Maruyama et al. BMC Genomics 2010, 11:363 http://www.biomedcentral.com/1471-2164/11/363 glue-like adhesive webbing), non-orb-weaving spider. Interestingly, these clades are the most defined in the dendogram, as may be observed through the proximity among members of these clades. Another feature of clades 1 and 2 is that each is formed by contigs from a single species of tick: clade 2 presented contigs only from A. cajennense and clade 1 contained contigs only from R. microplus, probably for this reason the clades showed better proximity among the members. This finding is compatible with our hypothesis that metastriate ticks presenting with different biological characteristics rely on different types of GRPs. Noteworthy is the fact FSP is an elastic and glue -like adhesive silk [15], a characteristic which could be important for a Brevirostrata monoxe- nous tick. The third distinct clade has showed similarity with an unknown protein (UK) from R. haemaphysa- loides and contains contigs from only Brevirostrata ticks, i.e., R. microplus and R. sanguineus, again a finding that is compatible with our hypothesis. Futhermore, although distinct clades were not formed by the remaining GRP contigs, we noted that contigs segregated into two differ- ent patterns: those which presented matches with spider silk proteins (filled symbols) are concentrated in clades that are distant from those that have similarities with cement-like proteins of ticks (open symbols), with the exception of contigs Rs54, Rm388 and Ac52. This finding suggests that these two "major types" of GRPs (silks ver- sus cements) may have different roles during attachment ticks on the host. The similarities found with spider GRPs were also with Masps and FSPs, albeit from different gen- era of spiders. Interestingly, A. cajennense was repre- sented only twice among the cement-like (open symbol) contigs, while two other cement-like contigs from this species grouped with spider silk-like contigs. A multiple alignment analyses of the three distinct clades (clades 1-3 from Figure 2) showed the level of con- servation among contigs, as can be observed in Figure 3. Clade 1, formed by contigs from SGFRm showed the greatest degree of conservation. Sequence alignment of Rm34 and Rm37 show an identity of 78.9% and 81.1% similarity. Rm33 and Rm36 presented the lowest conser- vation, 33% identity and 35.5% similarity. Contigs of clade 2 formed by SGFAc sequences also displayed low diver- gence, even the least similar transcript Ac14; Ac13 pre- sented a reasonable equivalence between sequences (25.1% identity and 27% similarity) whereas Ac15 and Ac16 had better conservation, presenting an identity of 56,5% and similarity of 61.4%. Clade 3 did not did not present the same level of conservation as clades 1 and 2, except for contigs Rs 402 and Rm31, which presented a high identity and similarity of 63.4% and 65.9%, respec- tively. It was interesting to note the large conserved region visualized in each of the sequence alignments of clades 1 Page 8 of 17 and 2. Five conserved regions are encountered in SGFRm contigs of clade 1 that are identical in the 6 contigs: 1) QLGPS (position 7-11), 2) SGSLG (position 13-17), 3) GVLPSG (position 56-61), 4) SGVGRG (position 82-87) and 5) TGFVLPG (position 89-95); some of these con- served regions could be extend if the charge and chemical proprieties of the residues from different contigs are taken into consideration. In the alignment for clade 1, aspartic acid (D) could change to glutamic acid (E) at res- idue 5, leucine (L) to isoleucin (I) at residue 6 (both hydrophobic), glycine (G) to serine (S) at residue 12 (both uncharged) and valine (V) to alanine (A) at residue 19 (both hydrophobic) and these amino acids present similar characteristics among each other. The same aspects can be observed for residues 43-61 and 89-98. In addition, when the contigs of clade 1 are compared with the com- position of flageliform silk proteins, positions of impor- tant residues of silk proteins such as glycine, serine and proline are conserved among them (Additional file 2). Regarding clade 2, two conserved regions are found in all SGFAc contigs, one composed of 5 residues, FGSGF (position 134-138), and a second one with 10 residues, SGLGGGYGSG (position 140-149). It is noteworthy that both regions have glycine (the majority) and serine resi- dues. Again, conserved regions contain glycine and serine residues, two abundant amino acids in spider silk. Align- ment of Ac contigs and Masp proteins showed similarity in most positions containing glycine and serine residues, but not in positions with proline residues (Additional file 3). Sheets are formed in secondary structures of silk pro- teins with repeats containing glycine, serine and alanine, which confer their elastic and strength proprieties [16]. The presence of a proline residue between serine and gly- cine, as happens in sequences of clade 1, could be impor- tant to "interrupt" secondary structures determined by glycine, serine and alanine, promoting acquisition of more elastic and less stiff properties. The mechanical property of elasticity is greater in flagelliform silk pro- teins of orb-weaving spiders (e.g., the Nephila genus) that are made to capture flying prey than in major ampullate spidroin silk proteins (Masp) used in capture threads in less mobile spiders [15,17]. The multiple alignments of clade 3 sequences, which are homologous with an "unknown" protein from Rhipicephalus haemaphysa- loides did not present conserved regions, perhaps owing to divergence among contigs and many gaps that could not allow long conserved regions. However, it can be observed though shading of the alignments that they present similarities as described before, with regions abundant in glycine and serine. In addition to the contigs derived from R. microplus, R. sanquineus and A. cajennense analyzed herein and in order to increase the stringency of the test for our hypothesis, we performed a multiple alignment using