Jonathan C. Banks

Molecular genetic tools for environmental monitoring of New Zealand's aquatic habitats, past, present and the future

The assessment of biological samples is critical for measuring the ‘health’ of New Zealand aquatic environments. Analysis of these samples commonly requires species identification and enumeration, which usually involves microscopy or microbiological methods. These techniques can be time-consuming, laborious, and are dependent on taxonomic expertise. Recent advances in molecular methods provide promising tools for assessing environmental samples. A range of molecular techniques are now used in New Zealand including: fluorescent in situ hybridisation; automated ribosomal intergenic spacer analysis; quantitative polymerase chain reaction; and, most recently, next-generation sequencing. The organisms (or targets) and environments monitored are equally diverse, ranging from cyanobacteria, rotifers and invasive fish in lakes, to macroinvertebrates, and biofilm communities in rivers, to bacteria, micro- and macro-algae and invertebrates in marine ecosystems. Despite research and validation demonstrating their potential, the application of these tools by monitoring agencies has been limited. Legislative requirements, costs, and a reluctance to change methodologies, are the most likely reasons for this. This review examines molecular tools that have been previously or are currently used for monitoring aquatic environments in New Zealand, and explores how these, and new techniques, may be applied in the future.

Early detection of eukaryotic communities from marine biofilm using high-throughput sequencing: an assessment of different sampling devices.

Marine biofilms are precursors for colonization by larger fouling organisms, including non-indigenous species (NIS). In this study, high-throughput sequencing (HTS) of 18S rRNA metabarcodes was used to investigate four sampling methods (modified syringe, sterilized sponge, underwater tape and sterilized swab) for characterizing eukaryotic communities in marine biofilms. PerspexTM plates were sampled in and out of water. DNA collected with tape did not amplify. Otherwise, there were no statistical differences in communities among the remaining three sampling devices or between the two environments. Sterilized sponges are recommended for ease of use underwater. In-depth HTS analysis identified diverse eukaryotic communities, dominated by Metazoa and Chromoalveolata. Among the latter, diatoms (Bacillariophyceae) were particularly abundant (33% of reads assigned to Chromalveolata). The NIS Ciona savignyi was detected in all samples. The application of HTS in marine biofilm surveillance could facilitate early detection of NIS, improving the probability of successful eradication.

Comparing plasma and faecal measures of steroid hormones in Adelie penguins Pygoscelis adeliae

Physiological measurements of both stress and sex hormones are often used to estimate the consequences of natural or human-induced change in ecological studies of various animals. Different methods of hormone measurement exist, potentially explaining variation in results across studies; methods should be cross-validated to ensure that they correlate. We directly compared faecal and plasma hormone measurements for the first time in a wild free-living species, the Adelie penguin (Pygoscelis adeliae). Blood and faecal samples were simultaneously collected from individual penguins for comparison and assayed for testosterone and corticosterone (or their metabolites). Sex differences and variability within each measure, and correlation of values across measures were compared. For both hormones, plasma samples showed greater variation than faecal samples. Males had higher mean corticosterone concentrations than females, but the difference was only statistically significant in faecal samples. Plasma testosterone, but not faecal testosterone, was significantly higher in males than females. Correlation between sample types was poor overall, and weaker in females than in males, perhaps because measures from plasma represent hormones that are both free and bound to globulins, whereas measures from faeces represent only the free portion. Faecal samples also represent a cumulative measure of hormones over time, as opposed to a plasma ‘snapshot’ concentration. Our data indicate that faecal sampling appears more suitable for assessing baseline hormone concentrations, whilst plasma sampling may best define immediate responses to environmental events. Consequently, future studies should ensure that they select the most appropriate matrix and method of hormone measurement to answer their research questions.

Testing use of mitochondrial COI sequences for the identification and phylogenetic analysis of New Zealand caddisflies (Trichoptera)

Abstract We tested the hypothesis that cytochrome c oxidase subunit 1 (COI) sequences would successfully discriminate recognised species of New Zealand caddisflies. We further examined whether phylogenetic analyses, based on the COI locus, could recover currently recognised superfamilies and suborders. COI sequences were obtained from 105 individuals representing 61 species and all 16 families of Trichoptera known from New Zealand. No sequence sharing was observed between members of different species, and congeneric species showed from 2.3 to 19.5% divergence. Sequence divergence among members of a species was typically low (mean = 0.7%; range 0.0–8.5%), but two species showed intraspecific divergences in excess of 2%. Phylogenetic reconstructions based on COI were largely congruent with previous conclusions based on morphology, although the sequence data did not support placement of the purse‐cased caddisflies (Hydroptilidae) within the uncased caddisflies, and, in particular, the Rhyacophiloidea. We conclude that sequence variation in the COI gene locus is an effective tool for the identification of New Zealand caddisfly species, and can provide preliminary phylogenetic inferences. Further research is needed to ascertain the significance of the few instances of high intra‐specific divergence and to determine if any instances of sequence sharing will be detected with larger sample sizes.

Latitudinal distribution and mitochondrial DNA (COI) variability of Stereotydeus spp. (Acari: Prostigmata) in Victoria Land and the central Transantarctic Mountains

Abstract We examined mitochondrial DNA (COI) variability and distribution of Stereotydeus spp. in Victoria Land and the Transantarctic Mountains, and constructed Neighbour Joining (NJ) and Maximum Likelihood (ML) phylogenetic trees using all publicly available COI sequences for the three Stereotydeus species present (S. belli, S. mollis and S. shoupi). We also included new COI sequences from Miers, Marshall and Garwood valleys in southern Victoria Land (78°S), as well as from the Darwin (79°S) and Beardmore Glacier (83°S) regions. Both NJ and ML methods produced trees which were similar in topology differing only in the placement of the single available S. belli sequence from Cape Hallett (72°S) and a S. mollis haplotype from Miers Valley. Pairwise sequence divergences among species ranged from 9.5–18.1%. NJ and ML grouped S. shoupi from the Beardmore Glacier region as sister to those from the Darwin with pairwise divergences of 8%. These individuals formed a monophyletic clade with high bootstrap support basal to S. mollis and S. belli. Based on these new data, we suggest that the distributional range of S. shoupi extends northward to Darwin Glacier and that a barrier to dispersal for Stereotydeus, and possibly other arthropods, exists immediately to the north of this area.

Report of a mummified leopard seal carcass in the southern Dry Valleys, McMurdo Sound, Antarctica

The wide spread occurrence of mummified seal and penguincarcasses tens of kilometres from the open ocean is aninteresting phenomenon occurring in the Antarctic DryValleys. Mummified seal carcasses were first reported byScott’s expedition in 1903 (Scott 1969), and live seals andseal carcasses have since been reported many kilometresfrom the nearest ice-free ocean. For example, Stirling &Rudolph (1968) reported a live crabeater seal near MountSaunders, Marie Byrd Land, 113km from the open ocean.Seal carcasses found in the McMurdo Dry Valleys arepredominantly crabeater seals (

Environmental influences on Adelie penguin breeding schedules, endocrinology, and chick survival

To understand how the social and physical environment influences behaviour, reproduction and survival, studies of underlying hormonal processes are crucial; in particular, interactions between stress and reproductive responses may have critical influences on breeding schedules. Several authors have examined the timing of breeding in relation to environmental stimuli, while others have independently described endocrine profiles. However, few studies have simultaneously measured endocrine profiles, breeding behaviour, and offspring survival across seasons. We measured sex and stress hormone concentrations (oestrogens, testosterone, and corticosterone), timing of breeding, and chick survival, in Adelie penguins (Pygoscelis adeliae) at two colonies in two different years. Clutch initiation at Cape Bird South (CBS; year 1, ~14,000 pairs) occurred later than at Cape Crozier East (CCE; year 2, ~ 25,000 pairs); however, breeding was more synchronous at CBS. This pattern was probably generated by the persistence of extensive sea ice at CBS (year 1). Higher corticosterone metabolite and lower sex hormone concentrations at CBS correlated with later breeding and lower chick survival compared to at CCE - again, a likely consequence of sea ice conditions. Within colonies, sub-colony size (S, 50-100; M, 200-300; L, 500-600; XL, >1000 pairs) did not influence the onset or synchrony of breeding, chick survival, or hormone concentrations. We showed that the endocrine profiles of breeding Adelie penguins can differ markedly between years and/or colonies, and that combining measures of endocrinology, behaviour, and offspring survival can reveal the mechanisms and consequences that different environmental conditions can have on breeding ecology.

A new, subalpine species of Daphnia (Cladocera, Anomopoda) in the D. carinata species complex, in the South Island, New Zealand

Until recently, only one native and three apparently introduced Daphnia species were known from New Zealand. We demonstrate that (1) Daphnia in subalpine habitats in southern New Zealand differ morphologically and genetically from the native taxon previously labelled Daphnia carinata to merit species nova status and (2) the name of the latter should revert to D. thomsoni, used by Sars (1894) for Daphnia described from New Zealand mud. We compare some key characteristics and cytochrome c oxidase subunit 1 (CO1) sequences of the New Zealand native and other morphologically similar species. Distinctive characteristics of subalpine populations, described as Daphnia tewaipounamu sp. nov., are a wide cephalic shield with lateral flanges curving dorsally via rounded fornices, dorsal cervical depression variably expressed as a ‘step’ in the cephalic shield exuviae and retention of ephippia within shed carapace exoskeletons long after ecdysis. CO1 sequences revealed that D. tewaipounamu sp. nov. belongs to the D. carinata complex but is highly divergent (>14%) from other known members of this complex. New Zealand D. thomsoni is divergent (>15%) from D. carinata s.s. However, it is not endemic to New Zealand, as we confirmed its presence in Tasmania, and some Australian populations are closely related to it.