Phylogeography and population genetic structure of the cardinal tetra (Paracheirodon axelrodi) in the Orinoco basin and Negro River (Amazon basin): evaluating connectivity and historical patterns of diversification

The Neotropics contain one of the most diverse assemblages of freshwater fishes worldwide. Part of this diversity is shared between the Orinoco and Amazon basins. These basins have been separated for a long time due to the Vaupes Arch, rising between 10 - 11 Ma. Today, there is only one permanent connection between the Orinoco and Negro (Amazon) basins, known as the Casiquiare Canal. However, alternative corridors allowing fish dispersion between both basins have been proposed. The cardinal tetra (Paracheirodon axelrodi), the most important fish in the ornamental world market, is distributed in both basins. Here we investigated P. axelrodi phylogeography, population structure, and potential routes of migration and connectivity between the two basins. A total of 468 bp of the mitochondrial gene (COI), 555 bp of the nuclear gene fragment (MYH6), and 8 microsatellite loci were analyzed. As a result, we found two major genetic clusters as the most likely scenario (K=2), but they were not discreetly distributed between basins. A gradient of genetic admixture was observed in Cucui and São Gabriel da Cachoeira, between the upper Negro River and the upper Orinoco. Samples from the middle-lower Negro River were highly structured. Cucui (Negro basin) was more similar to the Orinoco than to the rest of the Negro basin populations. However, substructure was also observed by the discriminant analysis, fixation indices and other hierarchichal structure analyses (K=3-6), showing three major geographic clusters: Orinoco, Cucui, and the remaining of the Negro basin. Unidirectional migration patterns were detected between basins: via Cucui toward Orinoco and via the remaining of the Negro basin toward Orinoco. Results from the Relaxed Random Walk analysis support a very recent origin of this species in the headwater Orinoco basin (Western Guiana Shield, at late Pleistocene) with a later rapid colonization of the remaining Orinoco basin and almost simultaneously the Negro River via Cucui, between 0.115 until about 0.001 Ma. Historical biogeography and population genetic patterns observed here for Cardinal tetra, seem to be better explained by river capture, physical, or ecological barriers than due to the geographic distance.

Methods
Muscle tissue samples (one per individual) were collected along the Orinoco and Amazon basins on the distribution area for P. axelrodi. Once the fish were received, they were euthanized by rapid chilling (hypothermic shock) on ice. Then, all individuals and tissue samples were preserved in ethanol at 95% and stored at 4°C. Genomic DNA was extracted.

新热带区（Neotropics）拥有全球多样性最为丰富的淡水鱼类类群之一。奥里诺科河与亚马孙河盆地共享了其中部分物种多样性。两大盆地因瓦普斯拱（Vaupes Arch）长期处于分隔状态，该构造拱的隆升时间为1000万至1100万年前。如今两者间仅存一处永久性连通通道，即卡西基亚雷运河（Casiquiare Canal）。不过，已有研究提出了其他可供鱼类在两盆地间扩散的潜在廊道。

红绿灯鱼（Paracheirodon axelrodi）作为观赏鱼市场中最重要的经济鱼类，在两大盆地均有分布。本研究针对该物种的系统地理学特征、种群结构以及两盆地间潜在的迁移与连通路径展开了调查。我们共分析了线粒体基因COI的468 bp序列、核基因片段MYH6的555 bp序列，以及8个微卫星位点。

分析结果显示，最符合数据的遗传聚类场景为两个主要类群（K=2），但此类群并未严格按照流域分布。在内格罗河上游与奥里诺科河上游之间的库库伊（Cucui）和圣加布里埃尔达卡绍埃拉（São Gabriel da Cachoeira）区域，观察到了明显的遗传混合梯度。内格罗河中下游的样本呈现出高度的种群遗传结构。库库伊种群（隶属于内格罗河盆地）相较于内格罗河盆地的其他种群，与奥里诺科河种群的遗传亲缘关系更近。不过，判别分析、固定指数及其他层级结构分析（K=3至6）也揭示了种群亚结构，显示出三个主要的地理聚类单元：奥里诺科河种群、库库伊种群，以及内格罗河盆地的其余种群。

研究检测到了两盆地间的单向迁移模式：一是经由库库伊区域向奥里诺科河迁移，二是经由内格罗河盆地其余区域向奥里诺科河迁移。宽松随机游走（Relaxed Random Walk）分析结果表明，该物种起源于更新世晚期的奥里诺科河上游源头盆地（圭亚那地盾西部），随后快速扩散至奥里诺科河盆地其余区域，并几乎同时经由库库伊区域拓殖至内格罗河，这一拓殖过程发生于0.115至约0.001百万年之间。本研究观察到的红绿灯鱼历史生物地理学与种群遗传模式，相较于地理距离因素，更能用河流袭夺、物理或生态屏障来解释。

方法
研究人员在红绿灯鱼的自然分布范围内，沿奥里诺科河与亚马孙河盆地采集肌肉组织样本，每个个体采集1份样本。样本送达实验室后，通过冰上快速降温（低温休克）的方式对实验鱼实施安乐死。随后将所有个体及组织样本置于95%乙醇中保存，并于4℃条件下储存。后续完成基因组DNA的提取。