Recently, transcriptomics and proteomics approaches have been extensively used to analyze the global composition of venoms from viper snakes. These studies provide a comprehensive knowledge of primary structure data based on sequences available in databanks. They are very important for an accurate description of the protein families expressed allowing evolutionary inferences about the generation of venom diversity and the toxin arsenal present in snake venoms [27, 28]. In most of the referred studies, SVMPs appear as the major component in venoms of viper snakes and representatives of P-I, P-II and P-III classes were detected in all of them. However, these approaches are not the most appropriate for inferences of functional diversity since sequence characterization is usually based on molecular fragments of varying sizes that may omit structural rearrangements or motifs involved in important functions of a particular toxin. In this study, thirty-one cDNAs coding for mature SVMPs were completely sequenced from a gland of one specimen of B. neuwiedi snake. Internal alignments and phylogenetic inferences with databank SVMPs revealed new aspects of the evolution of functional diversity of this protein family not evidenced before by high throughput approaches.
Sequences of SVMPs from classes P-I, P-II and P-III were amplified from B. neuwiedi venom gland with particular abundance of cDNA sequences from class P-II SVMPs (BnMP-II). Representatives of each class were not identical, with at least two distinct copies of P-I cDNAs (with 98% identity) and three of P-III (with % identity values above 88). In opposition, great variability was observed amongst class P-II sequences with six distinct clones presenting a greater number of nucleotide substitution and divergence (identity from 73%). High sequence variability is not surprising since, considering that toxins evolved by accelerated evolution, a number of non-synonymous substitutions is predicted in coding regions driving to diversity of venom proteins, including SVMPs. Indeed, nucleotide substitutions were apparently required for the emergence of new functions as disclaimed for disintegrins . Moreover, toxin genes contain in their exons a greater number of unstable triplets that were subjected to a faster point-mutation rate than constitutive genes . In this regard, we suggest that point mutations are occurring in SVMPs with a higher frequency in representatives of class P-II, reflecting a higher plasticity in this class of genes that reflects in the generation of new functions in the venoms.
Besides the diversity of class P-II SVMPs generated by nucleotide substitutions, evidences of recombination between P-II disintegrin domain with catalytic domains of P-I or P-III SVMPs were also noted. When B. neuwiedi sequences were clustered by cladistic analysis, three distinct types of P-II sequences were detected with distinct branching: BnMP-IIa grouped with P-I SVMPs, BnMP-IIx with P-III, and BnMP-IIb in a third group, composed only of P-II SVMPs. This evidence suggests that recombination between genes encoding SVMPs might have occurred after the emergence of the primary gene copies coding for each scaffold. In their recent publication, Casewell et al.  suggest that different scaffolds of SVMPs were generated by minimization of genes with domain loss, which have occurred several times during evolution of this protein family. The loss of cysteine rich domain would have occurred once to generate P-II SVMPs and the loss of disintegrin domains would have occurred at least 8 times in P-II to generate P-I SVMPs. Indeed, this is a very plausible hypothesis already supported by other reports in the literature [8, 24]. The data presented in this study is not in disagreement with this theory. However, we add an alternative mechanism to explain the diversity of precursors coding for SVMPs. The topology of the trees shown in this study indicates recombination of P-II genes with members from P-I and P-III classes both at DNA and RNA levels after the emergence of the three different scaffolds. The hypothetical recombination between P-I and P-II SVMPs might occur at the post-transcriptional level, supported by the striking identity (100%) between the 5'-terminal 365 bp fragment of BnMP-I and BnMP-IIa cDNA sequences that apparently were alternatively joined with two distinct fragments at 5', which diverged by nucleotide substitutions and the inclusion in only one of the fragments the region coding for the disintegrin domain. According to the previous papers [8, 24, 25], P-I SVMPs were derived from a PII ancestor and this would imply high similarity between such precursors. However, considering that genes coding for SVMPs are subjected to accelerated evolution and that most of substitutions accumulate in coding regions, it is very unlike to expect 100% identity in cDNA regions of genes that diverged previously, even though the time of divergence between P-I and P-II SVMPs is not reported in previous papers [8, 24, 25]. In this regard, post-transcriptional recombination is an appealing hypothesis to explain, together with genetic events, the generation of class P-I SVMPs in different events of SVMPs evolutionary history, as reported previously . Corroborating this hypothesis we found complementary regions in B. neuwiedi cDNA sequences that could be involved in loop assembly in the RNAs enabling them for alternative trans-splicing leading to the formation of "hybrid" mRNAs. Evidences for alternative splicing have been shown in Bungarus fasciatus acetylcholinesterase gene generating different proteins in liver or venom gland . This was also suggested by Siigur and colleagues , when studying the generation of diversity of serineproteinases from Vipera lebetiba venom and, more recently, Zeng et al.  provided evidences of trans-splicing for generation of toxin diversity in the venom of Buthus martensii scorpion. Alternative splicing may explain the intriguing distribution of class P-I SVMPs in previous phylogenetic studies of SVMPs, also grouped with class P-IIa SVMPs in different branches of their phylogenetic tree .
The other evidence of recombination between different classes of SVMPs was deduced from the phylogenetic inferences of independent domains. Several studies report the phylogenetic relationships of SVMPs isolated from closely-related species, or of specific classes or domains [8, 23, 24, 33–36]. Usually, these studies deal with a large number of sequences with different degrees of similarity. In this study, the hypothesis of interclass recombination arose from experimental data, during sequence alignments. Thus, the analyses were carried out using a different approach driven to identify such recombination in other snake species. For this purpose, we selected by megablast  only the most similar sequences (>85% identity) of independent domains from each BnMP sequences and carried out the phylogenetic analysis with this selected group of toxins. This approach may not represent the evolutionary history of the protein family, but allowed the characterization of closest relationships between catalytic domains from different classes of SVMPs, with statistically supported clusters enclosing P-I and P-IIa (99%) or including P-IIx and P-III (100%) catalytic domains. Considering the disintegrin domain, P-IIa, P-IIb and P-IIx sequences were monophyletic (94%). We understand that this is strong evidence that the multiple domain structure observed in SVMPs has undergone different evolutionary history, and the protein variability of this protein family may be dependent on recombination at different points of the evolution of this protein family. In the case of BnMP-IIx and P-III SVMPs, recombination events might occur at the genomic level with the assembly of P-II disintegrin domains with catalytic regions from genes of non-hemorrhagic SVMPs class P-III. The possibility that genomic recombination of independent domains occurs by exon shuffling cannot be ruled out since this is an accepted mechanism for evolution of modular molecules, particularly plasma and extracellular matrix proteins [38, 39]. In venoms, segment switches in exons (ASSETS) have been demonstrated and correlated to targeting diversification . Moreover, it has also been reported by different authors the occurrence of SVMP structures that might have been assembled by P-III catalytic domain with P-IIa disintegrin domain  or by PIIa catalytic domains with P-III disintegrin-like domains lacking the cysteine-rich domain . These evidences supporting recombination between P-III catalytic and P-II disintegrin domains indicate that the loss of cysteine rich domain may be a more frequent mechanism and may have occurred in different events during evolution of SVMPs. Unfortunately, these mechanisms of recombination are still speculative since, up to the moment, genomic sequences coding for SVMPs were not completely disclosed and the exon/intron distribution at catalytic domain is still unknown.
Regardless of the mechanisms used for generation of new genes/mRNAs encoding class P-II SVMPs, this is certainly the class under highest adaptative pressure and positive selection. Soto et al.  showed that accumulation of mutations is less frequent in class P-III SVMPs than in class P-II. In this study, we noted in a single snake three independent P-II structures, coding for distinct proteins. BnMP-IIa sequence shares catalytic domain with P-I SVMPs and representatives enclosed in this cluster generate, by processing, the P-I SVMPs (only catalytic domains) and the classical RGD-disintegrins; BnMP-IIb sequences grouped with other class P-IIb SVMPs corresponding to the non-proteolytic processed P-II SVMPs; BnMP-IIx group corresponds to a new subclass of P-II SVMP precursor which predicts proteins with catalytic domain of non-hemorrhagic P-III SVMPs and a disintegrin domain with a higher number of cysteines, an extra pair at C-termini that may be involved in a new disulfide bond reported here for the first time. This domain association has not been reported yet and the presence of these molecules in venoms in the unprocessed form is still unknown and the possibility that this precursor is a pseudogene is not ruled out up to the moment. This distribution is in agreement to the gene tree reported by Casewell et al.  with a few differences: the authors include a fourth group with dimeric P-IId SVMP and did not evidence the P-IIx SVMPs. It is important to note the large number of sequences enclosed on their P-I/P-IIa group. In our case, BnMP-IIa was the most abundant cDNA in the gland of B. neuwiedi, but in the pool of B. neuwiedi venom, the most abundant proteins are two isoforms of a class P-I SVMP, BnP1 and BnP2, which were partially sequenced revealing approximately 90% identity with either BnMP-I or BnMP-IIa cDNAs . Taken together, the great number of P-I/P-IIa SVMPs described and their high expression levels in venom glands point to the advantage of this form of precursor since it generates both P-I SVMPs and free disintegrins from a single precursor. The possible recombination between P-I and P-II precursors shown in this study is of great relevance for the evolution of SVMPs and the generation of the functionally active representatives of this family of proteins.