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Diversidad y control biológico de insectos - CyberTesis UACh ...

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products were precipitated adding ethanol. The sequencing reactions were suspen<strong>de</strong>d in<br />

<strong>de</strong>ionized formami<strong>de</strong>, heat <strong>de</strong>natured and run on an ABI 3100 Genetic Analyzer (Applied<br />

Systems, Foster City, CA).<br />

The resulting chromatographs were imported into Sequencher 4.1 (Gene Co<strong>de</strong>s Corp., Ann<br />

Arbor, Michigan) for visual inspection and editing. Multiple sequence alignments were<br />

constructed with the MegAlign module of DNASTAR 5 (Lasergene, Madison, WI).<br />

Data analysis.<br />

Phylogenetic approaches.<br />

The ninety seven sequences were aligned using the Clustal W module of MEGA version<br />

3.1 (Kumar et al., 2004) and collapsed into haplotypes using the DNASP 4.10 software<br />

(Rozas et al., 2003). The phylogenetic relationships among the whole sequences and the<br />

extracted haplotypes were evaluated by using neighbour-joining (NJ), maximum parsimony<br />

(MP) and minimum evolution (ME) methods. All analyses were conducted using the<br />

Phylogeny module of MEGA version 3.1 (Kumar et al., 2004).<br />

The NJ tree was constructed from the matrix of pairwise p distances. This distance measure<br />

was chosen because it has low variance and then it is suitable to <strong>de</strong>al with low variation<br />

sequences (Nei and Kumar, 2000). Both MP and ME trees were constructed using<br />

unweighted data. Bootstrapping values (600 pseudoreplicates) were used to <strong>de</strong>termine<br />

nodal support. The trees were rooted using a sequence obtained from the rice blast fungus<br />

Magnaporthe rosea, retrieved from Genbank (XM_368948,<br />

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleoti<strong>de</strong>&list_uids=3<br />

9975114&dopt=GenBank). We consi<strong>de</strong>red gapped positions as unreliable characters and<br />

exclu<strong>de</strong>d them from further analysis (Swofford et al, 1996). To further analyze the genetic<br />

relatedness of haplotypes, we constructed a haplotype network using the median-joining<br />

network method as implemented in Network software (Ban<strong>de</strong>lt et al., 1999; www.fluxus-<br />

engineering.com).<br />

Analyses of <strong>de</strong>mographic history.<br />

The mismatch analysis (the distribution of the observed number of differences between<br />

pairs of haplotypes) was adopted to examine the <strong>de</strong>mographic history of the species<br />

23

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