3. Results
3.1. UCE sequencing
All combined, the Miseq runs produced on average 526,308 (SD ± 356,015)
reads per specimen. The average number of loci per specimen after
assembly was 7398 (SD ± 5099). After retaining only loci matching the
UCE reference file and filtering for a 75% matrix completeness, the
number of retrieved loci varied between 686 and 1860 across the five
species complexes (Supplementary table S3). Resulting concatenated reads
were on average 899,690 bp long; the shortest reads were obtained forN. goodeniana/succincta (i.e. 429,820 bp) and the longest forA. amieti/bicolor (i.e. 1,459,230 bp). In total, seven specimens
did not pass the 90% missing data filter (Supplementary information S1)
and were therefore excluded from downstream analyses. No significant
departure form Hardy-Weinberg equilibrium was observed. The bi-allelic
SNPs screening recovered on average 18,545 bi-allelic SNPs (SD ± 23,430)
across all species complexes (Supplementary information S3).
3.2. Comparison of mitochondrial and nuclear
analyses
3.2.1 Species complex 1: Andrena
allosa/amieti/bicolor/montana
The phylogenetic trees performed on the COI and UCEs datasets provided
very similar topologies for A. bicolor (Figure 1). Both trees
showed distinct monophyletic clades with strong bootstrapping support
(hereafter BS values) for the UCE tree. Only one specimen (i.e.
“GBIFCH00135933”) sampled in Southern France was misplaced in the UCE
tree compared to its position in the corresponding mitochondrial tree.
For A. amieti , all specimens formed one monophyletic clade in the
UCE tree, contrasting with the two paraphyletic clades in the COI tree.
Furthermore, there was no apparent structuring in the UCE tree among
mitochondrial lineages sampled in the alpine region. Specimens with
large amounts of missing data (≥ 60%) exhibited longer branches in the
UCE tree (e.g. “GBIFCH00131686”, “GBIFCH00136250”). Strong isolation
by distance was found between the southern Italian and Alpine
populations (R2 = 0.7221, p-value = 0.039;
Supplemental information S4). These two individuals formed a distinct
monophyletic clade sister to all alpine specimens in the UCE
phylogenetic tree.
The DAPC with a prior knowledge on species assignments correctly
reassigned membership for the majority of specimens (Supplementary
information S5). The plotted cumulative variance of the eigenvalues
suggested to retain the first eight principal components (conserving
70.3% of the total variance; Supplementary information S6). All
specimens were correctly reassigned with a 100% membership probability
for three taxa (i.e. A. allosa , Andrena sp3, A.
montana ). For A. bicolor ML1, only one specimen (i.e.
“GBIFCH00135933” from Southern France) revealed mixed membership
probability, with 45.64% posterior probability (hereafter “pp”) to
belong to ML1 and 54.36% to ML2. For A. bicolor ML2, one
specimen (GBIFCH00117401) also showed mixed membership with a slight
probability (i.e. 3.8%; Supplementary information S5) of belonging toA. bicolor ML1. The mixed membership probability for those two
specimens are congruent with the PCA results where both specimens are
found marginally away from the main A. bicolor ML2 aggregation
(Supplementary information S7).
Between both lineages of A. amieti , genetic cluster assignments
were much less supported and only the two specimens sampled in south
Italy were assigned with a 100% probability to ML1. The lack of a clear
separation between mitochondrial lineages for the alpine specimens is
suggesting considerable levels of admixture.
The AMOVA (Table 1) depicted strong genetic difference (i.e. 43.43%;
p-value ≤ 0.0001) between the two A. bicolor lineages but no
significant difference between the two A. amieti lineages. The
lowest, yet significant fixation index (Table 2) was obtained between
both lineages of A. bicolor (Fst = 0.138). Nei’s genetic distance
between both lineages within A. bicolor (i.e. 0.00231) was
slightly higher that between A. allosa and A. amieti ML1
and ML2 (i.e. 0.00261 and 0.00299, respectively).
The GMYC analysis computed on all specimens identified nine clusters
(Supplementary information S8): two clusters corresponding to the
mitochondrial lineages found within A. bicolor , one cluster with
with A. amieti and A. allosa merged together, and six
clusters composed of only one specimen. The bGMYC analyses identified
the same 9 clusters with a high probability (p = 0.95 - 1; Supplementary
information S8). All other scenarios had very low posterior
probabilities (pp = 0 - 0.05). The two parallel BPP analyses
converged and were highly congruent (Supplementary information S9). Both
runs depicted: (i) one tree model [((A. allosa, A. amietiML1+ML2), ((A. bicolor ML1, A. bicolor ML2), Andrena
sp3 ))] with a posterior probability of ≥ 0.99; (ii) 5 delimited
species (i.e. Andrena sp3 , A. bicolor ML1, A.
bicolor ML2, A. allosa , A. amieti ML1+ML2), all with a
posterior probability of 1; (iii) and a posterior probability of 1 for
having 5 species present in the dataset. Finally, the DAPC analyses
performed without a prior knowledge on species identifications
identified K = 3 and K = 4 as best solution for A. amieti ML1+ML2
and A. bicolor ML1+ML2, respectively. Table 3 summarized the
number of clusters found for each analysis.
3.2.1. Species complex 2: Andrena
barbareae/cineraria
Mitochondrial and nuclear phylogenies were discordant, with no clear
separation between both species in the COI tree and two well-supported
monophyletic clades corresponding to the two morphological species in
the UCE tree (100% BS values; Figure 1). Results from the UCE
phylogenetic tree were corroborated by the PCA in which both species
were clearly separated (Supplementary information S7). The DAPC analyses
correctly reassigned membership for all specimens with 100% probability
(Supplementary information S5). The AMOVA revealed that 52.74% of the
total observed variance could be explained by the species level. The
GMYC and bGMYC provided similar results (Supplementary information S8).
Both analyses grouped all specimens morphologically identified asA. cineraria in one clade. For A. barbareae , both analyses
suggested the presence of three distinct clades. Both BPP runs were
congruent and highly supported the presence of three species (pp = 1;
Supplementary information S9). The runs however disagreed with respect
to the phylogenetic relationships among the three species. The first run
depicted three different possibilities for the species trees, with the
most likely (pp = 1) tree being: [(A. barbareae , (A.
cineraria , A. vaga)) ]. The second run depicted only one tree
[((A. barbareae , A. cineraria) , A. vaga)],corresponding to the expected tree based on the phylogeny. In the first
run this solution was supported at 75%. Congruent with the BPP
analyses, the DAPC identified K = 3 (with outgroup) as the best solution
(Supplementary information S10, Table 3). Clustering of all specimens
corresponded to the morphological identifications.
3.2.2. Species complex 3: Andrena
carantonica/trimmerana/rosae
Mitochondrial phylogenies recovered well-supported (BS 72-100%)
monophyletic clades for A. carantonica and A. rosae , but
not for A. trimmerana , which was composed of two clades forming a
paraphyletic unit from which A. carantonica arose. One clade was
composed of two specimens of A. trimmerana sampled in western
Switzerland and was sister to the A. carantonica clade; support
for this sister relationship was high (BS 93%; Figure 1). In contrast,
all three species appeared as strongly supported monophyletic clades in
the UCE tree (≥ 90% BS; Figure 1). Spring and summer generations ofA. rosae and of A. trimmerana were intermixed in both
mitochondrial and UCE trees, supporting the view that A.
stragulata and A. spinigera constitute the morphologically
differentiated spring generation of A. rosae and A.
trimmerana , respectively. Genetic distance between A.
carantonica and A. trimmerana was relatively low (Nei’s D =
0.00061), although AMOVA and pairwise Fst depicted significant
difference between both species (Table 1-2). The PCA with all three
species showed no difference between A. carantonica and A.
trimmerana , however when removing A. rosae from the analyses,
both species were separated on the first two components (Supplementary
information S7). The GMYC and bGMYC analyses failed to separate bothA. carantonica and A. trimmerana. In the DAPC both species
were also not separated with K = 2 and K = 3 but were with K = 4
(Supplementary information S10). All three clustering scenarios had very
closed BIC values. The BPP analyses however highly supported the
presence of three distinct species, with the following tree topology
[((A. carantonica , A. trimmerana ), A.
rosae )].
3.2.3. Species complex 4: Andrena
dorsata/propinqua
Strong mito-nuclear discordances were observed within this species
complex. In mitochondrial trees, Swiss specimens formed two clusters
corresponding to morphological identifications (Figure 1), but one
specimen of A. propinqua (GBIFCH00133244) collected in southern
France was sister to a well-supported clade containing all other
specimens of A. dorsata and of A. propinqua (Figure 1).
Our sampling also included one specimen of A. dorsata from this
French site (GBIFCH00133243). Phylogenetic trees and PCAs based on UCEs
recovered both species as separated clusters (Figure 1, Supplementary
information S7); the French specimens were not particularly divergent.
Both GMYC and bGMYC analyses, the BPP analyses and DAPC analysis
successfully separated both species (Table 3, Supplementary information
S8-S10). Taken together, these results indicate that A. dorsataand A. propinqua are valid species.
3.2.4. Species complex 5: Lasioglossum
alpigenum/bavaricum/cupromicans
Comparison of mitochondrial and nuclear trees revealed
mitochondrial-nuclear discordance for L. bavaricum and L.
cupromicans (Figure 1). Both taxa were well delimited with highly
supported monophyletic clades (95% bootstrap value) in the UCE tree but
shared the same COI barcode. All L. alpigenum specimens clustered
in a single monophyletic clade sister to both other taxa in both trees.
Beside the GMYC analysis that over-clustered L. bavaricum andL. cupromicans (Supplementary information S8), all other analyses
were congruent (Table 3) and supported the hypothesis of three distinct
species as previously postulated based on morphology.
3.2.5. Species complex 6: Nomada
goodeniana/succincta
COI and UCE trees depicted two well defined monophyletic clades (Figure
1). The bootstrap support for monophyly of N. goodeniana was low
in the mitochondrial trees due to the presence of two slightly divergent
specimens of N. goodeniana collected south of the Alps. In the
nuclear trees, these two specimens clustered with other specimens ofN. goodeniana with high support values. The species delimitation
tests also highly supported the hypothesis of two separated species
(Table 3, Supplementary information S8-S10).