Effects of landscape features and demographic history on the genetic structure of Testudo marginata populations in the southern Peloponnese and Sardinia

Testudo marginata, the largest European land tortoise, is suffering habitat degradation and destruction. Some populations, in markedly degraded habitats, are characterized by divergent morphotypes. However, the evolutionary significance of these morphotypes is of debate. Using 11 polymorphic microsatellites, we studied: (1) marginated tortoises from Sardinia that display a divergent morphotype – this population was potentially introduced from Greece; and (2) an area in the southern Peloponnese that includes a small and degraded zone in which marginated tortoises are dwarf. Genetic analyses run without any a priori assignment clearly acknowledge the specimens sampled in the territory of the dwarf form as a single group whilst Sardinian specimens are clustered with other specimens from the northern part of the area sampled in Greece. Demographic analyses suggest that Sardinian tortoises originated recently from some of the populations sampled in the northern part of the area sampled in Greece. Over locations sampled in Greece, a landscape‐genetic analysis allowed us to detect potential landscape features that may reduce gene flow between the dwarf form territory and surrounding areas. Our results suggest that the territory of the dwarf form is particularly propitious for marginated tortoises and that conservation regulations in Greece should be reinforced to protect this area from increasing impact of human activities changing from traditional agriculture to mechanization and extensive use of chemicals. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 591–606.     

Phase determination from direct sequencing of length‐variable DNA regions

For diploid organisms, haplotype determination usually requires sequencing cloned polymerase chain reaction (PCR) products or comparing the genotypes of several individuals. We found out that phase could be reconstructed from direct sequencing of mixed PCR products by combining for each individual the complementary information contained in its forward and reverse chromatograms, provided these products had different lengths. When applied to the internal transcribed spacer 2 (ITS2) from corals of the genus Pocillopora, this new method allowed us to identify two dominant sequence types in some specimens; however, sequencing cloned PCR products from the same specimens yielded more variants, including possible PCR‐generated recombination artifacts.     

Impacts of climate change and urban development on the spotted marsh frog (Limnodynastes tasmaniensis)

Climate change and urbanization are among the most serious threats to amphibians, although little is known about their combined effects. We used a predictive spatial habitat suitability model to explore the potential impacts of climate change and urban development on the spotted marsh frog (Limnodynastes tasmaniensis) on the urban‐fringe of Melbourne, Australia. The CSIRO climate‐change predictions for the region indicate likely temperature increases of 3°C, and annual rainfall reductions of around 200 mm by the year 2070. Much of the study area overlaps a region that has been identified as one of the city's growth corridors. We used Bayesian logistic regression modelling to estimate current and future habitat suitability of pond sites in the Merri Creek catchment, exploring a range of best‐ to worst‐case scenarios through the use of hydrological and urbanization models. Our predictions for 2070, even under a moderate climate‐change scenario, suggest that the majority of ponds in the study area will be dry throughout much of the year. This has obvious implications for L. tasmaniensis, which is an aquatic breeding species. However, in the short term, urbanization is likely to have a more significant effect on the distribution of L. tasmaniensis in the Merri Creek catchment, particularly if development moves beyond the current urban growth boundary. The combined effects of climate change and urbanization could have a profound impact on the species, potentially causing it to disappear from within the study area. We provide recommendations for including such predictive models in urban planning and restoration activities to prepare for future conservation challenges.     

Interactive effects of drought and N fertilization on the spatial distribution of methane assimilation in grassland soils

Soil methanotrophic bacteria constitute the only globally relevant biological sink for atmospheric methane (CH₄). Nitrogen (N) fertilizers as well as soil moisture regime affect the activity of these organisms, but the mechanisms involved are not well understood to date. In particular, virtually nothing is known about the spatial distribution of soil methanotrophs within soil structure and how this regulates CH₄ fluxes at the ecosystem scale. We studied the spatial distribution of CH₄ assimilation and its response to a factorial drought × N fertilizer treatment in a 3‐year experiment replicated in two grasslands differing in management intensity. Intact soil cores were labelled with ¹⁴CH₄ and methanotrophic activity mapped at a resolution of ～100 μm using an autoradiographic technique. Under drought, the main zone of CH₄ assimilation shifted down the soil profile. Ammonium nitrate (NH₄NO₃) and cattle urine reduced CH₄ assimilation in the top soil, but only when applied under drought, presumably because NH₄⁺ from fertilizers was not removed by plant uptake and nitrification under these conditions. Ecosystem‐level CH₄ fluxes measured in the field did show no or only very small inhibitory effects, suggesting that deeper soil layers fully compensated for the reduction in top soil CH₄ assimilation. Our results indicate that the ecosystem‐level CH₄ sink cannot be inferred from measurements of soil samples that do not reflect the spatial organization of soils (e.g. stratification of organisms, processes, and mechanisms). The autoradiographic technique we have developed is suited to study methanotrophic activity in a relevant spatial context and does not rely on the genetic identity of the soil bacterial communities involved, thus ideally complementing DNA‐based approaches.