A comparative approach for species delimitation based on multiple methods of multi-locus DNA sequence analysis: A case study of the genus Giraffa (Mammalia, Cetartiodactyla)

Molecular data are now commonly used in taxonomy for delimiting cryptic species. In the case of giraffes, which were treated as a single species (Giraffa camelopardalis) during half of a century, several molecular studies have suggested a splitting into four to seven species, but the criteria applied for taxonomic delimitation were not fully described. In this study, we have analysed all multi-locus DNA sequences available for giraffes using multispecies coalescent (MSC: *BEAST, BPP and STACEY), population genetic (STRUCTURE, allelic networks, haplotype network and bootstrapping, haplowebs and conspecificity matrix) and phylogenetic (MrBayes, PhyML, SuperTRI) methods to identify the number of species. Our results show that depending on the method chosen, different taxonomic hypotheses, recognizing from two to six species, can be considered for the genus Giraffa. Our results confirm that MSC methods can lead to taxonomic over-splitting, as they delimit geographic structure rather than species. The 3-species hypothesis, which recognizes G. camelopardalis sensu strico A, G. giraffa, and G. tippelskirchi, is highly supported by phylogenetic analyses and also corroborated by most population genetic and MSC analyses. The three species show high levels of nucleotide divergence in both nuclear (0.35–0.51%) and mitochondrial sequences (3–4%), and they are characterised by 7 to 12 exclusive synapomorphies (ES) detected in nine of the 21 nuclear introns analysed for this study. By contrast, other putative species, such as G. peralta, G. reticulata, G. thornicrofti or G. tippelskirchi sensu stricto, do not exhibit any ES in the nuclear genes. A robust mito-nuclear conflict was found for the position and monophyly of G. giraffa and G. tippelskirchi, which is interpreted as the result of a mitochondrial introgression from Masai to southeastern giraffe during the Pleistocene and nuclear gene flow mediated by male dispersal between southern populations (subspecies G. g. giraffa and G. g. angolensis).如今分子数据已广泛应用于分类学领域，用于界定隐存种（cryptic species）。以曾被视为单一物种（长颈鹿Giraffa camelopardalis）长达半个世纪的长颈鹿属为例，既往多项分子研究曾提出将其划分为4至7个物种，但相关分类界定（taxonomic delimitation）标准并未得到完整阐述。本研究利用多物种溯祖（multispecies coalescent, MSC：*BEAST、BPP与STACEY）、群体遗传学（STRUCTURE、等位基因网络、单倍型网络与自举检验、单倍型网与物种一致性矩阵）以及系统发育（MrBayes、PhyML、SuperTRI）方法，对所有已发表的长颈鹿属多位点DNA序列（multi-locus DNA sequences）展开分析，以确定该属的物种数量。研究结果显示，根据所选分析方法的差异，针对长颈鹿属可提出支持2至6个物种的不同分类学假说。本研究证实，多物种溯祖方法可能导致分类学上的过度划分，因为此类方法界定的是地理结构而非物种界限。其中，将狭义G. camelopardalis A、G. giraffa与G. tippelskirchi视为独立物种的三物种假说，得到了系统发育分析的强力支持，同时也得到多数群体遗传学与多物种溯祖分析的佐证。这三个物种在核基因（0.35%~0.51%）与线粒体序列（3%~4%）中均表现出较高的核苷酸分歧度，且在本研究分析的21个核内含子中的9个里，检测到7至12个专属共有衍征（exclusive synapomorphies）。与之形成鲜明对比的是，其他假定物种，如G. peralta、G. reticulata、G. thornicrofti或狭义G. tippelskirchi，在核基因中未表现出任何专属共有衍征。本研究还发现，G. giraffa与G. tippelskirchi的系统发育位置及单系性存在显著的核质冲突（mito-nuclear conflict），这一现象被解读为更新世（Pleistocene）期间马赛长颈鹿向东南非长颈鹿发生线粒体渐渗（mitochondrial introgression），以及南部种群（亚种G. g. giraffa与G. g. angolensis）间由雄性扩散介导的核基因交流所致。