Introduction

General description

Project description

Sampling methods

Step description: 

Collection description

The 530 specimens used in this study originated from three sources: 1) 412 from the UMR DYNAFOR collection stored at INP-ENSAT (Ollivier et al. 2024); 2) 88 from the private collection of the bee expert, Rémi Rudelle and 3) a set of 30 Bombus specimens from the CRBE (Centre de Recherche sur la Biodiversité et l‘Environnement) collection. Metadata with detailed information related to each specimen (geographic location, identifiers, sex etc.) can be found in Suppl. material 1.

1. Sample collection

For the UMR DYNAFOR collection, three coloured pan traps (blue, white and yellow) were set in the grassy strip boarding the crop. Each pan trap contained 250 ml of water with Teepol (3 drops/l). After 3 days of exposure, the insects were filtered and placed in ethanol (EtOH) 70% until identification. A panel of 61 specimens was captured with nets and euthanised in ethyl acetate vapour (Suppl. material 1). For the private collection of Rémi Rudelle and the CRBE collection, the specimens were captured with nets.

All of the specimens were morphologically identified using mostly the Insecta Fauna Helvetica reference (Amiet 1996, Amiet 1999, Amiet et al. 2001, Amiet et al. 2004, Amiet et al. 2007, Amiet et al. 2010, Amiet et al. 2017) and others (Schmid-Egger and Scheuchl (1997), Wood (2023) for the Andrenidae family; Ortiz-Sanchez and Jimenez-Rodriguez (1991), Terzo et al. (2007), Rasmont (2014), Smit (2018), Aubert (2020), Le Divelec (2021), Rasmont et al. (2021) for the Apidae family; Ornosa and Ortiz-Sanchez (2004) for the Colletidae family; Pauly (2019) for the Halictidae family and Benoist (1931), Benoist (1941), Pauly (2015) for the Megachilidae family) by one of the following entomologists: Rémi Rudelle, David Genoud, Romain Carrié, Léa Frontero and Dominique Pelletier. They are conserved, pinned in insect boxes and stored at room temperature. To prevent parasite infestation, specimens are frozen twice a year at -20°C for at least 48 hours for the UMR DYNAFOR collection.

2. Sequencing and processing

DNA was extracted from a portion or an entire leg of dried specimens using the Chelex method (see Casquet et al. (2012) for a detailed protocol). Two sequencing technologies were used: 275 specimens were sequenced using high-throughput Illumina technology (MiSeq Sequencing System) and 255 were sequenced with Sanger technology. The list of all species with the corresponding sequencing method is included in Suppl. material 1.

MiSeq sequencing and processing

For the set of 275 specimens processed with MiSeq sequencing, two microlitres of DNA were used as template for PCR. 16S primers ins16S_1R/ins16S_1F (R: TRRGACGAGAAGACCCTATA; F: TCTTAATCCAACATCGAGGTC, Clarke et al. (2014)) were chosen to amplify a 250 bp region of the mitochondrial 16S gene. PCR was performed in a 20 µl total volume containing 5.84 µl of purified water, 10 µl of 2x ampliTaq Gold 360 master mix (Thermo Scientific LSG Life Technologies) including dNTP and ampliTaq Gold, 0.16 µl BSA, 1 µl of each primer (5 µM) and 2 µl of DNA. PCR was carried out under the following conditions: hot-start at 95°C for 10 min followed by 40 cycles (denaturation at 95°C for 30 s, primer annealing at 49°C for 30 s and primer extension at 72°C for 30 s); and final extension at 72°C for 7 min. Primers were 5′ labelled with a set of 8 bp tags to identify samples in bioinformatics analysis. 16S PCR products were visualised on 1% TAE agarose gels quantified using PicoGreen dsDNA Quantitation Reagent and mixed aiming at equimolar pools. The pool was then purified using beads contained in the Illumina TruSeq Nano kit (part #15041758) and libraries were generated following the manufacturer’s guide for the Illumina TruSeq Nano kit, except that no sonication was performed. Libraries were sequenced on a single run of an Illumina MiSeq (2 × 250 paired-end reads), using the NGS core facility at the Génopole Toulouse Midi-Pyrénées. We obtained 22,540,200 demultiplexed reads (R1 and R2 reads). 16S rDNA amplicon sequences were analysed using the FROGS pipeline (version 3.1, Escudié et al. (2017)). Amplicons were processed according to their size (150 - 490 nucleotides) and clustered into ASVs (Amplicon Sequence Variant) using Swarm (aggregation distance: d = 1) (Mahé et al. 2014). For each sample, the most abundant ASV was kept for the procedure of barcode validation.

Sanger sequencing and processing

DNA barcoding using Sanger sequencing technology was performed on 255 specimens. Specific primers were used for each genus. All primer sequences and PCR conditions are given in Fig. 1. For each PCR reaction, 3 µl of extracted DNA was amplified in 25 µl final volume, 1 µM for each primer, 1 x PCRBIO Reaction Buffer (including Mg and dNTPs) and 0.25 µl of PCRBIO Taq DNA Polymerase (5 u/μl) (PB10.11-20; Eurobio). Prior to sequencing, a volume of 2.5 µl from each PCR product was examined on a 2% agarose gel electrophoresis to check the success and specificity of the PCR amplification. The sequencing reaction was subsequently prepared as follows: 2.5 µl of each PCR product was purified by adding 1 µl of each Exonuclease (M0293L; Ozyme) and TSAP (Thermo Sensitive Alcaline Phosphatase) (M9910; Promega) in a final volume of 18 µl. The sequencing reaction mixture was split in two volumes and 1 µl of 10 mM of each primer (forward and reverse) was added. PCR products were sent to a private company for Sanger sequencing in both directions. The sequences produced were manually checked for base calling using ChromasPro 2.1.10.1. (Technelysium Pty Ltd, Tewantin, Australia) and unreadable sequences were removed. For the 30 Bombus sequences from CRBE laboratory, the PCR was performed with the forward primer: CGCTGTTATCCCTAAGG and the reverse primer CTGTACAAAGGTAGCATAATC.

Figure 1.  

Primer sequences and PCR conditions used for Sanger sequencing per Genus. For all Sanger samples, primary denaturation was performed at 95°C for 5 minutes and final elongation was performed at 72°C for 20 minutes.

Amongst the 171 species included in our study, 43 were represented by a single specimen, 25 by two specimens, 51 by three specimens and 52 by four to ten specimens, corresponding to a total of 530 specimens (Fig. 2, step 1).

Figure 2.  

16S mini-barcode library workflow, from sampling to validation. There are four main steps represented by different colours in the left panel, from top to bottom. The middle part of the figure represent the workflow. The right panel indicates the final barcode status.

For sequence validation, we used : sequence assignation by BLASTn (Altschul et al. 1990) on GenBank nt database (Sayers et al. 2021); Neighbour-joining (NJ) distance-tree inferences using the K2P model and the Muscle algorithm (Edgar 2004) for alignment implemented in the BOLD toolkit; Sequence alignment using multalin software to visualise allelic variations (Corpet 1988). For some species, the default BLAST parameters were adjusted to take into account for the high AT content of the region in this genus.

The global success rate after MiSeq sequencing was very high, reaching 99%, with only a single specimen failing. The sequencing success with the Sanger technology was lower due to negative PCR or unreadable chromatograms. Indeed, no sequence could be obtained for 66 specimens. However, as replicate samples were included for most species, only seven species were excluded (Suppl. material 3) at this step (Fig. 2, step 2).

3. Sequence validation

The 463 sequences corresponding to the remaining 164 species were searched in GenBank (nt database) using BLASTn (Altschul et al. 1990) and contaminants (sequences which do not match with a wild bee reference) were removed from data. Fifty-three non-bee and Apis sequences were eliminated and the 410 remaining sequences (163 species) were analysed by cross validation with two filtering rounds (Fig. 2, step 3). The first round allows the detection of potential misidentifications as incongruence between the morphologically and the genetically-based species identification through Neighbour-Joining Trees. For species with only one specimen (singleton), the barcode was validated if the sequence belonged to the corresponding genus. Potentially misidentified species, as well as species that are known to be part of a species complex, were submitted to an entomologist for a second observation taking into account possible identification key updates. The second filtering round allowed sequences validation and confirmation as accurate barcodes. As a result, 16 additional species were excluded (Suppl. material 3).

In total 348 sequenced samples corresponding to 148 unique species were successfully analysed and validated and 97 species were represented by at least two specimens (Fig. 2, step 4).

Geographic coverage

Description: 

The wild bees presented in this study were collected from the French region of Occitanie (Fig. 3). The 412 specimens coming from the UMR DYNAFOR collection were collected in 17 sites located in south-west of France, in the long term socio-ecological research site Zone Atelier Pyrénées-Garonne (ZA PYGAR, Ouin et al. (2021)) over a period of 7 years (2013-2019). The ZA PYGAR takes place in the Pyrénées foothills and is characterised by a mosaic of landscapes with crops and small forests (Carrié et al. 2018, Rivers-Moore et al. 2020). Eighty-eight specimens were captured by Rémi Rudelle in different sites of the Aveyron, French Department and 30 Bombus specimens from the CRBE collection were sampled in the Pyrénées Orientales, French Department (personal communication Nathalie Escaravage).

Figure 3.  

Geographic distribution of the 16S database specimens collected in Occitanie (red points). Nine specimens are not mapped as they were collected outside of Occitanie, although they represent species that can be found within the region.

Taxonomic coverage

Description: 

Specimen records are reported for the 348 sequences (148 species) confirmed with the above workflow. Fig. 4 displays the sequences and species covered by the herein presented 16S library for each genus.

Figure 4.  

Taxonomic coverage of the 16S mini-barcode library compared to existing sequences in GenBank. nb seq: Number of sequences ; nb spe: Number of species. As a point of reference, the sequences available on the public database GenBank before the project start are given in the second column (only for species recorded in the area of collection ZA PYGAR). The last column indicates the number of new sequences added in GenBank and BOLD. Number of species corresponding to specimen records are indicated in brackets.

For 55 of the species included in our dataset, 16S partial or full-length sequences were already available in GenBank (Fig. 4). The list and the accession number of these is reported in Suppl. material 4. The Bombus genus is the most represented with 22 species and 118 sequences. For Andrena, 26 16S sequences corresponding to 15 species originated from the mitochondrion sequencing project were available. Seven species (22 sequences) of Lasioglossum were available at the time of writing the manuscript. At the end, there were no 16S data for 10 genera of wild bees. Thus, we provide 204 new 16S mini-barcodes for wild bees belonging to 93 species. For the most abundant species of France belonging to the Andrena and Lasioglossum genera sets, 71 new sequences (32 species) and 37 new sequences (18 species) were respectively added in the public databases.

Traits coverage

Temporal coverage

Collection data

Usage licence

Data resources

Additional information

Sequencing and barcoding results

The within-genus global sequencing success including MiSeq and Sanger technologies varies from 75% to 100%, except for Sphecodes (33%) (Fig. 5). For the most represented genus of our dataset, namely Andrena and Lasioglossum, the sequencing success rates were 84% and 78%, respectively. The barcoding success rate after all successive filtering steps was different according to genera. It was 100% for Nomada, Tetralonia, Antophora and Colletes, 90% for Bombus, 88% for Hylaeus, 77% for Osmia, 76% for Halictus, 72% for Andrena and it was under 65% for the other genera. For Lasioglossum, the low barcoding success rate (38%) is related to low sequencing success, being as low as 42.7% with MiSeq and 28% with Sanger (Suppl. material 1) and to high level of contaminated sequences (54). An in-depth analysis of Lasioglossum sequences showed that the end of the amplified fragment contains many stretches of AT nucleotide repeats (Suppl. material 2) which are known to disrupt the polymerase activity during the sequencing process. Amongst the 171 species of our dataset, no barcode could be obtained for 23 species including nine species of Lasioglossum (Suppl. material 3). Suppl. material 5 provides detailed sequencing and barcoding success per species.

Figure 5.  

Sequencing and barcoding success per family and genus.

Analysis of genetic distances

Examination of the general normalised divergence histogram performed with BOLD analyses toolkit on all species (replicates) indicates the existence of a DNA barcoding gap (maximal intraspecific distance > minimal interspecific distance), allowing reliable molecular identification of specimens (Fig. 6). However, a more in-depth analysis of each genus reveals two scenarios: There was an overlap between intraspecific and interspecific genetic distances in six genera: Andrena, Bombus, Eucera, Halictus, Lasioglossum and Nomada, whereas the barcoding gap was clearly existing for the five others genera: Xylocopa, Seladonia, Hylaeus, Osmia and Ceratina (Fig. 6). The tables with detailed intraspecific and interspecific genetic distances are given in Suppl. materials 6, 7.

Figure 6.  

Distribution of intraspecific and interspecific genetic distances per genus. The global normalised distance distribution for all specimens is shown at bottom right corner. Blue arrows indicate species that show an intraspecific distance > 1%. Red arrows indicate group of species that show an interspecific distance < 1%.

Genetic distance analyses per family and genus

• Andrenidae: (Taxon ID tree is given in Suppl. material 8).

For the Andrena genus, which has been reported difficult to barcode with the COI Folmer primers (Schmidt et al. 2015, Villalta et al. 2021), the 16S mini-barcode offers a good alternative. Amongst the 111 validated barcodes (47 species), the 16S mini-barcode allows us to discriminate all the species in accordance with the morphological subgenera classification (Suppl. material 8). Interestingly, the 250 bp of the 16S gene used in this study is sufficiently divergent to separate complex groups previously described in literature. For example, the barcoding of Andrena distinguenda species group (A. nitidula and A. distinguenda) with COI showed the existence of two bins (Schmidt et al. 2015). With the 16S mini-barcode, the minimum divergence between these two species was 1.13% supporting the existence of two species (Suppl. material 7). Similar to that described by Wood et al. (2021), we found a clear separation (3.11% minimum divergence) in the Andrena angustior group between Andrena impressa and Andrena fulvata (Suppl. material 7). A complex situation remains with Andrena trimmerana; our molecular data on eight specimens including two males and six females clearly show two groups (0% divergence within groups) with an intergroup divergence of 1.29% (Suppl. material 7). Interestingly, we observed allelic variation (1 SNP) between Andrena dorsata originating from our data and the two Andrena dorsata sequences provided in GenBank originated from the UK (KT16433.1 and OV815490.1). Elsewhere, two sequences of Andrena fulva were available in GenBank (KT164623.1 and OX276334.1). Alignment of these two A. fulva with our specimens reviewed by entomologists show that KT16423.1 is 100% homologous with our Andrena fulvago, whereas OX276334.1 aligns with Andrena fulva. The sequences of these two species diverge 5.9% with the 16S mini-barcode.

• Apidae: (Taxon ID tree is given in Suppl. material 9).

Bombus: The distance tree inferred from the 16S mini-barcodes of the 23 Bombus species (73 specimens) reveals genetic divergence that is consistent with the known subgenus classification (Cameron et al. 2007, Cejas et al. 2019, Sun et al. 2021). Interestingly, the 16S mini-barcode classifies without ambiguity each specimen of the following species complex: Bombus pascuorum/muscorum; Bombus sylvarum/ruderarius; Bombus ruderatus/hortorum; Bombus pyreaneus/pratorum and Bombus terrestris/lucorum. As some of the specimens in the Bombus terrestris group in our dataset were not morphologically identified at the species level, the 0% divergence between specimens named Bombus gr. terrestris and Bombus terrestris suggests that all of them are Bombus terrestris. Elsewhere, amongst the 10 Bombus pascuorum specimens barcoded with the 16S mini-barcode, some allelic variation was observed, with a minimum intraspecific divergence of 0% and a maximum intraspecific divergence of 0.98% (Suppl. material 6). In Switzerland, Amiet et al. (2017) reported the presence of two subspecies of Bombus pascuorum.

Eucera: In the Apidae family, the 16S mini-barcodes are not discriminant for two species belonging to the Eucera genus: Eucera nigrifacies and Eucera nigrescens, whereas it efficiently delineates the six others species, especially Eucera longicornis which was confused in the past with Eucera nigrescens (Dorchin et al. 2018). Allelic variations are observed for Eucera longicornis (0.26% maximum intraspecific divergence) and Eucera numida (0.26% maximum intraspecific divergence) (Suppl. material 6).

Nomada: Regarding the Nomada genus, recently reexamined by Odanaka et al. (2022) and Straka et al. (2024), our data shows that the 16S mini-barcode distinguishes the 21 specimens (13 species), except Nomada striata versus Nomada sexfasciata and Nomada fucata versus Nomada melathoracica. However, more specimens need to be analysed to conclude definitively that three of these species are singletons.

Ceratina: The molecular phylogeny of these small carpenter bees has been recently achieved by Sless et al. (2024). The two species of our dataset are extremely divergent (31.12% min interspecific divergence and 51.49% max interspecific divergence, Suppl. material 7). One belongs to the subgenus Euceratina (Eucera cyanea) and the other to the subgenus Ceratina sensu stricto (Ceratina cucurbitinia).

Xylocopa: The five specimens of Xylocopa from our dataset correspond to the three species: Xylocopa valga, Xylocopa iris and Xylocopa violacea exhibit 0.46% maximum intraspecific divergence and 7.19% minimum interspecific divergence (Suppl. materials 6, 7).

• Colletidae (Taxon ID tree is given in Suppl. material 10).

In the present study, five species belonging to Hylaeus (Almeida and Danforth 2009) genus were successfully barcoded with the 16S mini-barcode. Amongst them, four were singletons and Hylaeus brevicornis had three replicates with intraspecific divergence of 0% (Suppl. material 6). The divergence between species ranges from 10.47% to 28.66% (Suppl. material 7).

• Megachilidae (Taxon ID tree is given in Suppl. material 11).

Osmia: As Apis mellifera or Bombus, Osmia are commercially reared for pollination services. A complete phylogeny of the Palearctic Osmiine bee is available on the website of Müller (2024). Molecular data using UCEs or Elongation factor 1-α or LW-rhodopsin and Conserved ATPase domain have been reported by Praz et al. (2008) and Branstetter et al. (2021). In this work, the 16S mini-barcode is clearly efficient to separate the seven species of our dataset: 0.51% maximum intraspecific divergence and 6.22% minimum interspecific divergence were observed (Suppl. materials 6, 7).

• Halictidae (Taxon ID tree is given in Suppl. material 12).

Lasioglossum: Molecular phylogeny of Lasioglossum is poorly documented (Danforth 1999, Gibbs et al. 2012, Gibbs 2018, Pauly et al. 2019). For the 25 species belonging to genus Lasioglossum successfully barcoded in this study, the intraspecific divergence is < 1% for all species, except for Lasioglossum xanthopus which is > 2%. Interestingly, the 16S mini-barcode allows a clear separation (min interspecific divergence > 2%) for complex groups. Thus, for the specimens of Lasioglossum medinai/Lasioglossum villosulum species, the min interspecific divergence is 4.38%. It is 6.84% for Lasioglossum malachurum/subhirtum/calceatum/pauxillum/laticeps species; 5.70% for Lasioglossum morio/nitidulum species and 5.23% for Lasioglossum pauperatum/pygmaeum/truncaticolle/crassepunctatum species (Suppl. materials 6, 7).

Halictus: In the Halictus simplex group, Halictus simplex and Halictus langobardicus females are extremely difficult to distinguish morphologically. Unfortunately, the 16S mini-barcode did not allow for the discrimination of the two species, whereas Halictus compressus exhibited a 0.78% minimum interspecific divergence with the rest of the group (Suppl. material 7). Interestingly, two specimens from the Halictus simplex group diverged slightly from the others. It would be interesting to barcode more specimens including species belonging to the simplex group which were not included in our dataset. Sequencing complete mitochondrion in the future would help to clarify the status of this group. Elsewhere, we observed allelic variation amongst specimens of Halictus quadricinctus and we suspected that one of them could be Halictus brunnescens.

Acknowledgements

Author contributions

References

Alajmi R, Abdel-Gaber R, Alfozana L (2019)
Molecular insights of mitochondrial 16S rDNA genes of the native honey bees subspecies Apis mellifera carnica and Apis mellifera jementica (Hymenoptera: Apidae) in Saudi Arabia
.
Genetics and Molecular Research
18
(
1
):
1
‑
16
.
Almeida EA, Danforth BN (2009)
Phylogeny of colletid bees (Hymenoptera: Colletidae) inferred from four nuclear genes
.
Molecular Phylogenetics and Evolution
50
(
2
):
290
‑
309
. https://doi.org/10.1016/j.ympev.2008.09.028
Altschul S, Gish W, Miller W, Myers E, Lipman D (1990)
Basic local alignment search tool
.
Journal of Molecular Biology
215
(
3
):
403
‑
410
. https://doi.org/10.1016/s0022-2836(05)80360-2
Amiet F (1996)
Apidae 1 (Apis, Bombus, Psithyrus).
In: CSCF (Ed.)
Insecta Helvetica Fauna
.
12
.
Neuchâtel
.
Amiet F (1999)
Apidae 2. Colletes, Dufourea, Hylaeus, Nomia, Nomioides, Rhophitoides, Rophites, Sphecodes, Systropha
. In: faune CSdcdl (Ed.)
Fauna Helvetica
.
4
.
Neuchâtel
. URL: https://cscf.abacuscity.ch/de/chf/A~11FHE04X/0~0~Coll/Apidae-2
Amiet F, Herrmann M, Müller A, Neumeyer R (2001)
Apidae 3. Halictus, Lasioglossum
. In: faune CSdcdl (Ed.)
Fauna Helvetica
.
Neuchâtel
.
Amiet F, Herrmann M, Müller A, Neumeyer R (2004)
Apidae 4: Anthidium, Chelostoma, Coelioxys, Dioxys, Heriades, Lithurgus, Megachile, Osmia, Stelis
. In: CSCF, SEG (Eds)
Fauna Helvetica
.
9
.
Neuchâtel
.
Amiet F, Herrmann M, Müller A, Neumeyer R (2007)
Apidae 5: Ammobates, Ammobatoides, Anthophora, Biastes, Ceratina, Dasypoda, Epeoloides, Epeolus, Eucera, Macropis, Melecta, Melitta, Nomada, Pasites, Tetralonia, Thyreus, Xylocopa
. In: CSCF, SEG (Eds)
Fauna Helvetica
.
20
.
Neuchâtel
. URL: https://cscf.abacuscity.ch/de/chf/A~11FHE20X/0~0~Coll/Apidae-5
Amiet F, Herrmann M, Müller A, Neumeyer R (2010)
Apidae 6: Andrena, Melitturga, Panurginus, Panurgus
. In: CSCF, SEG (Eds)
Fauna Helvetica
.
26
.
Neuchâtel
. URL: https://cscf.abacuscity.ch/de/chf/A~11FHE26D/0~0~Coll/Apidae-6
Amiet F, Mueller A, Praz C (2017)
Apidae 1: Allgemeiner Teil, Gattungen, Apis, Bombus
.
Fauna Helvetica
29
:
1
‑
185
.
Aubert M (2020)
Proposition de clé d’identification des Eucerini (Hymenoptera: Anthophila) de France continentale – version provisoire
.
Observatoire des Abeilles
. URL: https://oabeilles.net/projets/eucerini
Avalos G, Trott R, Ballas J, Lin CH, Raines C, Iwanowicz D, Goodell K, Richardson RT (2024)
Prospects of pollinator community surveillance using terrestrial environmental DNA metagenetics
.
Environmental DNA
6
:
492
. https://doi.org/10.1002/edn3.492
Benoist R (1931)
Les osmies de la faune française
.
Annales de la Société Entomologique de France
100
(
1/2
):
23
‑
60
. https://doi.org/10.1080/21686351.1931.12280120
Benoist R (1941)
Remarques sur quelques espèces de Mégachiles principalement de la faune française (Hymen Apidae)
.
Annales de la Société entomologique de France
109
:
41
‑
88
. https://doi.org/10.1080/21686351.1940.12279433
Biesmeijer JC, Roberts SPM, Reemer M, Ohlemüller R, Edwards M, Peeters T, Schaffers AP, Potts SG, Kleukers R, Thomas CD, Settele J, Kunin WE (2006)
Parallel declines in pollinators and insect-pollinated plants in Britain and the Netherlands
.
Science
313
(
5785
):
351
‑
354
. https://doi.org/10.1126/science.1127863
Branstetter MG, Müller A, Griswold TL, Orr MC, Zhu CD (2021)
Ultraconserved element phylogenomics and biogeography of the agriculturally important mason bee subgenus Osmia (Osmia)
.
Systematic Entomology
46
(
2
):
453
‑
472
. https://doi.org/10.1111/syen.12470
Cameron SA, Hines HM, Williams PH (2007)
A comprehensive phylogeny of the bumble bees (Bombus)
.
Biological Journal of the Linnean Society
91
(
1
):
161
‑
188
. https://doi.org/10.1111/j.1095-8312.2007.00784.x
Carrié R, Lopes M, Ouin A, Andrieu E (2018)
Bee diversity in crop fields is influenced by remotely-sensed nesting resources in surrounding permanent grasslands
.
Ecological Indicators
90
https://doi.org/10.1016/j.ecolind.2018.03.054
Casquet J, Thebaud C, Gillespie R (2012)
Chelex without boiling, a rapid and easy technique to obtain stable amplifiable DNA from small amounts of ethanol‐stored spiders
.
Molecular Ecology Resources
12
(
1
):
136
‑
141
. https://doi.org/10.1111/j.1755-0998.2011.03073.x
Cejas D, López-López A, Muñoz I, Ornosa C, De la Rúa P (2019)
Unveiling introgression in bumblebee (Bombus terrestris) populations through mitogenome‐based markers
.
Animal Genetics
51
:
10
‑
1111
. https://doi.org/10.1111/age.12874
Chua PS, Bourlat S, Ferguson C, Korlevic P, Zhao L, Ekrem T, Meier R, Lawniczak MN (2023)
Future of DNA-based insect monitoring
.
Trends in Genetics
39
(
7
):
531
‑
544
. https://doi.org/10.1016/j.tig.2023.02.012
Clarke L, Soubrier J, Weyrich L, Cooper A (2014)
Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias
.
Molecular Ecology Resources
14
(
6
):
1160
‑
1170
. https://doi.org/10.1111/1755-0998.12265
Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW (2016)
GenBank
.
Nucleic Acids Research
44
(
D1
):
67
‑
72
. https://doi.org/10.1093/nar/gkv1276
Corpet F (1988)
Multiple sequence alignment with hierarchical clustering
.
Nucleic Acids Research
16
(
22
):
10881
‑
10890
. https://doi.org/10.1093/nar/16.22.10881
Costa M, Del Lama M, Melo GR, Sheppard W (2003)
Molecular phylogeny of the stingless bees (Apidae, Apinae, Meliponini) inferred from mitochondrial 16S rDNA sequences
.
Apidologie
34
(
1
):
73
‑
84
. https://doi.org/10.1051/apido:2002051
Creedy T, Norman H, Tang C, Chin KQ, Andujar C, Arribas P, O’Connor R, Carvell C, Notton D, Vogler A (2020)
A validated workflow for rapid taxonomic assignment and monitoring of a national fauna of bees (Apiformes) using high throughput barcoding
.
bioRxiv
https://doi.org/10.1101/575308
Danforth B (1999)
Phylogeny of the bee genus Lasioglossum (Hymenoptera: Halictidae) based on mitochondrial COI sequence data
.
Systematic Entomology
24
:
377
‑
393
. https://doi.org/10.1046/j.1365-3113.1999.00087.x
de Carvalho M, Bockmann F, Amorim D, Brandão C, de Vivo M, de Figueiredo J, Britski H, de Pinna MC, Menezes N, Marques FL, Papavero N, Cancello E, Crisci J, McEachran J, Schelly R, Lundberg J, Gill A, Britz R, Wheeler Q, Stiassny MJ, Parenti L, Page L, Wheeler W, Faivovich J, Vari R, Grande L, Humphries C, DeSalle R, Ebach M, Nelson G (2007)
Taxonomic impediment or impediment to taxonomy? A commentary on systematics and the cybertaxonomic-automation paradigm
.
Evolutionary Biology
34
:
140
‑
143
. https://doi.org/10.1007/s11692-007-9011-6
Dorchin A, Danforth B, Griswold T (2018)
A new genus of eucerine bees endemic to southwestern North America revealed in phylogenetic analyses of the Eucera complex (Hymenoptera: Apidae: Eucerini
.
Arthropod Systematics and Phylogeny
76
:
10
‑
3897
.
Edgar RC (2004)
MUSCLE: multiple sequence alignment with high accuracy and high throughput
.
Nucleic Acids Research
32
(
5
):
1792
‑
1797
. https://doi.org/10.1093/nar/gkh340
Elbrecht V, Taberlet P, Dejean T, Valentini A, Usseglio-Polatera P, Beisel J, Coissac E, Boyer F, Leese F (2016)
Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects
.
PeerJ
4
https://doi.org/10.7717/peerj.1966
Escudié F, Auer L, Bernard M, Mariadassou M, Cauquil L, Vidal K, Maman S, Hernandez-Raquet G, Combes S, Pascal G (2017)
FROGS: Find, Rapidly, OTUs with Galaxy Solution
.
Bioinformatics
34
(
8
):
1287
‑
1294
. https://doi.org/10.1093/bioinformatics/btx791
Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994)
DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates
.
Molecular Marine Biology and Biotechnology
3
:
294
‑
299
.
Gibbs J, Brady SG, Kanda K, Danforth BN (2012)
Phylogeny of halictine bees supports a shared origin of eusociality for Halictus and Lasioglossum (Apoidea: Anthophila: Halictidae
.
Molecular Phylogenetics and Evolution
65
(
3
):
926
‑
939
. https://doi.org/10.1016/j.ympev.2012.08.013
Gibbs J (2018)
DNA barcoding a nightmare taxon: assessing barcode index numbers and barcode gaps for sweat bees
.
Genome
61
(
1
):
21
‑
31
. https://doi.org/10.1139/gen-2017-0096
Hajibabaei M, Alex S, Janzen D, Rodriguez J, Whitfield J, Hebert PN (2006)
A minimalist barcode can identify a specimen whose DNA is degraded
.
Molecular Ecology Notes
6
(
4
):
959
‑
964
. https://doi.org/10.1111/j.1471-8286.2006.01470.x
Herrera-Mesías F, Bause C, Ogan S, Burger H, Ayasse M, Weigand A, Eltz T (2022)
Double-blind validation of alternative wild bee identification techniques: DNA metabarcoding and in vivo determination in the field
.
Journal of Hymenoptera Research
93
:
189
‑
214
. https://doi.org/10.3897/jhr.93.86723
Hsieh C, Huang C, Wu W, Wang H (2019)
A rapid insect species identification system using mini‐barcode pyrosequencing
.
Pest Management Science
76
(
4
):
1222
‑
1227
. https://doi.org/10.1002/ps.5674
Janko Š, Rok Š, Blaž K, Danilo B, Andrej G, Denis K, Klemen Č, Matjaž G (2024)
DNA barcoding insufficiently identifies European wild bees (Hymenoptera, Anthophila) due to undefined species diversity, genus‐specific barcoding gaps and database errors
.
Molecular Ecology Resources
24
(
5
). https://doi.org/10.1111/1755-0998.13953
Kek SP, Chin N, Tan SW, Yusof YA, Chua LS (2017)
Molecular identification of honey entomological origin based on bee mitochondrial 16S rRNA and COI gene sequences
.
Food Control
78
:
10
‑
1016
.
Le Divelec R (2021)
The West Palaearctic Epeolini Linsley & Michener, 1939 housed in the Muséum national d’Histoire naturelle (Paris) with some taxonomic notes (Hymenoptera: Apidae: Nomadinae).
Annales de la Société entomologique de France
N.S.
(
57
):
1
‑
33
. https://doi.org/10.1080/00379271.2021.1942206
Levesque-Beaudin V, Miller M, Dikow T, Miller S, Prosser S, Zakharov E, McKeown J, Sones J, Redmond N, Coddington J, Santos B, Bird J, deWaard J (2023)
A workflow for expanding DNA barcode reference libraries through ‘museum harvesting’ of natural history collections
.
Biodiversity Data Journal
11
https://doi.org/10.3897/bdj.11.e100677
Little D (2014)
A DNA mini‐barcode for land plants
.
Molecular Ecology Resources
14
(
3
):
437
‑
446
. https://doi.org/10.1111/1755-0998.12194
Liu M, Clarke L, Baker S, Jordan G, Burridge C (2019)
A practical guide to DNA metabarcoding for entomological ecologists
.
Ecological Entomology
45
(
3
):
373
‑
385
. https://doi.org/10.1111/een.12831
Magnacca K, Brown MF (2012)
DNA barcoding a regional fauna: Irish solitary bees
.
Molecular Ecology Resources
12
(
6
):
990
‑
998
. https://doi.org/10.1111/1755-0998.12001
Mahé F, Rognes T, Quince C, de Vargas C, Dunthorn M (2014)
Swarm: robust and fast clustering method for amplicon-based studies
.
PeerJ
https://doi.org/10.7717/peerj.593
Marconi M, Ushiñahua Ramírez D, Cerna Mendoza A, Vecco-Giove CD, Ormeño Luna J, Baikova L, Di Giulio A, Mancini E (2023)
An updated molecular phylogeny of the stingless bees of the genus Trigona (Hymenoptera, Meliponini) of the northern Peruvian forests
.
Journal of Hymenoptera Research
96
:
751
‑
760
. https://doi.org/10.3897/jhr.96.105311
Marquina D, Andersson A, Ronquist F (2018)
New mitochondrial primers for metabarcoding of insects, designed and evaluated using in silico methods
.
Molecular Ecology Resources
19
(
1
):
90
‑
104
. https://doi.org/10.1111/1755-0998.12942
Michener CD (2007)
The bees of the world
.
Johns Hopkins University Press
https://doi.org/10.56021/9780801885730
Müller A (2024)
Palaearctic Osmiine Bees, ETH Zürich
. http://blogs.ethz.ch/osmiini
Newton J, Bateman P, Heydenrych M, Kestel J, Dixon K, Prendergast K, White N, Nevill P (2023)
Monitoring the birds and the bees: Environmental DNA metabarcoding of flowers detects plant–animal interactions
.
Environmental DNA
5
(
3
):
488
‑
502
. https://doi.org/10.1002/edn3.399
Odanaka K, Branstetter M, Tobin K, Rehan S (2022)
Phylogenomics and historical biogeography of the cleptoparasitic bee genus Nomada (Hymenoptera: Apidae) using ultraconserved elements
.
Molecular Phylogenetics and Evolution
170
https://doi.org/10.1016/j.ympev.2022.107453
Ollivier M, Rivers-Moore J, Pichon M, Andrieu E, Carrié R, Rudelle R, Sarthou J, Ouin A (2024)
Wild bees (Apoidea, Anthophila) of south-west France: more than 10 years of inventories in mosaic landscapes of "Vallées et Coteaux de Gascogne" (ZA-PYGAR)
.
Biodiversity Data Journal
https://doi.org/10.3897/BDJ.12.e135157
Ollivier M, Rudelle R, Genoud D, Marquisseau A, Rougerie R, Perrard A, Pichon M (In prep.)
A national reference CO1 DNA barcode database covering 80% of the French wild bees fauna (Apoidea antophila)
.
BMC Ecology & Evolution
.
Ornosa C, Ortiz-Sanchez F (2004)
Hymenoptera: Apoidea I
. In: Ramos MA, et al. (Ed.)
Fauna Ibérica
.
23
.
Museo Nacional de Ciencias Naturales, Consejo Superior de Investigaciones Cientificas
,
Madrid
.
Ortega A, Geraldi N, Díaz-Rúa R, Ørberg S, Wesselmann M, Krause-Jensen D, Duarte C (2020)
A DNA mini-barcode for marine macrophytes
.
Molecular Ecology Resources
20
(
4
):
920
‑
935
. https://doi.org/10.1111/1755-0998.13164
Ortiz-Sanchez FJ, Jimenez-Rodriguez AJ (1991)
Actualizacion del catalogos de las especies españolas de Anthophorini (Hymenoptera, Anthophoridae)
.
Boletin de la Asociacion española de Entomologia
.
Ouin A, Andrieu E, Vialatte A, Balent G, Barbaro L, Blanco J, Ceschia E, Clement F, Fauvel M, Gallai N, Hewison AJM, Jean-François D, Kephaliacos C, Macary F, Probst A, Probst J, Ryschawy J, Sheeren D, Sourdril A, Tallec T, Verheyden H, Sirami C (2021)
Chapter Two - Building a shared vision of the future for multifunctional agricultural landscapes. Lessons from a long term socio-ecological research site in south-western France
. In: Bohan D, Dumbrell A, Vanbergen A (Eds)
Advances in Ecological Research
.
Vol. 65
.
Academic Press
https://doi.org/10.1016/bs.aecr.2021.05.001
Packer L, Ruz L (2017)
DNA barcoding the bees (Hymenoptera: Apoidea) of Chile: species discovery in a reasonably well known bee fauna with the description of a new species of Lonchopria (Colletidae)
.
Genome
60
(
5
):
414
‑
430
. https://doi.org/10.1139/gen-2016-0071
Pauly A (2015)
Clé illustrée pour l’identification des abeilles de Belgique. II. Megachilidae
.
Document de travail du projet BELBEES
.
Pauly A (2019)
Abeilles de Belgique et des régions limitrophes (Insecta Apoidea). Famille Halictidae
.
Institut royal des Sciences naturelles de Belgique
Pauly A, Noël G, Sonet G, Notton D, Boevé J-L (2019)
Integrative taxonomy resuscitates two species in the Lasioglossum villosulum complex (Kirby, 1802) (Hymenoptera: Apoidea: Halictidae).
European Journal of Taxonomy
541
https://doi.org/10.5852/ejt.2019.541
Piper AM, Batovska J, Cogan NOI, Weiss J, Cunningham JP, Rodoni BC, Blacket MJ (2019)
Prospects and challenges of implementing DNA metabarcoding for high-throughput insect surveillance
.
GigaScience
8
(
8
). https://doi.org/10.1093/gigascience/giz092
Powney G, Carvell C, Edwards M, Morris RA, Roy H, Woodcock B, Isaac NB (2019)
Widespread losses of pollinating insects in Britain
.
Nature Communications
10
(
1
). https://doi.org/10.1038/s41467-019-08974-9
Praz C, Müller A, Danforth B, Griswold T, Widmer A, Dorn S (2008)
Phylogeny and biogeography of bees of the tribe Osmiini (Hymenoptera: Megachilidae).
Molecular Phylogenetics and Evolution
49
:
185
‑
197
. https://doi.org/10.1016/j.ympev.2008.07.005
Rasmont P (2014)
Clef des Melectini de France (Apidae)
.
Université de Mons, Belgique
.
Rasmont P, Ghisbain G, Terzo M (2021)
Bumblebees of Europe and neighbouring regions
.
NAP Editions
,
Verrières-le-Buisson
.
Ratnasingham S, Hebert PDN (2007)
bold: The Barcode of Life Data System (http://www.barcodinglife.org).
Molecular ecology notes
7
(
3
):
355
‑
364
. https://doi.org/10.1111/j.1471-8286.2007.01678.x
Ratnasingham S, Hebert PN (2013)
A DNA-based registry for all animal species: The Barcode Index Number (BIN) System
.
PLOS One
8
(
7
). https://doi.org/10.1371/journal.pone.0066213
Ratnasingham S, Wei C, Chan D, Agda J, Agda J, Ballesteros-Mejia L, Boutou HA, El Bastami ZM, Ma E, Manjunath R, Rea D, Ho C, Telfer A, McKeowan J, Rahulan M, Steinke C, Dorsheimer J, Milton M, Hebert PD (2024)
BOLD v4: A centralized bioinformatics platform for DNA-based biodiversity data
.
Methods in Molecular Biology
2744
:
403
‑
441
. https://doi.org/10.1007/978-1-0716-3581-0_26
Remmel N, Buchner D, Enss J, Hartung V, Leese F, Welti EA (2024)
DNA metabarcoding and morphological identification reveal similar richness, taxonomic composition and body size patterns among flying insect communities
.
Insect Conservation and Diversity
17
(
3
):
449
‑
463
. https://doi.org/10.1111/icad.12710
Rhodes C (2018)
Pollinator decline – An ecological calamity in the making?
Science Progress
101
(
2
):
121
‑
160
. https://doi.org/10.3184/003685018x15202512854527
Rivers-Moore J, Andrieu E, Vialatte A, Ouin A (2020)
Wooded semi-natural habitats complement permanent grasslands in supporting wild bee diversity in agricultural landscapes
.
Insects
11
:
812
‑
10
. https://doi.org/10.3390/insects11110812
Santos B, Miller M, Miklasevskaja M, McKeown J, Redmond N, Coddington J, Bird J, Miller S, Smith A, Brady S, Buffington M, Chamorro ML, Dikow T, Gates M, Goldstein P, Konstantinov A, Kula R, Silverson N, Solis MA, deWaard S, Naik S, Nikolova N, Pentinsaari M, Prosser S, Sones J, Zakharov E, deWaard J (2023)
Enhancing DNA barcode reference libraries by harvesting terrestrial arthropods at the Smithsonian's National Museum of Natural History
.
Biodiversity Data Journal
11
https://doi.org/10.3897/bdj.11.e100904
Sayers EW, Bolton EE, Brister JR, Canese K, Chan J, Comeau D, Connor R, Funk K, Kelly C, Kim S, Madej T, Marchler-Bauer A, Lanczycki C, Lathrop S, Lu Z, Thibaud-Nissen F, Murphy T, Phan L, Skripchenko Y, Tse T, Wang J, Williams R, Trawick B, Pruitt K, Sherry S (2021)
Database resources of the national center for biotechnology information
.
Nucleic Acids Research
50
https://doi.org/10.1093/nar/gkab1112
Schmid-Egger C, Scheuchl E (1997)
Illustrierte Bestimmungstabellen der Wildbienen Deutschlands und sterreichs 3: Andrenidae
.
Self-publishing
Schmidt S, Schmid‐Egger C, Morinière J, Haszprunar G, Hebert PN (2015)
DNA barcoding largely supports 250 years of classical taxonomy: identifications for Central European bees (Hymenoptera, Apoidea partim)
.
Molecular Ecology Resources
15
(
4
):
985
‑
1000
. https://doi.org/10.1111/1755-0998.12363
Sheffield C, Hebert PN, Kevan P, Packer L (2009)
DNA barcoding a regional bee (Hymenoptera: Apoidea) fauna and its potential for ecological studies
.
Molecular Ecology Resources
9
:
196
‑
207
. https://doi.org/10.1111/j.1755-0998.2009.02645.x
Sheffield C, Heron J, Gibbs J, Onuferko T, Oram R, Best L, deSilva N, Dumesh S, Pindar A, Rowe G (2017)
Contribution of DNA barcoding to the study of the bees (Hymenoptera: Apoidea) of Canada: progress to date
.
The Canadian Entomologist
149
(
6
):
736
‑
754
. https://doi.org/10.4039/tce.2017.49
Sickel W, Kulow J, Krüger L, Dieker P (2023)
BEE-quest of the nest: A novel method for eDNA-based, nonlethal detection of cavity-nesting hymenopterans and other arthropods
.
Environmental DNA
5
:
1163
‑
1176
. https://doi.org/10.1002/edn3.490
Sless TJ, Branstetter MG, Mikát M, Odanaka KA, Tobin KB, Rehan SM (2024)
Phylogenomics and biogeography of the small carpenter bees (Apidae: Xylocopinae: Ceratina)
.
Molecular Phylogenetics and Evolution
198
https://doi.org/10.1016/j.ympev.2024.108133
Smit J (2018)
Identification Key to the European Species of the Bee Genus Nomada Scopoli, 1770 (Hymenoptera: Apidae), Including 23 New Species
.
Museum Witt
Straka J, Benda D, Policarová J, Astapenková A, Wood T, Bossert S (2024)
A phylogenomic monograph of West-Palearctic Nomada (Hymenoptera: Apidae)
.
Insect Systematics and Diversity
8
:
10
‑
1093
.
Sun C, Huang J, Wang Y, Zhao X, Su L, Thomas GC, Zhao M, Zhang X, Jungreis I, Kellis M, Vicario S, Sharakhov I, Bondarenko S, Hasselmann M, Kim C, Paten B, Penso-Dolfin L, Wang L, Chang Y, Gao Q, Ma L, Ma L, Zhang Z, Zhang H, Zhang H, Ruzzante L, Robertson H, Zhu Y, Liu Y, Yang H, Ding L, Wang Q, Ma D, Xu W, Liang C, Itgen M, Mee L, Cao G, Zhang Z, Sadd BM, Hahn MW, Schaack S, Barribeau SM, Williams PH, Waterhouse RM, Lockridge Mueller R (2021)
Genus-wide characterization of bumblebee genomes provides insights into their evolution and variation in ecological and behavioral traits
.
Molecular Biology and Evolution
38
(
2
):
486
‑
501
. https://doi.org/10.1093/molbev/msaa240
Terzo M, Iserbyt S, Rasmont P (2007)
Révision des Xylocopinae (Hymenoptera : Apidae) de France et de Belgique, Annales de la Société entomologique de France
.
International Journal of Entomology
43
(
4
):
445
‑
491
. https://doi.org/10.1080/00379271.2007.10697537
Trianto M, Purwanto H (2020)
Molecular phylogeny of stingless bees in the special region of Yogyakarta revealed using partial 16S rRNA mitochondrial gene
.
Buletin Peternakan
44
(
4
). https://doi.org/10.21059/buletinpeternak.v44i4.55539
Villalta I, Ledet R, Baude M, Genoud D, Bouget C, Cornillon M, Moreau S, Courtial B, Lopez-Vaamonde C (2021)
A DNA barcode-based survey of wild urban bees in the Loire Valley, France.
Scientific reports
11
(
1
):
4770
. https://doi.org/10.1038/s41598-021-83631-0
Vinarski M (2020)
Roots of the taxonomic impediment: Is the “integrativeness” a remedy?
Integrative Zoology
15
(
1
):
2
‑
15
. https://doi.org/10.1111/1749-4877.12393
Whitfield JB, Cameron SA (1998)
Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera
.
Molecular Biology and Evolution
15
(
ue 12
):
1728
‑
1743
. https://doi.org/10.1093/oxfordjournals.molbev.a025899
Wood T, Ghisbain G, Michez D, Praz C (2021)
Revisions to the faunas of Andrena of the Iberian Peninsula and Morocco with the descriptions of four new species The InBIO Barcoding Initiative Database: DNA barcodes of Iberian Bees 69 (Hymenoptera: Andrenidae
.
European Journal of Taxonomy
758
:
147
‑
193
. https://doi.org/10.5852/ejt.2021.758.1431
Wood T (2023)
The genus Andrena Fabricius, 1775 in the Iberian Peninsula (Hymenoptera, Andrenidae)
.
Journal of Hymenoptera Research
96
:
241
‑
484
. https://doi.org/10.3897/jhr.96.101873
Wood T, Gaspar H, Le Divelec R, Penado A, Silva TL, Mata V, Veríssimo J, Michez D, Castro S, Loureiro J, Beja P, Ferreira S (2024)
The InBIO Barcoding initiative database: DNA barcodes of Iberian bees
.
Biodiversity Data Journal
12
https://doi.org/10.3897/bdj.12.e117172
Yoon HJ, Kim SE, Kim YS, Lee SB (2004)
Colony developmental characteristics of the bumblebee queen Bombus ignitus by the first oviposition day
.
International Journal of Industrial Entomology
8
:
139
‑
143
.

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