Contrasting global genetic patterns in two biologically similar, widespread and invasive Ciona species (Tunicata, Ascidiacea)

Human-mediated dispersal interplays with natural processes and complicates understanding of the biogeographical history of species. This is exemplified by two invasive tunicates, Ciona robusta (formerly Ciona intestinalis type A) and C. intestinalis (formerly Ciona intestinalis type B), globally distributed and sympatric in Europe. By gathering new mitochondrial sequences that were merged with published datasets, we analysed genetic patterns in different regions, with a focus on 1) their sympatric range and 2) allopatric populations in N and S America and southern Europe. In the sympatric range, the two species display contrasting genetic diversity patterns, with low polymorphism in C. robusta supporting the prevalent view of its recent introduction. In the E Pacific, several genetic traits support the non-native status of C. robusta. However, in the NE Pacific, this appraisal requires a complex scenario of introduction and should be further examined supported by extensive sampling efforts in the NW Pacific (putative native range). For C. intestinalis, Bayesian analysis suggested a natural amphi-North Atlantic distribution, casting doubt on its non-native status in the NW Atlantic. This study shows that both natural and human-mediated dispersal have influenced genetic patterns at broad scales; this interaction lessens our ability to confidently ascertain native vs. non-native status of populations, particularly of those species that are globally distributed.

Contrasting global genetic patterns in two biologically similar, widespread and invasive Ciona species (Tunicata, Ascidiacea)

Human-mediated dispersal interplays with natural processes and complicates understanding of the biogeographical history of species. This is exemplified by two invasive tunicates, Ciona robusta (formerly Ciona intestinalis type A) and C. intestinalis (formerly Ciona intestinalis type B), globally distributed and sympatric in Europe. By gathering new mitochondrial sequences that were merged with published datasets, we analysed genetic patterns in different regions, with a focus on 1) their sympatric range and 2) allopatric populations in N and S America and southern Europe. In the sympatric range, the two species display contrasting genetic diversity patterns, with low polymorphism in C. robusta supporting the prevalent view of its recent introduction. In the E Pacific, several genetic traits support the non-native status of C. robusta. However, in the NE Pacific, this appraisal requires a complex scenario of introduction and should be further examined supported by extensive sampling efforts in the NW Pacific (putative native range). For C. intestinalis, Bayesian analysis suggested a natural amphi-North Atlantic distribution, casting doubt on its non-native status in the NW Atlantic. This study shows that both natural and human-mediated dispersal have influenced genetic patterns at broad scales; this interaction lessens our ability to confidently ascertain native vs. non-native status of populations, particularly of those species that are globally distributed.

Previous land use and climate influence differences in soil organic carbon following reforestation of agricultural land with mixed-species plantings

Reforestation of agricultural land with mixed-species environmental plantings (native trees and shrubs) can contribute to mitigation of climate change through sequestration of carbon. Although soil carbon sequestration following reforestation has been investigated at site- and regional-scales, there are few studies across regions where the impact of a broad range of site conditions and management practices can be assessed. We collated new and existing data on soil organic carbon (SOC, 0–30 cm depth, N = 117 sites) and litter (N = 106 sites) under mixed-species plantings and an agricultural pair or baseline across southern and eastern Australia. Sites covered a range of previous land uses, initial SOC stocks, climatic conditions and management types. Differences in total SOC stocks following reforestation were significant at 52% of sites, with a mean rate of increase of 0.57 ± 0.06 Mg C ha−1 y−1. Increases were largely in the particulate fraction, which increased significantly at 46% of sites compared with increases at 27% of sites for the humus fraction. Although relative increase was highest in the particulate fraction, the humus fraction was the largest proportion of total SOC and so absolute differences in both fractions were similar. Accumulation rates of carbon in litter were 0.39 ± 0.02 Mg C ha−1 y−1, increasing the total (soil + litter) annual rate of carbon sequestration by 68%. Previously-cropped sites accumulated more SOC than previously-grazed sites. The explained variance differed widely among empirical models of differences in SOC stocks following reforestation according to SOC fraction and depth for previously-grazed (R2 = 0.18–0.51) and previously-cropped (R2 = 0.14–0.60) sites. For previously-grazed sites, differences in SOC following reforestation were negatively related to total SOC in the pasture. By comparison, for previously-cropped sites, differences in SOC were positively related to mean annual rainfall. This improved broad-scale understanding of the magnitude and predictors of changes in stocks of soil and litter C following reforestation is valuable for the development of policy on carbon markets and the establishment of future mixed-species environmental plantings.

Genetic viability of Nabugabo lakes (LVR satellite lakes) fish species

Natural populations of fish species in Lake Victoria Region (LVR) have under gone dramatic changes including severe reduction in sizes, division of original stocks into disjunct subunits, and segregation into several isolated population units either within a single water body or even worse into separate waters. In addition, these changes have been either preceded or precipitated by introductions of non-indigenous species that out competed the native forms and in case of closely related species genetically swamped them through hybridisation. The latter is especially the case in Nabugabo lakes. Such events lead to fragmentation of populations, which results in reduction in genetic diversity due to genetic drift, inbreeding and reduced or lack of gene flow among independent units. Such phenomena make the continued existence of fisheries stocks in the wild precarious, more so in the face of the competition from exotic species. Species introductions coupled with growing exploitation pressure of the fisheries of these lakes have put the native stocks at risk. Nabugabo lakes harbor cichlid species that are unique to these lakes more so species of the cichlid complex. In this paper the ecological status and genetic viability of key Nabugabo lakes fish species is examined and management options are discussed.

The cellular and mechanistic study of nisin inhibition of bacillus anthracis spore outgrowth, nisin alteration of infection, and native producer immunity

A critical step during Bacillus anthracis infection is the outgrowth of germinated spores into vegetative bacilli that proliferate and disseminate rapidly within the host. An important challenge exists for developing chemotherapeutic agents that act upon and kill B. anthracis immediately after germination initiation when antibiotic resistance is lost, but prior to the outgrowth into vegetative bacilli, which is accompanied by toxin production. Chemical agents must also function in a manner refractive to the development of antimicrobial resistance. In this thesis we have identified the lantibiotics as a class of chemotherapeutics that are predicted to satisfy these two criteria. The objective of this thesis was to evaluate the efficacy of nisin, a prototypical lantibiotic, in prevention of outgrowth of germinated B. anthracis spores. Like all lantibiotics, nisin is a ribosomally translated peptide that undergoes post-translational modification to form (methyl)lanthionine rings that are critical for antimicrobial activity. Our studies indicate that nisin rapidly inhibits the in vitro outgrowth of germinated B. anthracis Sterne 7702 spores. Although germination initiation was shown to be essential for nisin-dependent antimicrobial activity, nisin did not inhibit or promote germination initiation. Nisin irreversibly killed germinated spores by blocking the establishment of a membrane potential and oxidative metabolism, while not affecting the dissolution of the outer spore structures. The membrane permeability of the spore was increased by nisin, but germinated spores did not undergo full lysis. Nisin was demonstrated to localize to lipid II, which is the penultimate precursor for cell wall biogenesis. This localization suggests two possible independent mechanisms of action, membrane pore formation and inhibition of peptidoglycan synthesis. Structure-activity studies with a truncated form of nisin lacking the two C-terminal (methyl)lanthionine rings and with non-pore forming mutants indicated that membrane disruption is essential for nisin-dependent inhibition of spore outgrowth to prevent membrane potential establishment. Finally, utilizing an in vitro infection model, it was shown that nisin reduced the viability of B. anthracis spores within an infection resulting in increased survival of immune cells while reducing infection-mediated cytokine expression. Fluorescence microscopy indicated that nisin localizes with spores within phagosomes of peritioneal macrophages in germinating conditions. These data demonstrate the effectiveness of nisin, as a model lantibiotic, for preventing spore outgrowth. It is speculated that nisin targeting of lipid II, resulting in membrane perturbations, may be effective at inhibiting the outgrowth of spores prepared from bacteria across a number of species.

Conversion of sub-tropical native vegetation to introduced conifer forest: Impacts on below-ground and above-ground carbon pools

Land-use change can have a major influence on soil organic carbon (SOC) and above-ground C pools. We assessed a change from native vegetation to introduced Pinus species plantations on C pools using eight paired sites. At each site we determined the impacts on 0–50 cm below-ground (SOC, charcoal C, organic matter C, particulate organic C, humic organic C, resistant organic C) and above-ground (litter, coarse woody debris, standing trees and woody understorey plants) C pools. In an analysis across the different study sites there was no significant difference (P > 0.05) in SOC or above-ground tree C stocks between paired native vegetation and pine plantations, although significant differences did exist at specific sites. SOC (calculated based on an equivalent soil mass basis) was higher in the pine plantations at two sites, higher in the native vegetation at two sites and did not differ for the other four sites. The site to site variation in SOC across the landscape was far greater than the variation observed with a change from native vegetation to introduced Pinus plantation. Differences between sites were not explained by soil type, although tree basal area was positively correlated with 0–50 cm SOC. In fact, in the native vegetation there was a significant linear relationship between above-ground biomass and SOC that explained 88.8% of the variation in the data. Fine litter C (0–25 mm diameter) tended to be higher in the pine forest than in the adjacent native vegetation and was significantly higher in the pine forest at five of the eight paired sites. Total litter C (0–100 mm diameter) increased significantly with plantation age (R2 = 0.64). Carbon stored in understorey woody plants (2.5–10 cm DBH) was higher in the native vegetation than in the adjacent pine forest. Total site C varied greatly across the study area from 58.8 Mg ha−1 at a native heathland site to 497.8 Mg ha−1 at a native eucalypt forest site. Our findings suggest that the effects of change from native vegetation to introduced Pinus sp. forest are highly site-specific and may be positive, negative, or have no influence on various C pools, depending on local site characteristics (e.g. plantation age and type of native vegetation).

Study on the inhibitory effects of native medicinal plants on growth and aflatoxin production by toxigenic Aspergilli isolated from rainbow trout feed

In the present study, natural occurrence of fungi and aflatoxin B1 (AFB1) in pellet feed and feed ingredients used for rainbow trout was investigated with emphasis to Aspergillus section Flavi members and medicinal plants inhibitory to Aspergillus growth and/or AF production. The feed samples were cultured on the standard isolation media including dichloran rosebengal chloramphenicol agar (DRCA) and Aspergillus flavus/parasiticus agar (AFPA) for 2 weeks at 28 °C. Identification of fungal isolates was implemented based on the macro- and microscopic morphological criteria. AFs were detected using high performance liquid chromatography (HPLC). Based on the results obtained, a total of 109 fungal isolates were identified of which Aspergillus was the prominent genus (57.0%), followed by Penicillium (12.84%), Absidia (11.01%) and Pseudallscheria (10.10%). The most frequent Aspergillus species was A. flavus (60.66%) isolated from all the feed ingredients as well as pellet feed. Among 37 A. flavus isolates, 19 (51.35%) were able to produce AFB1 on yeast extract-sucrose (YES) broth in the range of 10.2 to 612.8 [tg/g fungal dry weight. HPLC analyses of trout feed showed that pellet feed and all feed ingredients tested except gluten were contaminated with different levels of AFB1 in the range of 1.83 to 67.35 lig/kg. In order to finding natural inhibitors of fungal growth and/or AF production, essential oils (EOs) and extracts of 49 medicinal plants were studied against an aflatoxin-producing A. parasiticus using a microbioassay technique. The EOs was analyzed by gas chromatography/mass spectrometry (GC/MS). Based on the results obtained, Achillea millefolium sub sp. elborsensis, Ferula gummosa, Mentha spicata, Azadirachta indica, Conium maculatum and Artemisia dracunculus remarkably inhibited A. parasiticus growth without affecting AF production by the fungus. Besides of Thymus vulgaris and Citrus aurantifolia, the EO of Foeniculum vulgare significantly inhibited both fungal growth (-70.0%) and AFs B1 and G1 (-99.0%) production. The EO of Carum carvi and ethyl acetate extract of Platycladus orientalis suppressed AFs B1 and G1 by more than 90.0%, without any obvious effect on fungal growth. The IC50 values of bioactive plants for AFs B1 and G1 were determined in the ranges of 90.6 to 576.2 and 2.8 to 61.9 µg/ml, respectively. Overall, results of the present study indicate the importance of AF contamination of trout feed as a risk factor for fish farming and thus, an urgent necessity for constant monitoring of trout feed for any unacceptable levels of AF contamination. Likewise, antifungal activities of bioactive plants introduced here would be an important contribution to explain the use of these plants as effective antimicrobial candidates to protect feeds from toxigenic fungus growth and subsequent AF contamination.

An introduced competitor elevates corticosterone responses of a Native Lizard (Varanus varius).

Glucocorticoid hormone profiles are increasingly used as physiological markers to infer the strength of species interactions that can influence fitness and ensuing population dynamics of animals. Here we investigated two aims. First, we measured the effect of a 90-min capture stress protocol on the plasma corticosterone responses of a large native Australian lizard, the lace monitor (Varanus varius). Second, we compared the basal and postcapture stress corticosterone responses of lace monitors in habitats where they were exposed to high or low densities of the European red fox (Vulpes vulpes), an introduced competitor. Lace monitors responded to the capture stress protocol by significantly increasing plasma levels of corticosterone above basal at 45- and 90-min-postcapture blood-sampling intervals. In habitats with high fox densities, lace monitors produced a significantly greater basal and capture-stress-induced corticosterone response compared to individuals in low-fox density habitat. A significant interaction among fox density, time postcapture, and body condition was also found to influence plasma corticosterone values. These results suggest competition with red fox, perhaps via nutritional stress and increased hypersensitivity of the adrenocortical axis in lizards. At present, without further research, we do not understand whether such responses mediate lizard fitness or whether they have adaptive or maladaptive consequences for lizard populations in response to red fox competition. Nevertheless, our results help broaden understanding of the physiological implications arising from species interactions and specifically how introduced competitors could mediate diverse impacts on native biodiversity.

Limitations to the feasibility of using hypolimnetic releases to create refuges for riverine species in response to stream warming

Given predicted increases associated with human-induced climate change, stream temperatures are likely to approach upper tolerance limits of aquatic biota within the coming decades. Little information is available regarding thermal tolerance limits of lotic fauna or mechanisms allowing fauna to persist following high temperature events (e.g. use of thermal refuges). Cold-water refuges can facilitate survival of fish in the Northern Hemisphere, but little evidence of similar refuges exists elsewhere.Planned releases of hypolimnetic, or a mixture of top and bottom, waters from reservoirs have recently been touted as a novel method to potentially ameliorate extreme temperature events. However, the feasibility of this technique has not been fully discussed in the published literature. Therefore, we present a literature review, an analysis of thermal data for some large dams in southern Australia in relation to known thermal tolerances of native fauna, and an assessment of current management practices regarding the technique. We show that hypolimnetic releases have variable impacts on water temperatures downstream of a dam, depending on size, off-take infrastructure and management practices but, even where there is an effect, knowledge gaps are too numerous for this technique to be currently feasible. Furthermore, hypolimnetic releases generally evoke negative connotations among natural resource managers, due to the occurrence of cold-water shock in some species. If knowledge gaps and limitations can be addressed, it is possible that the technique may be considered in future, so we present potential tools for future assessment, capacities and limitations and discuss potential scenarios where environmental managers might consider this technique.

Species composition and hybridisation of mussel species (Bivalvia: Mytilidae) in Australia

Mussels belonging to the Mytilus edulis species complex have been the focus of numerous studies exploring the systematics and origin of this commercially and ecologically important genus. Species have wide geographical ranges and hybridise where their distributions overlap, making identification difficult. Several molecular markers have been used to distinguish between the species within the M. edulis species complex; however, no single marker system has been found to be completely diagnostic, and a combination of markers are used. Here, we used a combination of three nuclear genes and a mitochondrial gene region to assess the species composition of Mytilus mussels collected across its geographical range in Australia. Our results show that the majority (98.5%) of individuals sampled from Australian populations are Mytilus galloprovincialis, with 56.2% of them displaying a southern hemisphere haplotype, 10.3% displaying a putatively northern hemisphere haplotype, and 32% having M. galloprovincialis genotypes consistent with either northern or southern hemisphere M. galloprovincialis lineages. The taxonomic origin of the remaining 1.5% of samples (n ¼ 3) could not be conclusively determined. Our results suggest that there have been significant introductions of non-native M. galloprovincialis lineages into both southern and northern hemisphere populations.

Adaptation to urban environments promotes high reproductive success in the tawny frogmouth (Podargus strigoides), an endemic nocturnal bird species

The tawny frogmouth (Podargus strigoides) is an endemic, nocturnal bird species widespread throughout Australia with high numbers in urban environments but very limited information exists on its ability to cope with urban systems. We investigated the reproductive behaviour of this species in response to a continuum of urbanisation. Firstly, we asked does the degree of urbanisation influence the availability of suitable nesting locations. Secondly, does broad and/or local scale factors surrounding a nest influence reproductive success? And finally, does the degree of urbanisation influence time of breeding? We located 158 nest sites and monitored 189 breeding attempts across four breeding seasons (2010-2013). One hundred and thirty three of these attempts successfully fledged 177 chicks. We categorized 75 attempts as first known attempts for that season resulting in 77 fledged chicks. In some pairs, repeated attempts occurred after failure. We observed double brooding by three pairs, a strategy largely unknown in this species. Spatial modelling revealed that birds selected more vegetated areas of the gradient within which to locate their nests, avoiding more highly urbanized areas. We identified no association between land-use type and breeding success at both broad and local scales. Birds selected native rough-barked Eucalyptus tree species more frequently than other trees for nesting. The key drivers for the commencement of breeding were climatic variables, in particular rainfall. The ability of the species to synchronize breeding attempts to coincide with optimal environmental conditions resulted in extremely high reproductive success suggesting high individual fitness and an adaptation to local environmental conditions.

Previous land use and climate influence differences in soil organic carbon following reforestation of agricultural land with mixed-species plantings

Reforestation of agricultural land with mixed-species environmental plantings (native trees and shrubs) can contribute to mitigation of climate change through sequestration of carbon. Although soil carbon sequestration following reforestation has been investigated at site- and regional-scales, there are few studies across regions where the impact of a broad range of site conditions and management practices can be assessed. We collated new and existing data on soil organic carbon (SOC, 0-30 cm depth, N = 117 sites) and litter (N = 106 sites) under mixed-species plantings and an agricultural pair or baseline across southern and eastern Australia. Sites covered a range of previous land uses, initial SOC stocks, climatic conditions and management types. Differences in total SOC stocks following reforestation were significant at 52% of sites, with a mean rate of increase of 0.57 ± 0.06 Mg C ha-1 y-1. Increases were largely in the particulate fraction, which increased significantly at 46% of sites compared with increases at 27% of sites for the humus fraction. Although relative increase was highest in the particulate fraction, the humus fraction was the largest proportion of total SOC and so absolute differences in both fractions were similar. Accumulation rates of carbon in litter were 0.39 ± 0.02 Mg C ha-1 y-1, increasing the total (soil + litter) annual rate of carbon sequestration by 68%. Previously-cropped sites accumulated more SOC than previously-grazed sites. The explained variance differed widely among empirical models of differences in SOC stocks following reforestation according to SOC fraction and depth for previously-grazed (R2 = 0.18-0.51) and previously-cropped (R2 = 0.14-0.60) sites. For previously-grazed sites, differences in SOC following reforestation were negatively related to total SOC in the pasture. By comparison, for previously-cropped sites, differences in SOC were positively related to mean annual rainfall. This improved broad-scale understanding of the magnitude and predictors of changes in stocks of soil and litter C following reforestation is valuable for the development of policy on carbon markets and the establishment of future mixed-species environmental plantings.

Restoration rocks: integrating abiotic and biotic habitat restoration to conserve threatened species and reduce fire fuel load

With rapid urban expansion, biodiversity conservation and human asset protection often require different regimes for managing wildfire risk. We conducted a controlled, replicated experiment to optimise habitat restoration for the threatened Australian pink-tailed worm-lizard, Aprasia parapulchella while reducing fire fuel load in a rapidly developing urban area. We used dense addition of natural rock (30 % cover) and native grass revegetation (Themedatriandra and Poasieberiana) to restore critical habitat elements. Combinations of fire and herbicide (Glyphosate) were used to reduce fuel load and invasive exotic species. Rock restoration combined with herbicide application met the widest range of restoration goals: it reduced fire fuel load, increased ant occurrence (the primary prey of A. parapulchella) in the short-term and increased the growth and survival of native grasses. Lizards colonised the restored habitat within a year of treatment. Our study documents an innovative way by which conflicts between biodiversity conservation and human asset protection can be overcome.

Research, STEM Education, & Native American Programs in Montana

Oscillating electroosmotic flows in microchannels can be used as a separating mechanism for biological species. This MEMS related work specifically studies the separation of different sized DNA on the microscale as a pre-cursor to lab on a chip systems. In addition to this research, Dr. Thomas will present programs and activities that enable Native American students to obtain their STEM degrees. This involves working with middle school through graduate students at the 4 year institutions, Tribal colleges, and K-12 schools in Montana.

Life history of a native emydid turtle (Malaclemys terrapin centrata) on the remote oceanic islands of Bermuda

Diamondback terrapins (Malaclemys terrapin) are native to the remote oceanic islands of Bermuda and presently inhabit only four small brackish water ponds on a private golf course. The life history of this species is poorly understood on Bermuda and so the aim of this study was to fill these knowledge gaps, to compare the results with what is known from other areas in the North American range, and to inform the development of a local management plan. The results of a mark-recapture census revealed that ca. 100 individuals ≥81 mm straight carapace length live on Bermuda, of which nearly half (48.5%) were considered sexually mature. The population is dominated by females (sex ratio 2.9:1) and annual recruitment over the three year period was found to be extremely low (approximately two terrapins). Female diamondback terrapins in Bermuda nest almost exclusively within a limited number of sand bunkers on the golf course. Nesting commenced in late March or early April and ended in late August. Peak oviposition was observed in May and June. Clutch size averaged 5.1 eggs (range 0-10; SD 2.4) and the incubation period averaged 61.8 days (range 49-83; SD 10.5). Delayed emergence was documented, with 43.8% of the hatchlings remaining in their natal nests over the winter months. The mean annual hatching success rate was determined to be 19% (range 17.6-21; SD 1.9). Radio-telemetry was used to investigate the movements and survivorship of postemergent hatchling diamondback terrapins. The results indicated that mangrove swamps and grass-dominated marshes adjacent to the ponds are important developmental habitats for hatchlings. Yellow-crowned night herons (Nyctanassa violacea) were found to be significant predators of small terrapins during spring emergence. Small aquatic gastropods comprised 66.7% of the faecal samples analysed from the Bermudian population. Scavenged fish and vertebrate animal remains, terrestrial arthropods, polychaete worms and bivalves were consumed in lesser amounts. Sediment from the pond environment was found in 74% of the faecal samples analysed and is believed to have been incidentally ingested while foraging for the small benthic gastropods. Eco-toxicological analyses of the pond sediment, prey and terrapin eggs showed that the Bermudian diamondback terrapins live and feed in wetland habitats characterised by chronic, multifactorial contamination; principally total petroleum hydrocarbons, polycyclic aromatic hydrocarbons and a variety of heavy metals. This study found that some of those contaminants are accumulating in the gastropod prey as well as being transferred to terrapin eggs. This may be reducing the incidence of successful embryonic development for this species in Bermuda and may likely contribute to the observed low hatching rates. These collective findings indicate that the Bermudian population is very vulnerable to local extirpation.