Phylogeny of the supertribe Nebriitae (Coleoptera: Carabidae) based on analyses of DNA sequence data

The phylogeny of the carabid beetle supertribe Nebriitae is inferred from analyses of DNA sequence data from eight gene fragments including one nuclear ribosomal gene (28S), four nuclear-protein coding genes (CAD, topoisomerase 1, PEPCK and wingless) and three mitochondrial gene fragments (16S + tRNA-Leu + ND1, COI (“barcode” region) and COI (“Pat/Jer” region)). Our taxon sample included 264 exemplars representing 241 species and subspecies (25% of the known nebriite fauna), 39 of 41 currently accepted genera and subgenera (all except Notiokasis and Archileistobrius), and eight outgroup taxa. Separate maximum likelihood (ML) analyses of individual genes, combined ML analyses of nuclear, nuclear protein-coding and mitochondrial genes, and combined ML and Bayesian analyses of the eight-gene-fragment matrix resulted in a well-resolved phylogeny of the supertribe, with most nodes in the tree strongly supported. Within Nebriitae, 167 internal nodes of the tree (out of the maximum possible 255) are supported by maximum-likelihood bootstrap values of 90% or more. Tribes Notiophilini, Opisthiini, Pelophilini, Nebriini are well supported as monophyletic but relationships among these are not well resolved. Nippononebria is a distinct genus more closely related to Leistus than Nebria. Archastes, Oreonebria, Spelaeonebria, and Eurynebria, previously treated as distinct genera by some authors, are all nested within a monophyletic genus Nebria. Within Nebria, four major clades are recognized: (1) the Oreonebria Series, including eight subgenera arrayed in two subgeneric complexes (the Eonebria and Oreonebria Complexes); (2) the Nebriola Series, including only subgenus Nebriola; (3) the Nebria Series, including ten subgenera arrayed in two subgeneric complexes, the Boreonebria and Nebria Complexes, with the latter further subdivided into three subgeneric subcomplexes (the Nebria, Epinebriola and Eunebria Subcomplexes)); and (4) the Catonebria Series, including seven subgenera arrayed in two subgeneric complexes (the Reductonebria and Catonebria Complexes). A strong concordance of biogeography with the inferred phylogeny is noted and some evident vicariance patterns are highlighted. A revised classification, mainly within the Nebriini, is proposed to reflect the inferred phylogeny. Three genus-group taxa (Nippononebria, Vancouveria and Archastes) are given revised status and seven are recognized as new synonymies (Nebriorites Jeannel, 1941 and Marggia Huber, 2014 = Oreonebria Daniel, 1903; Pseudonebriola Ledoux and Roux, 1989 = Boreonebria Jeannel, 1937; Patrobonebria Bänninger, 1923, Paranebria Jeannel, 1937 and Barbonebriola Huber and Schmidt, 2017 = Epinebriola Daniel and Daniel, 1904; and Asionebria Shilenkov, 1982 = Psilonebria Andrewes, 1923). Six new subgenera are proposed and described for newly recognized clades: Parepinebriola Kavanaugh subgen. nov. (type species: Nebria delicata Huber and Schmidt, 2017), Insulanebria Kavanaugh subgen. nov. (type species: Nebria carbonaria Eschscholtz, 1829), Erwinebria Kavanaugh subgen. nov. (type species Nebria sahlbergii Fischer von Waldheim, 1828), Nivalonebria Kavanaugh subgen. nov. (type species: Nebria paradisi Darlington, 1931), Neaptenonebria Kavanaugh subgen. nov. (type species: Nebria ovipennis LeConte, 1878) and Palaptenonebria Kavanaugh subgen. nov. (type species: Nebria mellyi Gebler, 1847). Future efforts to better understand relationships within the supertribe should aim to expand the taxon sampling of DNA sequence data, particulary within subgenera Leistus and Evanoleistus of genus Leistus and the Nebria Complex of genus Nebria.

Methods
DNA was sequenced using both PCR/Sanger sequencing and Illumina sequencing.  Phylogenetic trees included in the submission were inferred using maximum likelihood in RAxML.

本研究基于8个基因片段的DNA序列数据，对步甲科Nebriitae总族的系统发育关系进行了推断。所选用的基因片段包括1个核核糖体基因（28S）、4个核蛋白编码基因（CAD、拓扑异构酶1、PEPCK及无翅基因），以及3个线粒体基因片段（16S+亮氨酸转运RNA+ND1、COI（"条形码区域"）及COI（"Pat/Jer区域"））。本研究的类群采样涵盖264个代表类群，对应241个物种及亚种（占已知Nebriitae类群的25%），涉及当前公认的41个属及亚属中的39个（除Notiokasis与Archileistobrius外），同时纳入8个外类群。
针对单个基因的单独最大似然（ML）分析、针对核基因、核蛋白编码基因及线粒体基因的联合最大似然分析，以及针对8个基因片段矩阵的联合最大似然与贝叶斯分析，均得到了分辨率良好的Nebriitae总族系统发育树，且树中绝大多数节点均得到了强力支持。在该系统发育树中，255个最大可能内部节点里有167个的最大似然自举值≥90%。Notiophilini族、Opisthiini族、Pelophilini族及Nebriini族均被支持为单系群，但这些族之间的系统发育关系分辨率仍欠佳。Nippononebria为独立属，其与Leistus属的亲缘关系较与Nebria属更近。此前部分学者将Archastes、Oreonebria、Spelaeonebria及Eurynebria视为独立属，本研究结果显示它们均嵌套于单系属Nebria之内。
在Nebria属内，共识别出4个主要演化支：（1）Oreonebria系，包含8个亚属，划分为两个亚属复合体（Eonebria复合体与Oreonebria复合体）；（2）Nebriola系，仅包含Nebriola亚属；（3）Nebria系，包含10个亚属，划分为两个亚属复合体（Boreonebria复合体与Nebria复合体），其中Nebria复合体进一步细分为3个亚属亚复合体（Nebria亚复合体、Epinebriola亚复合体及Eunebria亚复合体）；（4）Catonebria系，包含7个亚属，划分为两个亚属复合体（Reductonebria复合体与Catonebria复合体）。
本研究发现生物地理学特征与推断得到的系统发育具有高度一致性，并阐释了若干显著的异域分布分化格局。为反映本研究的系统发育结果，我们提出了修订的分类系统，主要针对Nebriini族。3个属级类群（Nippononebria、Vancouveria及Archastes）得到了分类地位修订；另有7个类群被确立为新异名：Nebriorites Jeannel, 1941与Marggia Huber, 2014均为Oreonebria Daniel, 1903的新异名；Pseudonebriola Ledoux and Roux, 1989为Boreonebria Jeannel, 1937的新异名；Patrobonebria Bänninger, 1923、Paranebria Jeannel, 1937及Barbonebriola Huber and Schmidt, 2017均为Epinebriola Daniel and Daniel, 1904的新异名；Asionebria Shilenkov, 1982为Psilonebria Andrewes, 1923的新异名。此外，我们为新识别的演化支提出并描述了6个新亚属：Parepinebriola Kavanaugh，新亚属（模式种：Nebria delicata Huber and Schmidt, 2017）；Insulanebria Kavanaugh，新亚属（模式种：Nebria carbonaria Eschscholtz, 1829）；Erwinebria Kavanaugh，新亚属（模式种：Nebria sahlbergii Fischer von Waldheim, 1828）；Nivalonebria Kavanaugh，新亚属（模式种：Nebria paradisi Darlington, 1931）；Neaptenonebria Kavanaugh，新亚属（模式种：Nebria ovipennis LeConte, 1878）；以及Palaptenonebria Kavanaugh，新亚属（模式种：Nebria mellyi Gebler, 1847）。未来为更好解析该总族内的系统发育关系，应扩大DNA序列数据的类群采样范围，尤其是在Leistus属的Leistus亚属与Evanoleistus亚属，以及Nebria属的Nebria复合体中。

材料与方法
本研究采用PCR/Sanger测序与Illumina测序两种技术获取DNA序列。本次提交的系统发育树均通过RAxML软件的最大似然法构建得到。