in 16 taxa including all 12 species currently recognised within the Ctenopseustis and Planotortrix complex. 106 polymorphic sites were detected that included some intraspecific variation between host and pheromone races of C. obliquana . A maximum parsimony tree constructed from this data set supports many groupings previously determined using morphological and pheromone characters. These include such features as the monophyly of the genus Ctenopseustis , with C. servana as ancestral, and the existence of an excessana group that includes P. excessana , P. avicenniae , P. octo , and P. octoides . The few incongruent relationships derived from the different morphological, pheromone and molecular data sets are discussed. The phylogeny inferred from cytochrome oxidase I variation suggests an order for the evolution of the pheromones used by these species, including a number of independent gains and losses of the same pheromone component. For example, it appears that the ability to produce ( Z )-5-tetradecenyl acetate has evolved twice within this complex.

Using genetic techniques to quantify reinvasion, survival and in situ breeding rates during control operations

Determining the origin of individuals caught during a control/eradication programme enables conservation managers to assess the reinvasion rates of their target species and evaluate the level of success of their control methods. We examine how genetic techniques can focus management by distinguishing between hypotheses of ‘reinvasion’ and ‘survivor’, and defining kin groups for invasive stoats (Mustela erminea) on Secretary Island, New Zealand. 205 stoats caught on the island were genotyped at 16 microsatellite loci, along with 40 stoats from the opposing mainland coast, and the age and sex were determined for each individual. Using these data, we compare and combine a variety of genetic techniques including genetic clustering, population assignment and kinship‐based techniques to assess the origin of each stoat. The population history and individual movement could be described in fine detail, with results indicating that both in‐situ survival and breeding, and reinvasion are occurring. Immigration to the island was found to be generally low, apart from in 1 year where around 8 stoats emigrated from the mainland. This increased immigration was probably linked to a stoat population spike on the mainland in that year, caused by a masting event of southern beech forest (Nothofagus sp.) and the subsequent rodent irruption. Our study provides an example of some of the ways genetic analyses can feed directly into informing management practices for invasive species.

Taxonomic revision of the marbled skink (Cyclodina oliveri, Reptilia: Scincidae) species complex, with a description of a new species

Abstract We have completed a taxonomic revision of the New Zealand marbled skink (Cyclodina oliveri) species complex. Morphological analyses and mitochondrial sequence data (ND2, ND4, Cytochrome b; Total 1933 bp) are used to describe a new taxon (commonly known as the “Mokohinau” skink) and redefine C. oliveri. The morphological and molecular data indicate that C. oliveri is distributed on the Poor Knights Islands, Mercury Islands and Aldermen Islands. The new species is restricted to the Mokohinau Islands, Hen and Chickens group, Little Barrier Island and Great Barrier Island. Our data demonstrate that there is no support for the separation of the Poor Knights Islands population of C. oliveri from those on the Mercury Islands and Aldermen Islands. The genetic data indicate that C. whitakeri is part of the C. oliveri species group. Divergence time estimates indicate that the C. oliveri species complex diverged during the late‐Miocene, with further divergences among island groups in C. oliveri including the origin of the new taxon during the late‐Pliocene and mid‐Pleistocene. We present a diagnostic key for Cyclodina.

Reliable discrimination of 10 ungulate species using high resolution melting analysis of faecal DNA.

Identifying species occupying an area is essential for many ecological and conservation studies. Faecal DNA is a potentially powerful method for identifying cryptic mammalian species. In New Zealand, 10 species of ungulate (Order: Artiodactyla) have established wild populations and are managed as pests because of their impacts on native ecosystems. However, identifying the ungulate species present within a management area based on pellet morphology is unreliable. We present a method that enables reliable identification of 10 ungulate species (red deer, sika deer, rusa deer, fallow deer, sambar deer, white-tailed deer, Himalayan tahr, Alpine chamois, feral sheep, and feral goat) from swabs of faecal pellets. A high resolution melting (HRM) assay, targeting a fragment of the 12S rRNA gene, was developed. Species-specific primers were designed and combined in a multiplex PCR resulting in fragments of different length and therefore different melting behaviour for each species. The method was developed using tissue from each of the 10 species, and was validated in blind trials. Our protocol enabled species to be determined for 94% of faecal pellet swabs collected during routine monitoring by the New Zealand Department of Conservation. Our HRM method enables high-throughput and cost-effective species identification from low DNA template samples, and could readily be adapted to discriminate other mammalian species from faecal DNA.

Experimental infection of self-cured Leiopelma archeyi with the amphibian chytrid Batrachochytrium dendrobatidis

The susceptibility of Archeys frog Leiopelma archeyi to Batrachochytrium dendrobatidis (Bd) is unknown, although one large population is thought to have declined sharply due to chytridiomycosis. As primary infection experiments were not permitted in this endangered New Zealand species, 6 wild-caught L. archeyi that naturally cleared infections with Bd while in captivity were exposed again to Bd to assess their immunity. These frogs were from an infected population at Whareorino, which has no known declines. All 6 L. archeyi became reinfected at low intensities, but rapidly self cured, most by 2 wk. Six Litoria ewingii were used as positive controls and developed heavier infections and clinical signs by 3 wk, demonstrating that the zoospore inoculum was virulent. Six negative controls of each species remained uninfected and healthy. Our results show that L. archeyi that have self cured have resistance to chytridiomycosis when exposed. The pattern is consistent with innate or acquired immunity to Bd, and immunological studies are needed to confirm this.