Floral hosts of leaf-cutter bees (Megachilidae) in a biodiversity hotspot revealed by pollen DNA metabarcoding of historic specimens

Results Summary statistics of the merged reads produced in this study are provided first (Table 2). Quality and adapter trimming of samples sequenced on the HiSeq 2500 resulted in a total number of 2,947,334 high-quality, merged reads obtained across all bee species. This resulted in a mean of 28,412 merged reads per sample (median = 13,816 and SD = 43,161). Twenty- four of the 105 samples (22.6%) produced less than 1,000 reads per sample and were discarded prior to further analyses. Pollen from 21 M. venusta specimens [26] produced a total of 1,124,324 reads across all samples, with a mean of 53,539 reads per pollen sample (SD = 57,314). When combined with samples sequenced on the HiSeq 2500, a total of 4,071,658 reads were produced across all six Megachile species, after 24 failed samples were removed. This results in a mean of 40,313 (median = 24,193; SD = 48,742) reads per pollen sample. The mean number of merged reads per Megachile species is provided in Table 2. ITS2 sequence classification resulted in 71.9% of total reads being confidently identified to family level across all samples. Samples with less than 1,000 reads confidently classified to fam- ily level, were discarded. Samples that passed the family-level read cut-off were all subject to species level taxonomic classification in utax. An additional read cut-off was not introduced at species level classification, as only reads classifying to angiosperm species were recorded. Nine samples did not pass the classifying criteria, leading to a total of thirty-three pollen samples

(26.6%) across all bee species being discarded. Only reads confidently identified to family level and reads classified to angiosperm species were used to draw rarefaction curves to determine whether sequence saturation was achieved. Rarefaction curves indicated that for all bee species examined, sufficient reads were sequenced to resolve all possible plant taxa present in the pol- len samples (S1 Fig). Fig 1 show the proportions of bee specimens sampled that visited certain plant families. Notably, Asteraceae was identified in 26 samples (83.9%) in the widespread bees but only in very low proportions of samples for the other two groups (2 samples; 6.4%) in the Succulent Karoo and 2 samples (6.7%) in the Savanna. Fabaceae was also identified in a much higher pro- portion in the widespread samples (20 samples, 64.5%) than those in the Savanna (6 samples, 20%) or Succulent Karoo (7 samples, 22.6%). Of the three bee groups, the widespread bee species visited the widest range of plant families with 30 families visited compared to 15 visited by the Savanna group and 18 visited by the Suc- culent Karoo group. After classification of reads into species OTUs, the widespread bee species again visited the most plant species, with a total of 39 different plant species identified. The Succulent Karoo bees visited a total of 25 plant species and the Savanna bees visited a total of Table 2. Summary statistics of merged reads (after quality and adapter trimming was performed, and subsequent merging of forward and reverse reads) of the six

Megachile species investigated in this study. Failed samples’ reads are excluded. Bee species Number of samples Sum of reads Mean of reads Median of reads Standard deviation Megachile felina 12 664,462 51,113 39,250 50,581 Megachile maxillosa 18 831,287 39,585 26,956 56,087 Megachile karooensis 15 362,278 22,642 9,954 26,406 Megachile murina 18 787,116 39,356 23,517 52,441 Megachile niveofasciata 10 302,191 30,219 20,800 21,917 Megachile venusta# 21 1,124,324 53,539 26,117 57,314 # The summary statistics represented for Megachile venusta are based on sequencing performed on an Illumina MiSeq, and disregards one sample which was excluded in the previous analysis (Gous et al., 2019). https://doi.org/10.1371/journal.pone.0244973.t002 PLOS ONE Floral hosts of Megachilidae revealed by metabarcoding PLOS ONE | https://doi.org/10.1371/journal.pone.0244973 January 21, 2021 6 / 16