Molecular evolution of Adh and LEAFY and the phylogenetic utility of their introns in Pyrus (Rosaceae)

<p>Abstract</p> <p>Background</p> <p>The genus <it>Pyrus </it>belongs to the tribe Pyreae (the former subfamily Maloideae) of the family Rosaceae, and includes one of the most important commercial fruit crops, pear. The phylogeny of <it>Pyrus </it>has not been definitively reconstructed. In our previous efforts, the internal transcribed spacer region (ITS) revealed a poorly resolved phylogeny due to non-concerted evolution of nrDNA arrays. Therefore, introns of low copy nuclear genes (LCNG) are explored here for improved resolution. However, paralogs and lineage sorting are still two challenges for applying LCNGs in phylogenetic studies, and at least two independent nuclear loci should be compared. In this work the second intron of <it>LEAFY </it>and the alcohol dehydrogenase gene (<it>Adh</it>) were selected to investigate their molecular evolution and phylogenetic utility.</p> <p>Results</p> <p>DNA sequence analyses revealed a complex ortholog and paralog structure of <it>Adh </it>genes in <it>Pyrus </it>and <it>Malus</it>, the pears and apples. Comparisons between sequences from RT-PCR and genomic PCR indicate that some <it>Adh </it>homologs are putatively nonfunctional. A partial region of <it>Adh1 </it>was sequenced for 18 <it>Pyrus </it>species and three subparalogs representing <it>Adh1-1 </it>were identified. These led to poorly resolved phylogenies due to low sequence divergence and the inclusion of putative recombinants. For the second intron of <it>LEAFY</it>, multiple inparalogs were discovered for both <it>LFY1int2 </it>and <it>LFY2int2</it>. <it>LFY1int2 </it>is inadequate for phylogenetic analysis due to lineage sorting of two inparalogs. <it>LFY2int2-N</it>, however, showed a relatively high sequence divergence and led to the best-resolved phylogeny. This study documents the coexistence of outparalogs and inparalogs, and lineage sorting of these paralogs and orthologous copies. It reveals putative recombinants that can lead to incorrect phylogenetic inferences, and presents an improved phylogenetic resolution of <it>Pyrus </it>using <it>LFY2int2-N</it>.</p> <p>Conclusions</p> <p>Our study represents the first phylogenetic analyses based on LCNGs in <it>Pyrus</it>. Ancient and recent duplications lead to a complex structure of <it>Adh </it>outparalogs and inparalogs in <it>Pyrus </it>and <it>Malus</it>, resulting in neofunctionalization, nonfunctionalization and possible subfunctionalization. Among all investigated orthologs, <it>LFY2int2-N </it>is the best nuclear marker for phylogenetic reconstruction of <it>Pyrus </it>due to suitable sequence divergence and the absence of lineage sorting.</p

Four Swedish long-term field experiments with sewage sludge reveal a limited effect on soil microbes and on metal uptake by crops

Purpose: This study aims to study the effect of sewage sludge amendment on crop yield and on microbial biomass and community structure in Swedish agricultural soils. Materials and methods Topsoil samples (0-0.20 m depth) from four sites where sewage sludge had been repeatedly applied during 14-53 years were analysed for total C, total N, pH and phospholipid fatty acids (PLFAs). Heavy metals were analysed in both soil and plant samples, and crop yields were recorded. Results and discussion At all four sites, sewage sludge application increased crop yield and soil organic carbon. Sludge addition also resulted in elevated concentrations of some heavy metals (mainly Cu and Zn) in soils, but high concentrations of metals (Ni and Zn) in plant materials were almost exclusively found in the oldest experiment, started in 1956. PLFA analysis showed that themicrobial community structure was strongly affected by changes in soil pH. At those sites where sewage sludge had caused low pH, Gram-positive bacteria were more abundant. However, differences in community structure were larger between sites than between the treatments. Conclusions: At all four sites, long-term sewage sludge application increased the soil organic carbon and nitrogen content, microbial biomass and crop yield. Long-term sewage sludge application led to a decrease in soil pH. Concentrations of some metals had increased significantly with sewage sludge application at all sites, but the amounts of metals added to soil with sewage sludge were found not to be toxic for microbes at any site