4. Discussion
4.1. Ultraconserved elements successfully delimit species in
all investigated cases
In all six complexes of wild bee species examined here, UCEs provided
robust species hypotheses and clearly outperformed COI for species
delimitation. The main results of our study can be summarized as
follows: First, we provide strong evidence of mitochondrial
introgression in two species pairs (Andrena barbareae andA. cineraria , Lasioglossum cupromicans and L.
bavaricum ): UCEs were in agreement with morphology but not with COI,
which suggests that barcode sharing occurs in these species pairs.
Second, three species complexes presented multiple mitochondrial DNA
barcodes in a single biological species (i.e. Andrena amieti ,A. propinqua , A. trimmerana ); for all three species UCEs
recovered strongly supported monophyletic groups which were in agreement
with morphology, while the two mitochondrial barcodes within each
species formed a paraphyletic assemblage from which another species
arose (A. allosa , A. dorsata and A. carantonica ,
respectively), resulting in the absence of a barcode gap and unresolved
mitochondrial species delimitation. Third, our results suggest that the
two mitochondrial clades observed within Andrena bicolor probably
represent two distinct cryptic species. In addition, UCE-based species
delimitation solved long-standing controversies in the taxonomy of
Central European bees; in particular, the two generations within each ofA. rosae and A. trimmerana , respectively, which do not
appear to represent distinct species; and the distinctiveness of the
other species pairs or triplets investigated here, which is strongly
confirmed by the UCE data.
4.2. DNA barcoding errors
COI-based barcoding is subject to two types of errors. The first error
(similar to type I error) occurs when one biological species is
associated with two distinct DNA barcodes, as observed for A.
amieti , A. propinqua and A. trimmerana . Type I errors
ultimately lead to erroneous detection of two hypothetical species
within a single biological species. Most often, these errors are
triggered by deep within-species divergences or artefacts such as
nuclear insertions (Song, Buhay, Whiting, & Crandall, 2008). The second
error (i.e. type II error) occurs when DNA barcoding fails to recognize
two distinct species because of barcode sharing, as observed between the
pairs Andrena barbareae/cineraria and Lasioglossum
cupromicans /barvaricum .
Identifying the exact biological mechanism behind these barcoding errors
can be tedious, but often they are linked to incomplete lineage sorting,
hybridization followed by introgressions, demographic disparities,Wolbachia infections or sex-biased asymmetries (i.e. male-biased
dispersal, mating behaviour or sex-biased offspring production) (Toews
& Brelsford, 2012). Most often these events occur in recently diverged
species and are not necessarily mutually exclusive (Mutanen et al.,
2016). In this study, the low number of specimens sampled and sequenced
render the investigation on the underlying mechanism difficult. A more
complete sampling across the entire distribution would be necessary to
separate incomplete lineage sorting from the other mechanisms. Indeed,
incomplete lineage sorting is most often not associated with any
biogeographical pattern (Funk & Omland, 2003; Toews & Brelsford,
2012). In contrast, events such as hybridization/introgression often
leave biogeographical footprints because they are unidirectional, which
implies that the gene flow is directed from the native taxon towards the
colonized taxon (Currat, Ruedi, Petit, & Excoffier, 2008; Nevado,
Fazalova, Backeljau, Hanssens, & Verheyen, 2011; Pons et al., 2014).
Therefore, introgression levels are highest at the hybridization zone
and fade away over the colonized distribution zone (Toews & Brelsford,
2012). Further work with a wider geographic coverage would be necessary
to unravel the cases of DNA barcoding errors documented here.
4.3. Could the UCEs have overlooked additional levels of
cryptic diversity?
With regard to the low rate of evolution of UCEs, an important question
in our study and more generally with the use of UCEs for species
delimitation is whether they can successfully uncover variation between
recently diverged species. It could be argued that the cases of
mitochondrial paraphyly (i.e. A. amieti , A. trimmerana andA. propinqua ) in fact represent additional, overlooked instances
of cryptic species, and that the UCEs rate of evolution is too low to
recover these divergences. At least for A. amieti , our sampling
across the entire known distribution of this species enables us to
exclude this scenario. We included specimens from the Alps and from the
Apennines in Southern Italy, some 600 km from the nearest Alpine
population; the Apennine specimens are morphologically slightly
divergent from the Alpine populations (Praz et al. 2019). In the COI
tree (Figure 1), the southern Italian specimens all clustered in one
mitochondrial clade, while the Alpine specimens were distributed over
both mitochondrial clades. The UCEs recovered two strongly supported
clades within A. amieti , one corresponding to the Southern
Italian population and the other including all alpine specimens (Figure
1). This result strongly contradicts the hypothesis of two separated
lineages corresponding to both mitochondrial clades. Rather, UCE results
agree with the strong geographic separation of the Alpine and Apennine
populations and with their slight morphological differentiation.
In the two other cases of mitochondrial paraphyly (i.e. A.
trimmerana and A. propinqua ) investigated in our study, the
presence of additional cryptic species can not completely be rejected.
We however deem this scenario as strongly unlikely since near-cryptic
species in bees are almost exclusively associated with some level of
morphological differentiation in highly variable character such as pile
colour or punctuation (McKendrick et al., 2017; Pauly et al. 2019; Praz
et al. 2019). In our study, such morphological variations were not
observed in the divergent specimens (it was admittedly also not observed
between the two clades within A. bicolor , although more specimens
of both genders and both generations are needed to address this question
thoroughly). In addition, these divergent specimens in mitochondrial
trees where nested within the clades of conspecific specimens in the UCE
trees. We speculate that such high within-species divergences in
mitochondrial barcodes will prove more common than previously expected
once barcoding with continental-scales sampling will be achieved
(Hinojosa et al., 2019; Schmidt et al., 2015).
4.4. Comparison of different species delineation
methods
The method of species delimitation that provided the results most in
agreement with current morphological hypotheses was BPP. By contrast,
results by both (b)GMYC were less congruent with morphological species
hypotheses, and in several cases had the tendency to inflate the number
of species. Compared to BPP, GMYC analyses can overestimation or
underestimations species delimitation rates (Carstens et al., 2013; Luo,
Ling, Ho, & Zhu, 2018), especially in the presence of high
intraspecific variation (Talavera, DincĒ, & Vila, 2013). In our
particular case, specimens with low-quality input DNA yield high levels
of missing values, which led to longer branches in the trees. In most
cases, the GMYC analyses split specimens harboring long branches
singleton species (Supplementary information S8) which ultimately
inflated the overall species number. Therefore, GMYC analyses should be
interpreted with caution when applied on UCE data.
4.5. Concluding remarks on the use of UCEs for species
delimitation
Our results confirm that UCEs can provide sufficient variation at
shallow time scale in insects to enable species discrimination, adding
to previous evidence gathered in vertebrates (Harvey, Smith, Glenn,
Faircloth, & Brumfield, 2016; Zarza et al., 2018). Harvey et al. (2016)
comprehensively compared the utility of sequence capture methods,
specifically using UCEs as in our study, and RAD-Seq for shallow
phylogenies. They found that both techniques resulted in similar
phylogenetic hypotheses and branch support values; and that RAD-seq
provided more overall information while sequence-capture provided higher
per-locus-information. They also suggested that the high amount of
information typical of RAD-seq was not necessarily an advantage when the
inherent question was phylogeography, phylogeny or species delimitation.
Harvey et al. (2016) concluded that sequence capture is more useful in
systematics because of its repeatability, the possibility to use
low-quality samples, the ease in read orthology assessment, and the
higher per-locus information.
Our results build upon this early work and largely confirm these
predictions. RAD-seq datasets would have been nearly impossible to
gather for the species investigated here due to low DNA quality or
quantity. The possibility of processing specimens belonging to three
different families simultaneously, and to iteratively assemble datasets,
represent particularly promising advantages of UCE capture methods for
species delimitation. Future work should focus on very recently diverged
taxa to further determine the level of divergences that can be recovered
with these conserved markers. In addition, whether UCEs will enable the
detection of hybrids, and to what extend the presence of these hybrids
impact the tree reconstruction or the species delimitation analyses
should be investigated. Lastly, our analyses strongly suggest the
presence of two cryptic species within one of the most common European
bee, Andrena bicolor . Enlarging our dataset to the entire
geographical range of A. bicolor will be necessary to further
untangle this remarkable case of cryptic species in bees.