The importance of being genomic: non-coding and coding sequences suggest different models of toxin multi-gene family evolution

Studies of multi-gene protein families, including many toxins, are crucial for understanding the role of gene duplication in generating protein diversity in general. However, many evolutionary analyses of gene families are based on coding sequences, and do not take into account many potentially confounding evolutionary factors, such as recombination and convergence due to selection. We illustrate this using snake venom gene sequences from the Phospholipase A2 (PLA2) subfamily. Novel gene sequences from 20 species of understudied Asian pitvipers were analyzed alongside available genomic PLA2 sequences from another four crotaline and several viperine species. In contrast to previous analyses of this toxin family based on cDNA sequences, we find that duplication events are concentrated at the tips of the tree, suggesting that major functions such as presynaptic neurotoxicity have evolved convergently multiple times in pitvipers. We provide evidence that this discrepancy is due to differing evolutionary patterns between introns and exons. The effects of several well-known sources of bias on the phylogeny were small, compared to the effect of analyses based on different partitions of the gene (whole gene sequence, non-coding regions, cDNA sequence). Switches of function were found to be largely associated with strong selection, and with duplication events. Use of coding sequences for phylogeny estimation potentially produces incorrect inferences in about the action of selection on individual lineages and sites. Our results have major implications for phylogenomic methods of functional inference as well as for our understanding of the evolution of multigene families.针对多基因蛋白质家族（涵盖众多毒素蛋白）的研究，对于阐明基因复制在普遍意义上推动蛋白质多样性产生的作用至关重要。然而，当前多数针对基因家族的进化分析均以编码序列为基础，未考量诸多可能干扰进化推断的关键因素，例如重组以及选择驱动的趋同演化。我们以磷脂酶A2（Phospholipase A2，PLA2）亚家族的蛇毒基因序列为例，阐明这一问题。我们对20种尚未被充分研究的亚洲蝮蛇的全新基因序列进行分析，并结合另外4种蝮亚科蛇类以及数种蝰亚科蛇类的已公开基因组PLA2序列开展联合分析。与此前基于互补DNA（cDNA）序列开展的该毒素家族分析结果不同，我们发现基因复制事件集中发生于系统发育树的末端分支，这表明蝮蛇体内诸如突触前神经毒性这类核心功能曾多次独立趋同演化而来。我们通过实证表明，这一差异源于内含子与外显子之间截然不同的进化模式。相较于基于基因不同分区（全基因序列、非编码区域、cDNA序列）开展的分析所带来的影响，数种已知偏倚来源对系统发育树构建的影响相对微弱。我们发现功能的转变大多与强烈的选择压力以及基因复制事件密切相关。利用编码序列进行系统发育树推断时，可能会对单个谱系及位点上的选择作用得出错误的推论。本研究结果不仅对基于系统基因组学的功能推断方法具有重要指导意义，同时也加深了我们对多基因家族演化的认知。