Validating the use of coloration patterns for individual recognition in the worm pipefish using a novel set of microsatellite markers

In studies of behaviour, ecology and evolution, identification of individual organisms can be an invaluable tool, capable of unravelling otherwise cryptic information regarding group structure, movement patterns, population size and mating strategies. The use of natural markings is arguably the least invasive method for identification. However, to be truly useful natural markings must be sufficiently variable to allow for unique identification, while being stable enough to permit long-term studies. Non-invasive marking techniques are especially important in fishes of the Family Syngnathidae (pipefishes, seahorses and seadragons), as many of these taxa are of conservation concern or used extensively in studies of sexual selection. Here, we assessed the reliability of natural markings as a character for individual identification in a wild population of Nerophis lumbriciformis by comparing results from natural markings to individual genetic assignments based on eight novel microsatellite loci. We also established a minimally invasive method based on epithelial cell swabbing to sample DNA. All pipefish used in the validation of natural markings, independently of sex or time between recaptures, were individually recognized through facial colouration patterns. Their identities were verified by the observation of the same multilocus genotype at every sampling event for each individual that was identified on the basis of natural markings. Successful recaptures of previously swabbed pipefish indicated that this process probably did not induce an elevated rate of mortality. Also, the recapture of newly pregnant males showed that swabbing did not affect reproductive behaviour.在行为学、生态学与进化生物学研究中，个体生物识别是极具价值的研究工具，能够揭示原本难以探明的关于种群群体结构、移动模式、种群规模以及交配策略的隐秘信息。利用自然斑纹进行个体识别，可说是侵入性最低的识别手段。然而，若要真正具备实用价值，自然斑纹需兼具足够的变异性以实现唯一个体识别，同时具备足够的稳定性以支持长期研究。非侵入式标记技术对于海龙科（Syngnathidae）鱼类（包括海龙、海马与叶海龙）尤为重要，因为该科多数类群均为保护关注物种，或被广泛应用于性选择相关研究。本研究以野生蚯蚓状海龙（Nerophis lumbriciformis）种群为研究对象，通过比对自然斑纹识别结果与基于8个新型微卫星位点的个体遗传归属结果，评估了自然斑纹作为个体识别性状的可靠性。此外，本研究还建立了一种基于上皮细胞拭子采样的微创DNA采样方法。所有用于自然斑纹有效性验证的海龙样本，无论性别或重捕间隔时长，均可通过其面部色斑模式实现个体识别。对于通过自然斑纹识别的每一个体，其在各次采样中均检测到一致的多位点基因型，这一结果验证了其个体身份。对已完成拭子采样的海龙的成功重捕结果表明，该采样过程大概率不会提高个体死亡率。此外，对新晋受孕雄性个体的重捕结果显示，拭子采样不会影响其繁殖行为。