IRTA-Institute for Food and Agricultural Research and Technology, Marine and Continental Waters Programme, Ctra de Poble Nou Km 5.5, E43540 Sant Carles de la Rápita, Catalonia, Spain; Departament de Geografia, Universitat Rovira i Virgili, C/Joanot Martorell 15, E43500 Vila-seca, Catalonia, Spain.
IRTA-Institute for Food and Agricultural Research and Technology, Marine and Continental Waters Programme, Ctra de Poble Nou Km 5.5, E43540 Sant Carles de la Rápita, Catalonia, Spain.
Sci Total Environ. 2020 Jul 20;727:138445. doi: 10.1016/j.scitotenv.2020.138445. Epub 2020 Apr 4.
Our study of 164 diatom samples from Catalonia (NE Spain) is the first to evaluate the applicability of DNA metabarcoding, based on high throughput sequencing (HTS) using a 312-bp rbcL marker, for biomonitoring Mediterranean rivers. For this, we compared the values of a biotic index (IPS) and the ecological status classes derived from them, between light microscope-based (LM) and HTS methods. Very good correspondence between methods gives encouraging results concerning the applicability of DNA metabarcoding for Catalan rivers for the EU Water Framework Directive (WFD). However, in 10 sites, the ecological status class was downgraded from "Good"/"High" obtained by LM to "Moderate"/"Poor"/"Bad" by HTS; these "critical" sites are especially important, because the WFD requires remedial action by water managers for any river with Moderate or lower status. We investigated the contribution of each species to the IPS using a "leave-one-out" sensitivity analysis, paying special attention to critical sites. Discrepancies in IPS between LM and HTS were mainly due to the misidentification and overlooking in LM of a few species, which were better recovered by HTS. This bias was particularly important in the case of Fistulifera saprophila, whose clear underrepresentation in LM was important for explaining 8 out of the 10 critical sites and probably reflected destruction of weakly-silicified frustules during sample preparation. Differences between species in the rbcL copy number per cell affected the relative abundance obtained by HTS for Achnanthidium minutissimum, Nitzschia inconspicua and Ulnaria ulna, which were also identified by the sensitivity analysis as important for the WFD. Only minor IPS discrepancies were attributed to the incompleteness of the reference library, as most of the abundant and influential species (to the IPS) were well represented there. Finally, we propose that leave-one-out analysis is a good method for identifying priority species for isolation and barcoding.
我们对来自西班牙东北部加泰罗尼亚的 164 个硅藻样本的研究是首次评估基于高通量测序（HTS）的 DNA 条形码技术在监测地中海河流中的适用性，该技术使用了 312-bp 的 rbcL 标记。为此，我们比较了基于光学显微镜（LM）和 HTS 方法的生物指数（IPS）及其衍生的生态状况类别之间的数值。方法之间非常好的一致性对于 DNA 条形码技术在加泰罗尼亚河流中应用于欧盟水框架指令（WFD）来说是令人鼓舞的结果。然而，在 10 个地点，生态状况类别从 LM 获得的“良好”/“高”等级降级为 HTS 的“中等”/“差”/“差”等级；这些“关键”地点尤为重要，因为 WFD 要求水管理部门对任何处于中等或更低状态的河流采取补救措施。我们使用“逐一剔除”敏感性分析调查了每个物种对 IPS 的贡献，特别关注关键地点。LM 和 HTS 之间 IPS 的差异主要归因于 LM 中少数物种的错误识别和忽略，而 HTS 更好地恢复了这些物种。在 Fistulifera saprophila 的情况下，这种偏差尤其重要，因为 LM 中该物种的明显代表性不足对解释 10 个关键地点中的 8 个非常重要，可能反映了样品制备过程中对弱硅化壳的破坏。每个细胞中 rbcL 拷贝数的差异会影响 HTS 获得的 Achnanthidium minutissimum、Nitzschia inconspicua 和 Ulnaria ulna 的相对丰度，这些物种也通过敏感性分析被确定为对 WFD 很重要。只有少数 IPS 差异归因于参考文库的不完整性，因为大多数丰富且有影响力的物种（对 IPS 而言）在参考文库中都有很好的代表。最后，我们提出逐一剔除分析是一种识别优先分离和条形码物种的好方法。
