New Caledonia Project

An important theme in the ecology of the southern hemisphere conifers has been their regeneration status in relation to competing angiosperms. A formerly popular view was that many of these conifers were not adequately regenerating and were being replaced by angiosperm trees (Womersley 1958, Schmithüsen 1960, Robbins 1962). This view was probably stimulated by two broader themes in ecology: (1) global-scale changes over evolu­tionary time in the dominance and diversity of conifers rela­tive to angio­sperm trees (e.g. Bond 1989, Midgley 1991); and (2) the dominance of equilib­rium ideas in traditional plant succession theory which did not adequately incorporate disturbance (major reviews of succession theory include; Whittaker 1953, Drury and Nisbet 1973, Connell and Slatyer 1977, Pickett and White 1985, Glenn-Lewin et al. 1993). Some 200 species (30%) of conifers have distributions which are southern hemisphere (Enright and Hill 1995), but the geo­graphic range of most is small and few dominate forests and treelines in the way that some northern hemisphere coni­fers do.

Despite its small area (19,000 km²) New Caledonia pos­sesses a rich and distinctive flora, totaling some 3,000 species of which 80% are endemic. Among these are 43 conifers, all endemic, from 4 fami­lies: Taxaceae, Podocarpaceae, Araucariaceae, and Cupressaceae. No other region of the world of similar size has such a diverse conifer flora. Many of these endemic species are growing on ultramafic soils and have restricted distributions within New Caledonia and have suf­fered from habitat reduction from forestry, mining and erosion of uplands. Vegetation on ultramafic soils is often highly distinctive in terms of species composition and community structure relative to that on adjacent, non-ultramafic soils. In New Caledonia ultramafic substrates cover approximately 5500 km² with fire-prone shrublands (maquis) occupying 80-90% of this area and closed rainforest most of the remainder (Jaffré 1980, Jaffré 1992). A few conifers, including Araucaria laubenfelsii, A. muelleri and Agathis ovata, occur as emergent trees within remnants of closed tropical forest, and in a unique structural assemblage as emergents within maquis. Recent research has begun to examine the successional processes resulting in this unique structural community on these ultramafic substrates (Jaffré 1995, Rigg et al. 1998, Enright and Goldblum 1998, McCoy et al. 1999). The environments of the rainforest understorey and maquis contrast greatly, especially in the intensity of incident radiation and the likely impacts of disturbance by fire (Enright et al. 2001) and other potential environmental variables.

Soil microbial communities probably are the most complex of natural communities with estimates of as many as 4000 species/g soil in the rhizosphere and can be regarded as a “hot spot” for microbial colonization and activity (Miethling et al. 2000, Normander and Prosser 2000, Torsvik et al. 1990). Microorganisms are important in soil humus formation, cycling of nutrients, soil tilth, and structure, and a myriad of other functions that can influence the overlying sediment, vegetation, and underlying aquifer (Kennedy and Smith 1995, Waldrop et al. 2000, Applegate 2001). Environmental factors that influence microbial community structure in soil are vegetation, soil moisture, temperature, pH, soil texture, organic carbon concentration, inorganic concentration, and soil redox potential (Pepper et al.1996).

Characterization of bacterial communities present in heavy metal-contaminated soils has been reported (Roane and Kellogg 1996, Baath et al. 1998, Brim et al. 1999, Sandaa et al. 1999, Mengoni et al. 2001).  It was found that high levels of heavy metals can affect both the qualitative and the quantitative structure of microbial communities, resulting in decreased metabolic activity and biomass as well as decreased diversity (Mengoni et al. 2001, Sandaa et al. 1999).  Other studies have found that bacteria that are resistance to nickel are also simultaneously resistant to Cr, Co, Zn, and Cu (Mengoni et al. 2001).  In ultramafic soils of New Caledonia, Schlegel et al. (1991) found a strong positive correlation between the proportion and maximal extent of nickel-resistant bacteria and the proximity of the hyperaccumulating tree, Sebertia acuminata. 

Vegetation is one environmental factor thought to be a major determinant on microbial community structure since it provides primary resources for growth (Nusslein and Tiedje 1999, Miethling et al. 2000). Decreases in microbial biomass were observed in the rhizosphere when vegetation was changed from forest to agriculture usage (Nusslein and Tiedje 1999, Waldrop et al. 2000). Previous studies in New Caledonia have examined the bacterial community of root nodules associated with the angiosperm Gymnostoma spp. (Navarro et al. 1999, Navarro et al. 1997).  They determined that the distribution of a nitrogen-fixing bacteria (Frankia) were associated with soil type and host-plant species. In other study, bacteria were isolated from the rhizosphere of Alyssum bertolonii in Italy and from serpentine soil at various distances from the plant.  Pseudomonas strains were found to predominant in the plant rhizosphere, whereas Streptomyces strains were mainly present in the soil (Mengoni et al. 2001). 

           The composition and structure of the microbial communities in the two contrasting environments of maquis and rainforest and the role they play in the ability of certain species to persist in both communities is unstudied. The boundary between closed forest (rainforest) and maquis may in part be a result of soil biological properties. It is hypothesized that conifers are able to better tolerate extreme edaphic conditions (Bond 1989, Midgley 1991) than angiosperm tree species. There may be differences in microbial community between closed forest and maquis associated with the unusual chemistry of ultramafic parent materials, which limit the exchange of certain vegetation species between the two communities.

           Our aim is for Dr. Melissa Lenczewski to join Drs. Lesley Rigg (Northern Illinois University, DeKalb, USA), Neal Enright (Dept. of Geography, University of Melbourne, Australia), and Tanguy Jaffré (Institute for Research and Development, New Caledonia) in New Caledonia to examine the potential for further research on this topic. Dr. Lenczewski will discuss the potential of the project in more detail with Dr. Enright in Melbourne (April 7-14, 2002) and complete some preliminary sampling and analysis during the one week visit (April 15-21, 2002) to New Caledonia.

Vegetation sampling methodology

Work in New Caledonia since 1995 has established many permanent vegetation plots in the south of the island, including several hectares of permanent plots on Mont Do, with over 2500 tagged Araucaria laubenfelsii individuals (seedlings, saplings and trees). In each plot, all conifers >30 cm tall have been tagged, their location mapped, diameter at breast height (dbh) and height recorded, and a sample of individuals >5 cm dbh cored using a Suunto increment-corer for age determination from tree-ring counts. Conifer seedlings (<30 cm tall) and saplings (>30 cm tall, but < 5 cm dbh) have been tagged in replicate random sub-plots of 100m². These individuals have been re-measured annually or semi-annually since the plots were established with the aim of tracing patterns of growth, survival and reproduction for use in demographic analysis (Rigg et al. 1998). This trip will provide the opportunity of an annual re-measurement and further quantification of the vegetation in the two community types.

Soil samples will be collected during the field site visit for obtaining preliminary microbial characterization data for future grants.  The soil samples will be immediately placed in sterile Naglene^® bottles or Whirl-Pak^® bags, put in a cooler, and transported to Northern Illinois University (NIU) and placed in a refrigerator or –80°C freezer until processed.  NIU has a soil permit from the USDA (permit number: S-51439) that authorizes shipment from all foreign sources. Dr. Rigg has worked in New Caledonia since 1995 and has applied successfully for sampling permission and is confident that such permission will be granted for this project.

Assessment of the microbial community structure

This task is to culture and describe novel strains of bacteria found in the subsurface soil at Mont Do.  In previous studies of the soils from Ni polluted ecosystems in New Caledonia Riviere Bleue area found microorganisms such as Actinobacteria, Burkholderia, Pseudomonas, Comamonas, Hafnia, and Arthrobacter (Stoppel and Schlegel 1995). We expected to find similar bacteria in the samples from the proposed field sites (i.e. Mont Do, Southern Province). Differences in microbial communities may be found in association with different vegetation types (rainforest vs. maquis) and according to soil characteristics.

Cultivation of soil bacteria from soil will be done by separation of bacteria from soil particulates by vortexing 1 part of soil in 9 parts of buffer with NaCl and SDS (Ovreas and Torsvik 1998).  Bacteria will be diluted and plated on various standard media such as nickel bacteria agar (Mengoni et al. 2001, Stoppel and Schlegel 1995), tryptic soy agar (general bacteria), Sands and Rovira medium (Gram negative), starch casein (Actinomycetes), Pseudomonas isolation agar (Pseudomonas), and R2A (low nutrient agar). Plates will be incubated at 15, 25, and 37°C that reflect the range of temperatures found throughout the pedosphere. 

Bacteria separated from soil particulates as describe above can also be examined using Biolog plates (Biolog, Hayward, CA).  Other studies have used Biolog to examine microbial communities in soil and the environment in the rhizosphere (Garland and Mills 1991, Zak et al. 1994, Ovreas and Torsvik 1998, Miethling et al. 2000), but none of these studies have examined the microbial communities in serpentine soils. Biolog plates such as the EcoPlate^TM could be used to determine the phenotypical characterization of the total microbial community in the environment and give a metabolic fingerprint (Zak et al. 1994, Buyer and Drinkwater 1997, Lindstrom et al. 1998).  This approach is based on measuring metabolism of the carbon substrates in the plates to generate distinctive patterns for a bacterial community.  Methods for using Biolog substrates or production profiles may also provide useful information on functional biodiversity (Zak et al.1994, Ovreas and Torsvik 1998). Enriched bacteria from different wells within the Biolog plate will also be isolated using the carbon source information from the plate. 

Extraction and purification of nucleic acids from soil

Current estimates indicate that less than 1% of the microorganisms present in the environment are readily culturable (Hurst et al. 1997) therefore molecular techniques will also be used to determine the microbial community structure. Nucleic acids will be extracted from soil by bead beating/soil homogenization or other similar methods described for heavy metal soils (Mengoni et al. 2001, Navarro et al. 1999, Navarro et al. 1997, Stoppel and Schlegel 1995).

Cloning/sequencing approach to examining microbial community structure

This technique adds to the information gathered from culturing of bacteria since it is better for recovery of the dominant prokaryotic population including non-culturable organisms (Nusslein and Tiedje 1999). Nucleic acid extracted either from soil samples or from cultured organisms will be used to construct clone libraries from 16S rDNA that is obtained via PCR amplification with universal or domain specific primers (Lenczewski 2001). The DNA will be cloned into E. coli using the TOPO Cloning Kit (Invitrogen, Carlsbad, CA) or by standard methods (Sambrook and Russell 2001).  The clone libraries will be constructed from community rDNA that is PCR-amplified with oligonucleotide forward primer 530F (5’-GTG CCA GCM GCC GCG GTA A-3’) and with the oligonucleotide reverse primer of 1392R (5’-ACG GGC GGT GTG TRC-3’).  The plasmids from individual colonies will be purified using the RPM^® AFS Kit from Bio101 (QBioGene, Carlsbad, CA). Clones may be screened prior to sequencing using random amplified polymorphic DNA (RAPD) or restriction linked polymorophism (RFLP) as described by Lenczewski (2001).  Plasmids will be sequenced at the Core DNA Synthesis and Sequencing Facility on the Northern Illinois University campus. The 16S rDNA sequence can then be analysed using the BLAST and FASTA programs at the NCBI, the GCG package of Accelrys and PC Gene.  Phylogenetic trees will be constructed using the CLUSTAL W alignment and PHYLIP Tree construction programs.  Collector’s curves or rarefraction analysis will be done to determine when complete diversity in the samples has been sequenced (McCaig et al. 1999). 

Research Significance and Implications

Conservation of biodiversity requires a thorough understanding of the natural processes that operate at both the population and community scales. The endemic plant species and unusual structural communities found in New Caledonia are of significant biological interest and yet their ecology is largely unstudied. This proposal is concerned with the maintenance of biodiversity and the survival of a rare species in one of the worlds most threatened biodiversity hot-spots and the structure of the associated microbial community. At the same time, it investigates fundamental processes in plant population dynamics and the role of the microbial community structure in vegetation succession within a unique vegetation context.  Monitoring of seedling survivorship is essential in providing baseline data for all aspects of population-based ecological analysis and assessing the bacteria community structure in these ultramafic soils will further our understanding of such extreme environments.