Investigating the Efficacy of Riparian Reserves for Maintaining Invertebrate Biodiversity in a Fragmented Tropical Landscape

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PhD Thesis


Global tropical forest cover continues to decline at an alarming rate, in part due to oil palm expansion in Southeast Asia. In an area of the world with high levels of biodiversity and endemism, the conversion of these natural systems into agricultural plantations is of grave concern for conservationists. Oil palm monoculture has been repeatedly demonstrated to have lower biodiversity and ecosystem functioning than old growth and logged forests. In many countries, buffers of forest are retained around rivers during the conversion of natural systems into agriculture. These strips of forest, or riparian reserves, are primarily designed to protect aquatic systems from runoff, however there is increasing evidence that these zones may also protect aquatic and terrestrial biodiversity.

Here we propose studying three functionally important groups of terrestrial invertebrates (moths, dung beetles and termites) to assess the conservational efficacy of riparian reserves in fragmented oil palm landscapes in Sabah, Malaysia. Specifically, we aim to assess the potential for riparian reserves to act as ecological corridors and refugial habitat for invertebrate species in wider oil palm landscapes. The connectivity afforded by riparian forest may be predicted to increase the persistence of species over time, through metapopulation dynamics and by promoting dispersal among subpopulations. We propose the use of both traditional sampling techniques and molecular approaches to investigate these processes. In addition we will assess the persistence of ecosystem functions in fragmented landscapes and the provision of ecosystem services to oil palm plantations by riparian reserve spillover effects.

Key Aims & Objectives
1. To investigate the role of riparian reserves as refugial habitats for several insect taxa (moths, dung beetles & termites) in comparison to other habitat types.
2. To investigate the efficacy of riparian reserves in oil palm plantations as dispersal corridors for these taxa.
3. To identify source-sink dynamics in fragmented forest landscapes and the impact that these may have on estimates of the conservation value of riparian reserves.
4. To investigate invertebrate-mediated ecosystem functions in riparian reserves.
5. To investigate the role of riparian reserves in providing ecosystem services to oil palm plantations.
6. To investigate the physiological and morphological traits that promote the persistence of invertebrate taxa in oil palm landscapes.


We aim to investigate the role of riparian reserves in the dynamics, dispersal and persistence of invertebrate populations in Malaysian fragmented tropical forests. We propose to characterize differences in communities and biodiversity levels between continuous forest, riparian reserves and oil palm plantation for selected invertebrate taxa. We will also use mark-release-recapture methods and molecular approaches to assess the efficacy of these reserves as corridors for dispersal within a fragmented landscape. Furthermore, we will investigate the impact that these reserves have on ecosystem services and disservices internally and externally into the oil palm plantations. Finally, we aim to study the morphological and physiological traits that allow certain species to persist in these corridors using assays of thermal tolerance, desiccation, and gene expression.

Supervision and Study Sites
The proposed research would be carried out under the supervision of: Prof. Stephen Rossiter - Head of Organismal Biology at Queen Mary University London and a co-PI LOMBOK consortium of the HTMF programme; Prof. Owen Lewis - Professor of Ecology at the University of Oxford and Lead PI of the LOMBOK consortium, Dr. Paul Eggleton - Merit Researcher at the Natural History Museum (London) and Co-Investigator of BALI and Dr. Eleanor Slade coordinator of LOMBOK and researcher at the University of Oxford. We will also benefit from collaboration with Dr. Arthur Chung – Senior Researcher at the Sabah Forestry Department and Dr. Matthew Struebig (University of Kent; co-PI for LOMBOK). Here we propose undertaking our research at Maliau Basin Conservation Area, Danum Valley Conservation Area, and the SAFE Project. Maliau Basin will offer old growth riparian forest as our control sites.

Study Taxa
Our three focal taxa for diversity assessments are Lepidoptera (moths and butterflies), Coleoptera (dung beetles), and termites. All three taxa are simple to survey, with lepidopterans and dung beetles easily baited and trapped, and termites studied using standardised transects used by Paul Eggleton and the BALI team. In addition, all three groups provide key ecosystem services with lepidopterans serving as pollinators and prey for many predatory groups (e.g. bats, birds, spiders), and dung beetles and termites as increasing decomposition rates and bioturbation.

Invertebrate Sampling Methods
To sample leptidopterans, we will use standard fruit-baited butterfly live traps. Bait is batch-prepared at the start of each field season by homogenising and freezing large quantities of rotten bananas sourced locally. 100g of bait will be used for each trap and left to attract lepidopterans for 24 hours. Individuals will be identified and released in the field where possible. If a positive identification is not possible, individuals will be killed using ethyl acetate soaked cotton wool in killing jars and brought back to the field lab. At the field lab they will be papered and pinned for identification at the field lab. For our 5 mark-release-recapture (MRR) species (see MRR section), individuals are uniquely numbered using a permanent marker on the underside of the left hind-wing before release. Thorax width may also be measured using callipers to investigate whether individuals with traits linked to high dispersal are found within corridors.

To sample dung beetles dung-baited pitfall traps are used. Locally sourced dung (cattle) will be suspended above a pitfall trap made from a plastic cup and a funnel. These traps are ‘dry’ to keep individuals alive for MRR methods. Leaf litter and soil is placed in the bottom to minimise induced stress in captured individuals. Traps will also be protected from rainfall and leaf litter using a paper plate and wooden skewers. Similarly to the butterfly baits, traps are left to collect for 24 hours. For our mark-release recapture species, individuals are given a unique code using spots of silver permanent marker on the elytra.

Termite sampling will be undertaken using Paul Eggleton's research group's standardised protocol. Firstly, a 100m transect is set up and subsequently divided into 20 5x2m sections. Each of these sections is then sampled for one hour of effort (usually two people for 30 mins) slowly and continuously. During each subsection of the transect, samplers search six separate zones and collect termites. These search zones are (1) the inside of dead wood (logs, branches, stumps), (2) leaf litter and soil at the base of trees, (3) beneath decomposing dead wood and in soil within dead wood, (4) trees up to 2m high for arboreal nests, (5) surface soil 12 times (12x12cm plots, 10cm deep) and (6) subterranean nests, carton sheeting, runways on vegetation and mounds (Jones & Eggleton 2000). Some species are only distinguishable using a microscope and so every population encountered has an individual sampled via collection in 80% ethanol (either a worker or soldier to aid identification). Samples will be exported for formal identification at the Natural History Museum, London.

If riparian reserves are acting as sink populations for insect species we might expect insect population density to decrease with distance from the source population. That is to say, the number of observed individuals in a transect away from a fragment of forest would be expected to attenuate towards zero. To assess whether this is the case we will use mark release recapture methods with specific lepidopteran and dung beetle species. Mark-release-recapture methods are used to estimate population densities for a wide number of taxa. Target species will be captured and marked using the methodology described in the previous section. Our MRR study species are all large-bodied and found in continuous forest and riparian reserves, with few individuals found in oil palm matrix. Our mark-release-recapture data will be collected on five moth species (Erebus caprimulgus, Erebus ephesperis, Erebus gemmans, Hypoprya lactipex, Ischyja spp.) in the Noctuidae (subfamily Catocalinae). Selected moth species were chosen due to their abundance and size allowing for ease of capture and marking without damaging individuals. Several dung beetle species (all belonging to Scarabaeoidea) that are large enough for marking will be investigated using MRR methods. Termites are not suitable for MRR methods and so will not be used in this part of the study.

Several rivers in the Virgin Jungle Reserve (VJR), an area of selectively logged continuous forest on the outskirts of the SAFE project, and in Maliau Basin Conservation area, an area of old growth continuous forest, will be used as our control rivers. Our riparian reserves will be rivers that extend outwards from the VJR and into the oil palm matrix that have previously been studied by Dr. Claudia Gray, and more recently by the LOMBOK consortium. These rivers will all be sampled at 200m intervals from the fragment-riparian reserve boundary for 2km. Further, transects will be carried out away from release points into the oil palm plantations and down corridors to assess whether these species preferentially move along corridors rather than into the oil palm matrix.

Population Genetics Analysis
To quantify dispersal across the landscape, we propose to use population genetic markers. Specifically we will determine (a) whether riparian reserves act as corridors for invertebrate species, and (b) whether source-sink dynamics are occurring in riparian reserves. For this work, we plan to study two Catharsius species, one of our MRR moth species, and one termite species. We intend to develop and study polymorphic microsatellite markers, which are frequently used to quantify gene flow in population genetics studies (Brinkmann et al. 1995). We hope to identify new panels of microsatellite loci for each of our study species markers using a MiSeq (Illumina) sequencing platform. We will endeavor to carry out this sequencing at UMS if possible. The subsequent genotyping will be performed on an ABI 3730 platform at the NERC Molecular Biology Facility, to which PI Joseph Williamson is entitled to free access as a NERC-funded student. Individuals will be collected every 500m along networks of riparian reserves and rivers in connected fragments such as the VJR for these analyses. Population genetics analyses using microsatellite markers have been performed routinely in the lab of Williamson’s supervisor Rossiter at QMUL.

Firstly, we will be able to identify any individuals which have migrated from one subpopulation to another, or potentially, the offspring of a successfully reproductive migrant. This will give us clear evidence that individuals are able to traverse the landscape. However, this will not tell us whether or not they came via a riparian corridor or via the oil palm landscape. If there is no effect of the structure of a landscape on the genetic distribution of a population, we may expect that genetic distance would be strongly correlated with geographic distance. Given that riparian reserves have a non-linear dendritic structure, we would expect that if they aid the dispersal of individuals through the oil palm matrix, genetic distance would not be strongly correlated with geographic distance; rather, genetic distance would be strongly correlated with riparian distance.

Ecosystem Function Methods
All ecosystem function assays will be collected at our sampling points in the riparian reserves, oil palm logged, and old growth forest sites. We have selected four functions, bioturbation, leaf litter decomposition, wood decomposition and resource removal as examples of invertebrate-mediated ecosystem services. Bioturbation is the mixing and disturbance of soil and other sediments through the movement and growth of organisms. This process prevents the compaction of soil over time, mixes nutrients, oxygenates soil and allows greater movement of water through soil. Bioturbation is largely carried-out by termites, ants, cicadas, worms and other soil-dwelling invertebrates (Scheu 1987; Nkem et al. 2000; Smith & Hasiotis 2008; Jouquet et al. 2011) . Leaf litter decomposition and especially wood decomposition is predominantly carried out by termites (Jouquet et al. 2011) and is an important part of nutrient recycling in an ecosystem. Ants are the primary source of invertebrate resource removal, which is, again, an important factor in nutrient recycling and availability.

A 25x25m grid is set up around each of our sampling sites, within which ten 1x1m quadrats selected for assays in a pseudo-random design. Leaf litter, aboveground soil and other decomposing humus are removed using gloves to create a flat surface in each quadrat. After 5 days, all new aboveground soil is photographed to identify the source of bioturbation and collected for drying at 60°C for 3 days and subsequent weighing.

Invertebrate Leaf Litter Decomposition
Recently senesced leaf litter will be collected using leaf litter traps under Shorea johorensis trees. Samples will be taken every 2 weeks either in Maliau Basin Conservation Area or in the logged forest control at the SAFE Project. Only Shorea johorensis leaves will be used in assays to prevent any differential rates of decomposition between plant species. Leaves are then dried at 60°C for three days and homogenized. 10g of homogenized leaves are placed in 20x20cm Plastock 300-micron nylon mesh ( bags. Small holes are cut in the sides of the treatment bags with a hole-punch to allow termites to enter, whilst control bags are left uncut allowing us to distinguish between fungal and termite-mediated decomposition rates. 10 leaf litter bags (5 control and 5 treatment) are labeled and set up at pseudo-random locations within the 25x25m grid specified in the bioturbation protocol by pegging them to the ground before leaving them for 224 days. Collected samples are carefully removed from mesh bags with a spatula and any soil brought in by termites is discarded. Samples are then dried at 60°C for three days again and weighed. Net change in leaf litter weight is then calculated by subtracting the decomposed weight from the initial 10g sample.

Termite Wood Decomposition
Planed and sanded blocks of 9x9x5cm Pinus radiata wood that contain few deformations and knots are selected for decomposition experiments. All wood is heated at 60°C for 5 days and weighed before use. BALI researchers have enough of these blocks at Maliau Basin field station for use in this study. Blocks are placed in the same 300-micron nylon mesh bags as used in the leaf litter assays and labeled. 5 control and 5 treatment blocks are placed in the same grid as the leaf litter and bioturbation protocols in a pseudorandom design. Before placement, the ground is cleared of leaf litter and dead wood so that the block is in contact with the humic layer. These blocks are pegged to the floor and then left for approximately a year to be decomposed. Samples are then collected, dusted for loose material and broken open to remove soil infilling by termites and any organisms inside. After drying at 60°C for five days again the blocks are weighed and their net decomposition calculated.

Thermal and Desiccation Tolerance Assays
Riparian reserves in oil palm plantations have higher temperatures and lower humidity than riparian systems in old growth continuous forest. Abiotic factors such as these could be one of the reasons that different community structures have been found in riparian reserves. We may expect to find that species able to persist in such environments are morphologically equipped to do so.

Here we propose investigating the maximum thermal tolerances of different invertebrate species. Individuals are placed in glass vials and with the bottom partially submerged in a water bath. Temperatures are slowly increased by 1°C per 5 minutes, long enough to decrease the chances of a delayed response to temperature increase but short enough to limit the chances of death being caused by other factors such as desiccation. The critical thermal maximum (CTMax) is recorded as the temperature at which muscles begin to spasm in individuals.

Further, we propose investigating desiccation tolerances of selected invertebrate species to determine whether it is associated with persistence in riparian reserves. Weighed groups of individuals are placed into either 50mL control falcon tubes (ambient humidity) or into 50mL falcon tubes containing 5g of silica gel to reduce humidity with a cardboard barrier to prevent contact with the silica. Humidity of both treatment and control tubes is measured using a probe before the assay takes place. At time points of 10, 30, 60, 120 and 180 minutes, water loss is measured indirectly through weighing the group and the number of dead are recorded.

Finally, to determine the likely physiological basis of thermal tolerance, we will quantify and compare expression patterns of candidate stress loci across species that show contrasting levels of persistence in oil palm plantations (e.g. Catharsius dung beetles). For this, equal samples of each taxon will be exposed to shaded and exposed conditions. Individuals will then be sacrificed at equal time-points, and the RNA isolated for library construction and sequencing on a HiSeq or MiSeq. These techniques are routinely used in the study of thermal tolerances and similar assays of gene expression are performed in the lab of Williamson’s supervisor Rossiter at QMUL.
Project members
ResearcherProject roleProject contact
Joseph WilliamsonLead Researcher
Eleanor SladeSupervisor
Owen LewisSupervisor
Paul EggletonSupervisor
Stephen RossiterSupervisor
Arthur ChungMalaysian Collaborator