Datasheet1_Evolution and functional role prediction of the CYP6DE and CYP6DJ subfamilies in Dendroctonus (Curculionidae: Scolytinae) bark beetles.DOCX

Dendroctonus-bark beetles are natural components and key ecological agents of coniferous forests. They spend most of their lives under the bark, where they are exposed to highly toxic terpenes present in the oleoresin. Cytochrome P450 (CYP) is a multigene family involved in the detoxification of these compounds. It has been demonstrated that CYP6DE and CYP6DJ subfamilies hydroxylate monoterpenes, whose derivatives can act as pheromone synergist compounds or be pheromones themselves in these insects. Given the diversity and functional role of CYPs, we investigated whether these cytochromes have retained their function throughout the evolution of these insects. To test this hypothesis, we performed a Bayesian phylogenetic analysis to determine phylogenetic subgroups of cytochromes in these subfamilies. Subgroups were mapped and reconciled with the Dendroctonus phylogeny. Molecular docking analyses were performed with the cytochromes of each subgroup and enantiomers of α-pinene and β-pinene, (+)-3-carene, β-myrcene and R-(+)-limonene. In addition, functional divergence analysis was performed to identify critical amino acid sites that influence changes in catalytic site conformation and/or protein folding. Three and two phylogenetic subgroups were recovered for the CYP6DE and CYP6DJ subfamilies, respectively. Mapping and reconciliation analysis showed different gain and loss patterns for cytochromes of each subgroup. Functional predictions indicated that the cytochromes analyzed are able to hydroxylate all monoterpenes; however, they showed preferential affinities to different monoterpenes. Functional divergence analyses indicated that the CYP6DE subfamily has experimented type I and II divergence, whereas the CYP6DJ subfamily has evolved under strong functional constraints. Results suggest cytochromes of the CYP6DE subfamily evolve to reinforce their detoxifying capacity hydroxylating mainly α- and β-pinene to (+) and (−)-trans-verbenol, being the negative enantiomer used as a pheromone by several Dendroctonus species; whereas cytochromes of the CYP6DJ subfamily appear to retain their original function related to the detoxification of these compounds.

齿小蠹属树皮甲虫（Dendroctonus-bark beetles）是针叶林的天然组成部分与关键生态作用类群。它们一生中的大部分时间都栖息于树皮之下，在此处会接触到植物油树脂（oleoresin）中含有的高毒性萜类物质（terpenes）。细胞色素P450（Cytochrome P450, CYP）是一类参与此类化合物解毒代谢的多基因家族。已有研究证实，CYP6DE与CYP6DJ亚家族可对单萜类物质（monoterpenes）进行羟基化修饰，其衍生物可作为信息素增效剂，或是直接作为此类昆虫的信息素发挥功能。
鉴于细胞色素P450家族的多样性及其功能重要性，本研究旨在探究此类细胞色素在齿小蠹属昆虫的演化历程中是否保留了其原始功能。为验证该假说，我们采用贝叶斯系统发育分析（Bayesian phylogenetic analysis）方法，明确了这两个亚家族中细胞色素的系统发育分支群。随后对各分支群进行图谱绘制，并与齿小蠹属的系统发育树开展整合分析。
针对各分支群的细胞色素，以及α-蒎烯、β-蒎烯、(+)-3-蒈烯、β-月桂烯与R-(+)-柠檬烯的对映异构体（enantiomers），本研究开展了分子对接分析（molecular docking analyses）。此外，还进行了功能分化分析，以鉴定影响催化位点构象及/或蛋白质折叠的关键氨基酸位点（amino acid sites）。
本研究共从CYP6DE亚家族中鉴定出3个系统发育分支群，从CYP6DJ亚家族中鉴定出2个。图谱绘制与整合分析结果显示，各分支群的细胞色素呈现出不同的基因获得与丢失模式。功能预测结果表明，本次分析的细胞色素均可对各类单萜类物质进行羟基化修饰，但对不同单萜类物质表现出差异化的结合偏好性。
功能分化分析结果显示，CYP6DE亚家族经历了I型与II型功能分化，而CYP6DJ亚家族则在较强的功能约束下演化。研究结果表明，CYP6DE亚家族的细胞色素通过将α-蒎烯与β-蒎烯主要羟基化为(+)与(-)-反式马鞭草烯醇（trans-verbenol），从而强化其解毒能力；其中(-)-反式马鞭草烯醇可作为多种齿小蠹属昆虫的信息素。而CYP6DJ亚家族的细胞色素似乎保留了其原始的此类化合物解毒功能。