Phylogénie Et Evolution Du Comportement Social Chez Les Blattes ...

An n e x e sMolecular data for all the species and behavioural data for Eublaberus distanti have beenacquired for the present study.The sampling effort for behaviour has been critically evaluated with respect to theprevious behavioural studies that were conducted and published on the same insect species(e.g., van Baaren et al., 2002). Accumulation curves for the occurrence of transitionsaccording to the number of observations have been computed to show whether the samplingeffort is large enough to observe the transitions, either uncommon or frequent, occurring ineach species.Character independence has also been evaluated by checking whether frequenciesof transitions involving a same behavioural act are not misleadingly correlated. This can beeasily tested with a χ 2 goodness-of-fit test (Chatfield and Lemon, 1970; Zar, 1999) whichverifies whether the frequency of a transition between two acts can be determined by thetotal frequency of each act involved. Basically, this test compares expected frequencies withobserved frequencies. Following Zar (1999), some data have been pooled together in order tohave an average expected frequency of at least six, which avoids bias in χ 2 computation.Pr i m e r s, PCR a n d s e q u e n c i n gLeg muscle tissue was excised from roaches specimens preserved in 100% ethanol. DNAwas extracted using the Qiagen DNeasy protocol for animal tissue. Mitochondrial ribosomalDNA large subunit (16S, ~ 385 bp), nuclear ribosomal DNA small subunit (18S, ~ 1875 bp),and nuclear ribosomal DNA large subunit (28S) domains A (~ 360 bp) and C (~ 330 bp) wereamplified. 18S was amplified and sequenced in four overlapping fragments corresponding toGA, AD1D2, BCE and EF domains. PCR reactions were lead on a DNA Engine DYAD TM ,Peltier Thermal Cycler with the following conditions: an initial heating step of 94°C for 2min followed by 40 cycles of 94°C for 60 s, 55°C for 60 s and 72°C for 75 s. Then a finalelongation at 72°C during 7 min was carried out. The different already published primers usedare listed in Table 1. Electrophoresis gel was used to visualize PCR products and to checkthat there was no contamination thanks to a negative control. PCR products were purifiedvia the Montage PCR 96Cleanup Kit (Millipore®) and sequenced using ABI Big Dye 3.1®with the following sequence profile: 27 cycles of 96°C for 10 s, 50°C for 5 s and 60°C for 4min. Sequencing reactions products were purified with Sephadex TM columns and fractionatedon an ABI 3730 XL DNA sequencer. Each sequence was edited using Sequencher® 4.0(Genecodes, 1999) and blasted on GenBank (http://www.ncbi.nlm.nih.gov/blast/) to checkfor contamination. All the sequences (16S/18S/28SA/28SC, respectively) were deposited onGenBank under the following accession numbers: Eublaberus distanti (XX/XX/XX/XX),Lanxoblatta emarginata (XX/XX/XX/XX), Phortioeca nimbata (XX/XX/XX/XX), Schultesialampyridiformis (XX/XX/XX/XX) and Thanatophyllum akinetum (XX/XX/XX/XX).285