Conservation value, biodiversity value and methods of assessment in regenerating and human disturbed tropical forest

Although the value of primary forests for biodiversity conservation is well known, the potential biodiversity and conservation value of regenerating forests remains controversial. Many factors likely contribute to this, including: 1. the variable ages of regenerating forests being studied (often dominated by relatively young regenerating forests); 2. the potential for confounding on-going human disturbance (such as logging and hunting); 3. the relatively low number of multi-taxa studies; 4. the lack of studies that directly compare different historic disturbances within the same location; 5. contrasting patterns from different survey methodologies and the paucity of knowledge on the impacts across different vertical levels of rainforest biodiversity (often due to a lack of suitable methodologies available to assess them). We also know relatively little as to how biodiversity is affected by major current impacts, such as unmarked rainforest roads, which contribute to this degradation of habitat and fragmentation. This thesis explores the potential biodiversity value of regenerating rainforests under the best of scenarios and seeks to understand more about the impact of current human disturbance to biodiversity; data comes from case studies from the Manu and Sumaco Biosphere Reserves in the Western Amazon. Specifically, I compare overall biodiversity and conservation value of a best case regenerating rainforest site with a selection of well-studied primary forest sites and with predicted species lists for the region; including a focus on species of key conservation concern. I then investigate the biodiversity of the same study site in reference to different types of historic anthropogenic disturbance. Following this I investigate the impacts to biodiversity from an unmarked rainforest road. In order to understand more about the differential effects of habitat disturbance on arboreal diversity I directly assess how patterns of butterfly biodiversity vary between three vertical strata. Although assessments within the canopy have been made for birds, invertebrates and bats, very few studies have successfully targeted arboreal mammals. I therefore investigate the potential of camera traps for inventorying arboreal mammal species in comparison with traditional methodologies. Finally, in order to investigate the possibility that different survey methodologies might identify different biodiversity patterns in habitat disturbance assessments, I investigate whether two different but commonly used survey methodologies used to assess amphibians, indicate the same or different responses of amphibian biodiversity to historic habitat change by people. The regenerating rainforest study site contained high levels of species richness; both in terms of alpha diversity found in nearby primary forest areas (87% ±3.5) and in terms of predicted primary forest diversity from the region (83% ±6.7). This included 89% (39 out of 44) of the species of high conservation concern predicted for the Manu region. Faunal species richness in once completely cleared regenerating forest was on average 13% (±9.8) lower than historically selectively logged forest. The presence of the small unmarked road significantly altered levels of faunal biodiversity for three taxa, up to and potentially beyond 350m into the forest interior. Most notably, the impact on biodiversity extended to at least 32% of the whole reserve area. The assessment of butterflies across strata showed that different vertical zones within the same rainforest responded differently in areas with different historic human disturbance. A comparison between forest regenerating after selective logging and forest regenerating after complete clearance, showed that there was a 17% greater reduction in canopy species richness in the historically cleared forest compared with the terrestrial community. Comparing arboreal camera traps with traditional ground-based techniques suggests that camera traps are an effective tool for inventorying secretive arboreal rainforest mammal communities and detect a higher number of cryptic species. Finally, the two survey methodologies used to assess amphibian communities identified contrasting biodiversity patterns in a human modified rainforest; one indicated biodiversity differences between forests with different human disturbance histories, whereas the other suggested no differences between forest disturbance types. Overall, in this thesis I find that the conservation and biodiversity value of regenerating and human disturbed tropical forest can potentially contribute to rainforest biodiversity conservation, particularly in the best of circumstances. I also highlight the importance of utilising appropriate study methodologies that to investigate these three-dimensional habitats, and contribute to the development of methodologies to do so. However, care should be taken when using different survey methodologies, which can provide contrasting biodiversity patterns in response to human disturbance.

FAM49 - A novel regulator of the protrusive behaviour and motility of cells

Most eukaryotic cell motility relies on plasma membrane protrusions, which depend on the actin cytoskeleton and its tight regulation. The SCAR/WAVE complex, a pentameric assembly comprising SCAR/WAVE, Nap1, CYFIP/Pir121, Abi and HSPC300, is a key driver of actin-based protrusions such as pseudopods. SCAR/WAVE is thought to activate the Arp2/3 complex, a crucial actin nucleator, after being itself activated by upstream signals such as active Rac1. Despite recent progress on the study of the SCAR/WAVE complex, its regulation is still incompletely understood, with Nap1’s role being particularly enigmatic. Upon screening for potential Nap1 binding partners in the social amoeba Dictyostelium discoideum – a well established model organism in the study of the actin cytoskeleton and cell motility – we found FAM49, a ~36 kDa protein of unknown function which is highly conserved in Metazoa (animals) and evolutionarily closer species such as D. discoideum. Interestingly, D. discoideum’s FAM49 and its homologs contain a DUF1394 domain, which is also predicted in CYFIP/Pir121 proteins and most likely involved in their direct binding to active Rac1, which in turn contributes to SCAR/WAVE’s activation. FAM49’s unknown role, apparent high degree of conservation and potential connections to SCAR/WAVE and Rac1 persuaded us to start investigating its function and biological relevance in D. discoideum, leading to the work presented in this thesis. Several pieces of our data collectively support a function for FAM49 in modulating the protrusive behaviour, and ultimately motility, of D. discoideum cells, as well as a regulatory link between FAM49 and Rac1. FAM49’s involvement in protrusion regulation was first hinted at by our observation that GFP-tagged FAM49 is enriched in pseudopods. The possibility of a link with Rac1 was then strengthened by two additional observations: first, pseudopodial GFP-FAM49 is substantially co-enriched with active Rac, both showing fairly comparable spatio-temporal accumulation dynamics; second, when dominant-active (G12V) Rac1 is expressed in cells, it triggers the recruitment and persistent accumulation of GFP-FAM49 at the plasma membrane, where both become highly co-enriched. We subsequently determined that fam49 KO cells differ from wild-type cells in the way they protrude and move, as assessed in under-agarose chemotaxis assays. In particular, our data indicate that fam49 KO cells tend to display a lower degree of global protrusive activity, their protrusions extend more slowly and are less discrete, and the cells end up moving at lower speeds and with higher directional persistence. This phenotype was substantially rescued by FAM49 re-expression. While re-expressing FAM49 in fam49 KO cells we generated putative FAM49 overexpressor cells; compared to wild-type cells, they displayed atypically thin pseudopods and what seemed to be an excessively dynamic, and perhaps less coordinated, protrusive behaviour. Additional data in our study suggest that pseudopods made by fam49 KO cells are still driven by SCAR/WAVE, which is clearly not being replaced by WASP (as is now known to be the case in D. discoideum cells lacking a functional SCAR/WAVE complex). Nonetheless, the peculiar dynamics of those pseudopods imply that SCAR/WAVE’s activity is regulated differently when FAM49 is lost, though it remains to be determined how. This thesis is the first report of a dedicated study on FAM49 and lays the foundation for future research on it.