Reference population and gametophyte culture genotypes

Many challenges arise when monitoring organisms with cryptic life
histories. For example, cryptic stages are hard to identify or sample due
to their microscopic nature, which creates unknowns surrounding an
organism’s population dynamics. Environmental DNA (eDNA) is a non-invasive
sampling technique used to monitor cryptic species when traditional survey
methods are challenging. Generally, eDNA has been used to quantify the
presence/absence of species in various habitats. However, recent advances
in next-generation sequencing have enabled researchers to detect
intraspecific species abundances with eDNA. In this study, we present two
complementary R packages that can be used to estimate the number of
individuals in an eDNA sample. The first package (Amplicomsat) cleans
high-throughput amplicon microsatellite sequences and counts the observed
alleles identified in eDNA. Our second package (GenotypeQuant) then uses a
numerical maximum likelihood estimator (NMLE) to estimate the number of
contributors most likely to have produced the sequenced panel of
microsatellite alleles amplified from eDNA. We first present simulations
to characterize the accuracy and precision of the method. We then
estimated densities of Nereocystis luetkeana (bull kelp)
microscopic gametophytes from eDNA collected from an experiment with a
manipulated number of gametophytes. Finally, we analyzed benthic eDNA from
kelp forest habitats. We found that gametophyte estimates produced by the
NMLE varied within +3/-2 individuals when processing eDNA from rocks with
8 seeded gametophytes. eDNA harvested from the field showed that
gametophyte estimates scaled with the sampling area, and total densities
in July ranged between 500 to 800 m2.

对具有隐蔽生活史的生物开展监测时，会面临诸多挑战。例如，隐蔽阶段因具有显微尺度特性而难以识别或采样，这给生物种群动态的相关研究带来了诸多未知。环境DNA（Environmental DNA, eDNA）是一种非侵入性采样技术，在传统调查方法难以实施时，可用于监测隐蔽物种。通常而言，eDNA被用于量化不同生境中物种的存在与否。然而，下一代测序技术的近期突破使得研究人员可借助eDNA检测物种的种内种群丰度。
本研究介绍了两款互补的R语言程序包，可用于估算eDNA样本中的个体数量。第一款程序包（Amplicomsat）可对高通量扩增子微卫星序列进行质控清洗，并统计eDNA中鉴定出的观测等位基因。第二款程序包（GenotypeQuant）则采用数值极大似然估计器（Numerical Maximum Likelihood Estimator, NMLE），估算最有可能贡献了从eDNA中扩增得到的微卫星等位基因测序组合的个体数量。
我们首先通过模拟实验对该方法的准确性与精确性进行了表征。随后，我们利用一组设置了不同配子体数量梯度的实验采集的eDNA，估算了Nereocystis luetkeana（公牛海带藻，bull kelp）的显微配子体密度。最后，我们对海带森林生境的底栖eDNA进行了分析。
我们发现，在处理接种了8个配子体的岩石样本的eDNA时，NMLE产出的配子体数量估算值与真实值的偏差范围为-2至+3个个体。野外采集的eDNA分析结果显示，配子体数量估算值随采样面积发生变化，7月的总密度范围为每平方米500至800个个体。