DISCUSSION
The number of DNA barcodes for oak-feeding Lepidoptera is lower in
southern Europe, despite the higher species richness. As expected, the
effect of the geographical scale on the genetic divergence depended on
the latitude. In pairwise sequence comparisons, for any given spatial
distance the genetic divergence was higher when at least one of the
sequences came from one of the southern European peninsulas included in
the study (Italy and Iberia). This made identification of some southern
query sequences problematic, as the genetic distance with respect to the
reference barcodes in BOLD was above the maximum intra-specific
threshold allowed. The effect of the latitude is due to the presence of
southern haplotypes with a reduced geographical distribution.
Accordingly, the COI gene-tree showed in different species monophyletic
clades exclusive from southern Europe (mainly Iberian). The GMYC single
threshold model classified some of those clusters as different OTUs, and
thus potentially cryptic species.
The lower availability of DNA barcodes in southern Europe cannot be
explained by any factor but the regional scarcity of DNA barcoding
initiatives. Before we started the project, only 26.36% of the DNA
barcodes available for the study species in BOLD came from the southern
European Peninsulas (Iberia, Italy and the Balkans). The Iberian
Peninsula was underrepresented according to its area and to its species
richness. In fact, for some species (Archips xylosteana ,Bena bicolorana , Dryobotodes eremita , Dryobotodes
monochroma , Ennomos quercaria , Eupithecia cocciferata ,Malacosoma neustria , Nycteola columbana , Orthosia
cruda , Tortricodes alternella and Tortrix viridana ) no
Iberian sample had ever been sequenced before the present study (January
2018) and others, restricted to the southwestern Mediterranean Basin or
Iberia (Dryobota labecula , Phycita torrenti ), were
sequenced for the first time. The function relating latitude and number
of barcodes peaked at 48 degrees north, because that is the latitude
around which the largest European barcoding initiative has been carried
out in Germany (Gemeinholzer et al . 2011).
Previous studies have shown that the larger the sampling scale the
greater the intra-specific divergence and the more likely finding
overlapping closely related taxa (Bergsten et al. 2012). In this study,
we did not analyze the effects on the barcoding gap, because the closest
relatives of the study species often feed on other host plants (Cates
1981; Thompson & Pellmyr 1991). Rather, we focused on intra-specific
genetic divergence but considering not only the effect of the spatial
distance alone, but its interaction with the latitude. Doing so we found
that, intra-specific genetic divergence, was higher in pairwise
comparisons that included at least one DNA sequence from a southern
peninsula than when both came from elsewhere in the continent.
The peninsulas of southern Europe are hotspots of species and genetic
diversity (Murienne & Giribert 2009; Pinto et al . 2012; Geigeret al . 2014), thus, when they are undersampled, intra-specific
genetic divergence is underestimated more than expected by the mere
reduction of the geographical scale. The low availability of DNA
barcodes or their reduced geographic distribution is a main concern in
DNA barcoding (Savolainen et al . 2005; Bergsten et al .
2012; Geiger et al . 2014; Dincă et al . 2015). Our results
show that, to capture as much intra-specifc genetic variability as
possible, sequencing efforts should be concentrated in southern European
genetic diversity hotspots (Murienne & Giribert 2009; Pinto et
al . 2012; Dincă et al . 2015) where, paradoxically, the number of
available DNA barcodes is lower.
The disproportionately strong effect of the Iberian samples is largely
related with the distribution patterns of genetic diversity determined
by Pleistocene glaciations (Hewitt 1996; Schmitt 2007). The southern
European Peninsulas were refugia that hosted a large number of plant and
animal taxa when the ice sheet covered large extensions of the
continent. This was the case of our study group (insects associated with
oaks and other species of broad-leaved trees). In the Iberian Peninsula,
where a greater geographic isolation is observed than, for example, in
the Italian peninsula, deciduous and evergreen oak forests were
restricted to a few refugia close to the coast or at the south-facing
slopes of some mountainous systems (Koster 2005; Magri et al .
2007). When the ice retreated, not all haplotypes spread northwards but
just some of them. This “founder effect” is responsible for the higher
species richness in the south and the genetic homogeneity of the
recently colonized areas in the central and northern parts of the
continent (Hewitt 1996; Taberlet, Fumagalli, Wurst-Saucy & Cosson 1998;
Hewitt 1999). Taking our study species as an example, there are many
examples of haplotypes shared by different central and northern European
countries, especially between Germany, the Netherlands, the United
Kingdom, Czech Republic, Austria or Finland. Similarly, a noteworthy
study including hundreds of Lepidoptera species showed little
intra-specific genetic variability between central (Austria) and
northern (Finland) in Europe (Huemer et al. 2014).
Due to the historical factors linked to the paleoclimate of the
continent, in 56 % of our study species we found monophyletic Iberian
clades. This was not the case for the Italian peninsula, which shared a
higher genetic similarity with the territories northwards, suggesting a
stronger effect of the Pyrenees as geographical barrier than that of the
Alps. Previous studies have reported similar results for organisms like
butterflies or freshwater fish (Geiger et al . 2014; Dincăet al . 2015). Some alpine butterflies, for example, show higher
intra-specific genetic distance among the populations in the Alps and
the nearby Pyrenees than among the Alps and Scandinavia (Dincă et
al . 2015). However, it is well known that the Alps have a deep impact
on the genetic variation of other groups of animals (Arntzen 2001;
Cornetti et al . 2015; Leys, Keller, Räsänen, Gattolliat &
Robinson 2016).
Four Iberian clades were retrieved as different OTUs by the GMYC single
threshold model, which confirmed its utility for species delimitation
within poorly inventoried biogeographic regions in Europe. According to
Fujisawa and Barraclough (2013), GMYC single threshold model is more
reliable than the multiple thresholds one, which overestimates the
number of OTUs. Moreover, ABGD and jMOTU retrieved identical or very
similar results to the single threshold GMYC, while the latter was
considered the most reliable of the three in a comparison of
performances (Ratnasingham & Herbert 2013).
The presence of different putative species in southern Europe conditions
the efficacy of species identification on the basis of DNA barcoding
(Derkarabetian & Hedin 2014; Geiger et al . 2014; Dincă et
al . 2015; Fossen, Ekrem, Nilsson & Bergsten 2016). In the hypothetical
case that there had not been any Iberian barcode in BOLD, in 7 out of 15
species (possible comparisons between Iberia and Europe, Table 2), there
would have been at least one haplotype that would have not been
determined to the species level (EUIB K2P distance above 1%). Even
including the Iberian barcodes available in BOLD before the present
study, the same still happened in 3 species. The case ofTortricoides alternella is specially remarkable: from 16 new
haplotypes recorded in this study, none of them could not be matched to
any reference sequence in BOLD using the 1% threshold (Table S4). This
lack of identification due to the absence of Iberian reference sequences
is not exclusive of the study species, having been reported for other
insect taxa as well (e. g. Cerambyx cerdo , Coleoptera)
(Torres-Vila & Bonal 2019).
If well the present data set shows a clear trend, we have to be cautious
before generalizing, as it is restricted to a limited number of species
of oak-feeding moths. The occurrence of putative cryptic species in
southern Europe in other taxa (Geiger et al. 2014, Dincă et al .
2015) suggests that the pattern may be widespread, but further studies
are needed to confirm it. Future large-scale DNA barcoding initiatives
in Europe should cover latitudinal gradients, rather than large
distances at the same latitude, to avoid neglecting genetic diversity
hotspots like the Iberian Peninsula.