Unveiling invasive insect threats to plant biodiversity: Leveraging eDNA metabarcoding and saturated salt trap solutions for biosurveillance

five IAS together were only obtained at the sites collected in 2019 and 2020. eDNA from four of these five IAS (all but Trichoferus campestris eDNA) was detected in the 2022 sites. All IAS detected in 2022, except Lymantria dispar, were successfully identified morphologically from the decanted specimens collected in that collection year. At the sample level, there were 25 eDNA- based individual detections in 2022 (Table 2), with 13 of them lacking morphological confirma- tion. Ten out of those thirteen detections missing morphology-based conformation came from the same collection site (Windsor). The only IAS that was genetically and morphologically con- firmed to be present at Windsor was Agrilus planipennis (see sample WR_21_7_22_G3; Table 2). The remaining three mismatches occurred for Popillia japonica and Daktulosphaira vitifoliae and were only seen in two traps belonging to the same site (PB_10_8_22_3 and PB_10_8_22_4) where the species were also morphologically confirmed. Lymantria dispar and Daktulosphaira vitifoliae eDNA detections at Windsor in 2022 represented the only cases where the species could not be morphologically confirmed at any collected trap in the given site. For the 2018 collected sites, eDNA from Agrilus planipennis, Lymantria dispar, Daktulo- sphaira vitifoliae was also found, while in the samples collected from sites in 2017, only eDNA from Daktulosphaira vitifoliae was detected (Fig 4). Morphological validation In the current study, the 2022 collection was the only one where trap contents, including speci-

mens and debris, were sent to the Hanner Laboratory for morphological validation. In 2022, four IAS species were detected, with a total of 25 individual detections over three sites, 14 traps, and four collecting dates (Table 2). Of those individual detections, 11 were confirmed by both molecular and morphological methods, one was morphological only, and the remainder (13) were molecular detections only. If assessed by site and date, there is an 89% congruence between molecular and morphological data, since IAS species not detected morphologically in one trap at a site were often detected in another trap at the same site. The Windsor site collec- tions from July 21, 2022, show consistently low congruence between molecular and morpho- logical identification, with only one of 11 morphologically validated detections, which suggests a sample-specific bias. Full filter analysis vs quarters The whole filter versus quarter analysis for the 2020 collection showed that all five IAS eDNA was detected in only one quarter (Quarter I; Fig 5). These results were consistent when com- paring IAS species recovered from half and whole filters (Fig 5). Agrilus planipennis eDNA was missing in three out of the four quarters belonging to the same filters (Fig 5). Additionally, species composition analysis demonstrated that quarters or halves alone did not necessarily reflect the species composition of the whole filter, with complete filter analysis consistently showing higher average species richness for all 2020 sampled sites (Fig 6). Canopy level sampling vs ground level sampling

Only eDNA from one IAS (Lymantria dispar) was detected at the canopy level, contrary to the five IAS that were seen at the ground sampling level during the whole filter analysis of the 2020 Fig 2. Rarefaction curves per collection year (2017 and 2018) constructed with three diversity indices. D0 is species richness, D1 approaches the exponential of the Shannon index, and D2 is inverted of the Simpson index [2, 29]. Coverage refers to the Good’s coverage index [2]. Rarefaction curves per collection year (2019 and 2020) constructed with three diversity indices. 50 is species richness, D1 approaches the exponential of the Shannon index, and D2 is inverted of the Simpson index [2, 29]. Coverage refers to the Good’s coverage index [2]. Sample types (canopy and ground) are distinguished in the 2020 collection. https://doi.org/10.1371/journal.pone.0290036.g002 PLOS ONE Unveiling invasive insect threats to plant biodiversity PLOS ONE | https://doi.org/10.1371/journal.pone.0290036 August 11, 2023 11 / 24