Miniaturization, genome size, and biological size in a diverse clade of salamanders

Genome size (C-value) can affect organismal traits across levels of biological organization, from tissue complexity to metabolism. Neotropical salamanders show wide variation in genome and body sizes, including several clades with miniature species. Because miniaturization imposes strong constraints on morphology and development, and genome size is strongly correlated with cell size, we hypothesize that body size has played an important role in the evolution of genome size in bolitoglossine salamanders. If this hypothesis is correct, then genome size and body size should be correlated in this group. Using Feulgen Image Analysis Densitometry (FIAD), we estimated genome sizes for 60 species of neotropical salamanders. We also estimated the “biological size” of species by comparing genome size and physical body sizes in a phylogenetic context. We found a significant correlation between C-value and physical body size using optimal regression with an Ornstein-Uhlenbeck model, and
report the smallest salamander genome found to date. Our index of biological size showed that some salamanders with large physical body size have smaller biological body size than some miniature species, and that several clades showed patterns of increased or decreased biological size compared to their physical size. Our results suggest a causal relationship between physical body size and genome size and show the importance of considering the impact of both on the biological size of organisms. Indeed, biological size may be a more appropriate measure than physical size when considering phenotypic consequences of genome size evolution in many groups.
Methods
We generated a Sanger sequence dataset by sequencing some taxa of Neotropical salamanders for two mitochondrial genes (cytochrome b and 16S) and combined these data with data from Genbank for these two genes and three nuclear genes (RAG1, SLC8A3, POMC). We concatenated these data and estimated a phylogeny using BEAST. We estimated the maximum clade credibility tree from the posterior distribution of this analysis as well as a file of 100 trees from the posterior distribution of this analysis. We also include a file of measurements of body size and head size taken from museum specimens using Vernier calipers.

基因组大小（C-value）可影响生物组织各级水平的有机体性状，从组织复杂度直至新陈代谢等各个层面均受其调控。新热带区蝾螈的基因组与体型存在广泛变异，其中包含多个带有微型物种的演化支。由于体型微型化对形态与发育存在强烈约束，且基因组大小与细胞大小呈强相关，我们提出假说：体型在无肺螈亚科（bolitoglossine）蝾螈的基因组大小演化中发挥了重要作用。若该假说成立，那么该类群的基因组大小与体型应存在相关性。

本研究采用孚尔根图像分析光度法（Feulgen Image Analysis Densitometry, FIAD），对60种新热带区蝾螈的基因组大小进行了估算。同时，我们在系统发育框架下比较基因组大小与实体体型，以此估算物种的“生物体型”。通过奥恩斯坦-乌伦贝克模型（Ornstein-Uhlenbeck）的最优回归分析，我们发现C值与实体体型存在显著相关性，并报道了迄今已知的最小蝾螈基因组。我们的生物体型指数显示，部分实体体型较大的蝾螈，其生物体型反而小于某些微型物种；且多个演化支相较于其实体体型，呈现出生物体型升高或降低的模式。研究结果表明实体体型与基因组大小之间存在因果关系，同时证实了同时考量二者对有机体生物体型的影响的重要性。事实上，在诸多类群中，当探讨基因组大小演化的表型效应时，生物体型或许是比实体体型更合适的衡量指标。

研究方法
本研究通过对新热带区蝾螈的部分类群进行测序，获得了两个线粒体基因（细胞色素b及16S rRNA）的桑格测序（Sanger sequence）数据集，并将这些数据与GenBank中上述两个线粒体基因以及三个核基因（RAG1、SLC8A3、POMC）的序列数据进行合并。我们对合并后的序列数据进行拼接，并采用BEAST软件构建系统发育树。从本次分析的后验分布中，我们获取了最大类群置信度树，以及包含100棵树的后验分布文件。此外，本数据集还包含通过游标卡尺对馆藏标本测量得到的体型与头长数据。