Transcriptome sequencing and phylogenomic resolution within Spalacidae (Rodentia)

Background: Subterranean mammals have been of great interest for evolutionary biologists because of their highly specialized traits for the life underground, however, the scarcity of genomic information limits our better understanding of their evolutionary history. Owing to the convergence of morphological traits and the incongruence of molecular data, the phylogenetic relationships among three subfamilies Myospalacinae (zokors), Spalacinae (blind mole rats) and Rhizomyinae (bamboo rats) within the family Spalacidae remain unresolved. Here, we performed de novo transcriptome sequencing of four RNA-seq libraries prepared from brain and liver tissues of a plateau zokor (Eospalax baileyi) and a hoary bamboo rat (Rhizomys pruinosus), and analyzed the transcriptome sequences alongside a published transcriptome of the Middle East blind mole rat (Spalax galili). We characterize the transcriptome assemblies of the two spalacids, and recover the phylogeny of the three subfamilies using a phylogenomic approach. Results: Approximately 50.3 million clean reads from the zokor and 140.8 million clean reads from the bamboo rat were generated by Illumina paired-end RNA-seq technology. All clean reads were assembled into 138,872 (the zokor) and 157,167 (the bamboo rat) unigenes, which were annotated by the public databases: the Swiss-prot, Trembl, NCBI non-redundant protein (NR), NCBI nucleotide sequence (NT), Gene Ontology (GO), Cluster of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 5,116 nuclear orthologous genes were identified in the three spalacids and mouse, which was used as an outgroup. Phylogenetic analysis revealed a sister group relationship between the zokor and the bamboo rat, which is supported by the majority of gene trees inferred from individual orthologous genes, suggesting subfamily Myospalacinae is more closely related to subfamily Rhizomyinae. The same topology was recovered from combined sequences of concatenated sequences of 5,116 nuclear genes (5,541,534 bp in length), fourfold degenerate sites of the 5,116 nuclear genes (715,762 bp) and concatenated sequences of 13 protein coding mitochondrial genes (11,307 bp). In this topology, notably, the branch leading to subfamilies Myospalacinae and Rhizomyinae is remarkably short, indicative of a burst of speciation within the family Spalacidae. Conclusions: This is the first report of transcriptome data in zokors and bamboo rats, representing a valuable resource for future studies of comparative genomics in subterranean mammals. Phylogenomic analysis provides a conclusive resolution of interrelationships of the three subfamilies within the family Spalacidae, and highlights the power of phylogenomic approach to dissect the evolutionary history of rapid radiations in the tree of life.

背景：地下栖息哺乳动物因其适应地下生活的高度特化性状，一直受到进化生物学家的广泛关注。然而，基因组信息的匮乏限制了我们对其进化历史的深入认知。由于形态性状趋同与分子数据不一致，鼹形鼠科（Spalacidae）内的三个亚科——鼢鼠亚科（Myospalacinae，鼢鼠）、盲鼹形鼠亚科（Spalacinae，盲鼹形鼠）以及竹鼠亚科（Rhizomyinae，竹鼠）之间的系统发育关系仍未明确。本研究对高原鼢鼠（Eospalax baileyi）和花白竹鼠（Rhizomys pruinosus）的脑与肝脏组织构建的4个RNA测序（RNA-seq）文库进行从头转录组测序，并结合已发表的中东盲鼹形鼠（Spalax galili）转录组数据进行分析。我们对两种鼹形鼠科动物的转录组组装结果进行了系统表征，并通过系统基因组学方法解析了三个亚科的系统发育关系。结果：通过Illumina双端RNA测序技术，我们分别获得了约5030万条高原鼢鼠清洁读段与1.408亿条花白竹鼠清洁读段。所有清洁读段分别组装为138872条（高原鼢鼠）与157167条（花白竹鼠）单基因（unigene），并通过多个公共数据库进行注释：瑞士Prot（Swiss-prot）、TrEMBL、NCBI非冗余蛋白数据库（NCBI non-redundant protein, NR）、NCBI核苷酸序列数据库（NCBI nucleotide sequence, NT）、基因本体论（Gene Ontology, GO）、直系同源簇数据库（Cluster of Orthologous Groups, COG）以及京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）。我们在三种鼹形鼠科动物以及作为外类群的小家鼠中，共鉴定出5116个核直系同源基因。系统发育分析显示，鼢鼠亚科与竹鼠亚科构成姊妹群，这一结果得到了绝大多数单个直系同源基因构建的基因树的支持，表明鼢鼠亚科与竹鼠亚科的亲缘关系更近。通过拼接5116个核基因的序列（总长5541534 bp）、5116个核基因的四重简并位点序列（总长715762 bp）以及13个线粒体蛋白编码基因的拼接序列（总长11307 bp）进行分析，均得到了一致的拓扑结构。值得注意的是，在该拓扑结构中，指向鼢鼠亚科与竹鼠亚科的分支长度极短，表明鼹形鼠科内曾发生过快速的物种形成事件。结论：本研究首次报道了鼢鼠与竹鼠的转录组数据，可为未来地下栖息哺乳动物的比较基因组学研究提供宝贵的资源。系统基因组学分析明确解析了鼹形鼠科内三个亚科的相互关系，并凸显了系统基因组学方法在解析生命之树中快速辐射类群进化历史方面的优势。