Integrative Taxonomy Reveals a New Melitaea (Lepidoptera: Nymphalidae) Species Widely Distributed in the Iberian Peninsula

Abstract

The Melitaea phoebe group is constituted by six species distributed throughout the Palearctic. One of the most widespread species is Melitaea ornata Christoph, 1893, present from France (Provence) to Central Asia. Recently, populations of M. ornata were discovered in a mountainous region of southeastern Iberia, although doubts about their taxonomy existed. To clarify the taxonomic status of these populations and to revise the distribution of this taxon in Iberia, we have sequenced mitochondrial (COI barcode region) and nuclear (wg, RPS5, MDH, and EF-1α) markers, and analyzed the male genitalia for 72 Iberian individuals and for all the species of the M. phoebe group. This information was complemented with phenological and ecological data. Our results unveiled that the Iberian M. ornata-like taxon is in fact distributed through most of the Iberian Peninsula, except for the southwest and northeast. In contrast to the univoltine M. ornata, the Iberian taxon can be bivoltine in the wild. The Iberian taxon was retrieved to be related to M. ornata, but the differences in the genetic markers and genitalia were comparable to those found between species in the group. Based on the evidence here presented and according to species delimitation results, we propose to consider the Iberian taxon as a novel species , tentatively named Melitaea pseudornata Muñoz Sariot & Sánchez Mesa, 2019, stat. nov.

Full text

1
Taxonomy
Integrative Taxonomy Reveals a New Melitaea
(Lepidoptera: Nymphalidae) Species Widely Distributed in
the Iberian Peninsula
JoanC.Hinojosa,1 JánosP.Tóth,2 Yeray Monasterio,3 LuisSánchez Mesa,4
MiguelG.MuñozSariot,5 RuthEscobés,3 and RogerVila1,6,
1Institut de Biologia Evolutiva (CSIC-UPF), Passeig Marítim de la Barceloneta 37-49, 08003, Barcelona, Spain, 2H-2373 Dabas, Hungary,
3Asociación Española para la Protección de las Mariposas y su Medio (ZERYNTHIA), Calle Madre de Dios 14-7D, 26004, Logroño,
Spain, 4Calle Santa Clara 8-4C, 18007, Granada, Spain, 5Avenida Paraíso 6, 1823, Atarfe (Granada), Spain, and 6Corresponding author,
e-mail: roger.vila@csic.es
Subject Editor: MarkoMutanen
Received 26 October 2021; Editorial decision 7 January 2022
Abstract
The Melitaea phoebe group is constituted by six species distributed throughout the Palearctic. One of the
most widespread species is Melitaea ornata Christoph, 1893, present from France (Provence) to Central Asia.
Recently, populations of M.ornata were discovered in a mountainous region of south-eastern Iberia, although
doubts about their taxonomy existed. To clarify the taxonomic status of these populations and to revise the dis-
tribution of this taxon in Iberia, we have sequenced mitochondrial (COI barcode region) and nuclear (wg, RPS5,
MDH, and EF-1α) markers, and analyzed the male genitalia for 72 Iberian individuals and for all the species of
the M.phoebe group. This information was complemented with phenological and ecological data. Our results
unveiled that the Iberian M.ornata-like taxon is in fact distributed through most of the Iberian Peninsula, except
for the south-west and north-east. In contrast to the univoltine M.ornata, the Iberian taxon can be bivoltine in
the wild. The Iberian taxon was retrieved to be related to M.ornata, but the differences in the genetic markers
and genitalia were comparable to those found between species in the group. Based on the evidence here pre-
sented and according to species delimitation results, we propose to consider the Iberian taxon as a novel spe-
cies, tentatively named Melitaea pseudornata Muñoz Sariot & Sánchez Mesa, 2019, stat. nov.
Resum
El grup Melitaea phoebe està format per sis espècies distribuïdes arreu del Paleàrtic. Una de les espècies més
esteses és la Melitaea ornata Christoph, 1893, present des de França (Provença) fins a l’Àsia Central. Recentment,
es descobriren poblacions de M.ornata en una regió muntanyosa del sud-est de la península Ibèrica, tot i que
existien dubtes sobre la seva taxonomia. Amb l’objectiu d’esclarir l’estatus taxonòmic d’aquestes poblacions i
revisar la distribució d’aquest tàxon a la península Ibèrica, hem seqüenciat els marcadors mitocondrials (regió
del codi de barres del COI) i nuclears (wg, RPS5, MDH i EF-1α) i hem analitzat la genitàlia masculina de 72
individus ibèrics i de totes les espècies del grup de M.phoebe. Aquesta informació s’ha complementat amb
dades fenològiques i ecològiques. Els nostres resultats revelaren que, de fet, el tàxon ibèric de tipus M.ornata
es distribueix per bona part de la península Ibèrica llevat del sud-oest i el nord-est. Adiferència de la M.ornata,
que és univoltina, el tàxon ibèric pot ser bivoltí a la natura. El tàxon ibèric està relacionat amb M.ornata, però
les diferències en els marcadors genètics i la genitàlia foren comparables a les trobades entre les diferents
espècies del grup. Degut a aquestes diferències i segons els resultats de la delimitació d’espècies, proposem
considerar el tàxon ibèric com a una espècie nova, provisionalment anomenada Melitaea pseudornata Muñoz
Sariot & Sánchez Mesa, 2019, stat. nov.
Key words: phylogeography, integrative taxonomy, hybridization, phylogenetics, butterfly
© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America.
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Insect Systematics and Diversity, (2022) 6(2): 1; 1–9
https://doi.org/10.1093/isd/ixac004
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2Insect Systematics and Diversity, 2022, Vol. 6, No. 2
The information available about the taxonomy (Wiemers et al.
2018), evolution (Dapporto etal. 2019, Wiemers etal. 2020, Dincă
etal. 2021), distribution (Kudrna etal. 2015) and ecology (Settele
etal. 2009) of the European butteries is massive even at local and
regional scale, which highlights them as probably the best-known
insect group in Europe. Nevertheless, the emergence of genetic
techniques revealed that this knowledge was not as complete as
believed due to the existence of cryptic diversity: taxa that have re-
mained unnoticed due to the morphological similarity with other
species. Indeed, novel buttery species are still being documented in
Europe (e.g., Vodolazhsky and Stradomsky, 2008, Dincă etal. 2011,
Hernández-Roldán etal. 2016, Hinojosa etal. 2021). This evidences
the existence of potential gaps in the taxonomy of some groups,
which brings uncertainty in actions linked to conservation and may
hamper a proper understanding of these organisms and their evolu-
tion (e.g., Gill et al. 2016, Sales et al. 2018). Thus, further studies
addressing the presence of hidden diversity are required, especially
in the groups with most debated taxonomy.
The buttery genus Melitaea Fabricius, 1807 comprises about
a hundred species distributed in the Palearctic (van Oorschot and
Coutsis, 2014). Its taxonomy and systematics have been tradition-
ally problematic since it comprises species characterized by being
polymorphic and by having similar adult and larval morphology.
In consequence, this genus is particularly affected by descriptions
of redundant species and subspecies, as well as by cases of long-
overlooked taxa. This scenario is especially true for the Melitaea
phoebe species group, which was recently reviewed using DNA and
morphological data (Tóth etal. 2014, 2017) and divided in six spe-
cies: M. abbas Gross & Ebert, 1975, M. ornata Christoph, 1893, M.
phoebe ([Denis & Schiffermüller], 1775), M. punica Oberthür, 1876,
M. scotosia Butler, 1878, and M. telona Fruhstorfer, 1908.
One of the most confusing species of the M.phoebe species group
is M. ornata. It was originally described as Melitaea phoebe var.
ornata by Christoph (1893) from an individual collected in Guberlya
(Orenburg Oblast, Russia). Its similarity with M.phoebeled to the
description of a large list of M.phoebe subspecies that corresponded,
in fact, to the taxon ornata. It was elevated to the species rank in
parallel and with two distinct names, Melitaea emipunica (Russell
et al. 2005) and Melitaea ogygia (Varga et al. 2005), which were
nally synonymized (Tóth and Varga 2011, Russell and Tennent
2016) to M.ornata. The most constant feature in M.ornata is the
head color of the 4th and later larval instars, which is typically brick
red (Russell etal. 2007, Russell and Tennent 2016) instead of black,
as in M.phoebe. The rst molecular analysis comparing both taxa
(Tóth etal. 2014) conrmed that M.phoebe and M.ornata are dis-
tinct species: they were monophyletic and showed considerable di-
vergence in nuclear DNA (nuDNA), albeit some populations share
mitochondrial DNA (mtDNA) haplotypes with M. phoebe (Tóth
etal. 2017).
M.ornata is known to be present from Kazakhstan, the Middle
East, and eastern Europe to the Italian Peninsula, Sicily (Russel
etal. 2007, Tóth et al. 2013, Tóth et al. 2017) and the Provence
(Lafranchis etal. 2015). Based on larval morphology, this species
was recently reported for the rst time in the Iberian Peninsula,
restricted to mountain areas of the south-east (Sánchez Mesa and
Muñoz Sariot, 2017, Muñoz Sariot and Sánchez Mesa, 2019a).
The same authors named these populations as the subspecies M.
ornata baetica (a name that was preoccupied) and, later, M. ornata
pseudornata, but they also suggested that it could be related to the
north African M. punica due to similarities in the caterpillars or
that it could even be a distinct species. Hence, in order to clarify
the placement of the Iberian M.ornata-like taxon in the frame of
the M.phoebe species group and to document its distribution and
ecology, we have sequenced nuDNA and mtDNA markers, per-
formed a geometric morphometry analysis of the male genitalia,
and gathered phenological, hostplant and parasitoid data. The re-
sults allowed to launch a taxonomic hypothesis grounded on mul-
tiple evidence: the Iberian taxon is considered as a distinct species,
which we tentatively name Melitaea pseudornata Muñoz Sariot &
Sánchez Mesa, 2019 stat.nov.
Material andMethods
DNA Extraction and Sequencing
The bodies of the specimens used in the DNA analyses were stored in
99% ethanol at –20°C and wings were kept separately as vouchers.
The DNA analyses were conducted employing sequences retrieved
from 191 individuals, including all the species of the M. phoebe
group. Sampling sites in the Iberian Peninsula are plotted in Fig. 1;
here, the specimens were identied using the wg phylogeny (Supp
Fig. S1 [online only]). In total, we used 160 COI and 180 wg, 111
RPS5, 87 MDH, and 90 EF-1α (Supp Table S1 [online only]).
Total genomic DNA was extracted using Chelex 100 resin, 100–
200 mesh, sodium form (Biorad), under the following protocol: one
leg was removed and introduced into 100μl of Chelex 10% and 5μl
of Proteinase K (20mg/ml) were added. The samples were incubated
overnight at 55°C in a shaker and were subsequently incubated at
100°C for 15 minutes.
Primers and PCR protocols used for the amplication of COI
(barcode region), wg, EF-1ɑ (three fragments), RPS5, and MDH
are written in Supp Tables S2 and S3 (online only). Universal tails
were included in all primers. PCR products were puried and Sanger
sequenced by Macrogen Inc. Europe (Amsterdam, the Netherlands).
All sequences have been deposited in GenBank (Supp Table S1 [on-
line only]).
Phylogenetic Reconstruction
Sequences were visualized, edited, and aligned in Geneious Prime
2019.0.3 (https://www.geneious.com). Ahaplotype network of the
COI barcode region was created in POPART v1.7 (Leigh and Bryant
2015) under the TCS method. The best-tting substitution model
was estimated in jModelTest (Darriba etal. 2012) under the Akaike
information criterion.
The alignments of the nuclear genes were concatenated and a
phylogeny was reconstructed using BEAST v2.5.0 (Bouckaert etal.
2014). Distinct partitions were selected for each gene using the best
model indicated by jModelTest and four rate categories if included
gamma and base frequencies were estimated. Parameters were esti-
mated using two independent runs of 30 million generations each
and convergence was checked with TRACER 1.7.1 (Rambaut 2018).
Aburn-in of 10% was applied.
A maximum likelihood (ML) inference was obtained with the
concatenated (partitioned) alignment of nuclear loci using RAxML
v8.2.11 (Stamatakis 2014). RAxML was also used to retrieve a
phylogeny for every individual nuclear gene, which was employed in
ASTRAL. Athorough bootstrapping was employed and we selected
a GTRGAMMA model and 1,000 bootstrap replicates.
Species Delimitation
Joint Bayesian species delimitation was conducted using the pro-
gram BPP (Yang 2015). The method uses the multispecies coalescent
model to compare different models of species delimitation (Yang and
Rannala 2010, Rannala and Yang 2013) in a Bayesian framework,
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3Insect Systematics and Diversity, 2022, Vol. 6, No. 2
accounting for incomplete lineage sorting due to ancestral poly-
morphism and gene tree-species tree discordance. Considering that,
in butteries, the median of the theta is 0.0160 (ranging from 0.004
to 0.043) mutations/site (Mackintosh etal. 2019), a sensible diffuse
theta prior would be IG(3,0.045).
Assuming neutrality, the mutation rate for butteries has been
estimated to be about 2.9×10–9 mutations/site/year (Keightley etal.
2015). Adivergence time between 3.3–9.6 Mya (95% credibility
interval) has been estimated for the M.phoebe species group (Tóth
etal. 2017). By multiplying these values, we considered a divergence
between the root of the species tree and the present time (tau) be-
tween 0.0096 and 0.0278 mutations/site and we assigned a diffuse
tau prior of IG (3,0.03).
The other divergence time parameters are specied by the uni-
form Dirichlet distribution (Yang and Rannala 2010: equation 2).
We selected 500,000 MCMC and 50,000 burn-in and the analysis
was run twice to conrm consistency between runs.
Genitalia Morphometry
Geometric morphometry on the processus posterior (male geni-
talia) was applied to determine the morphological relationships
between M.pseudornata and the other species of the M. phoebe
species group. In total, 315 specimens have been measured. These
individuals are partly identical with the material used in Tóth and
Varga (2011), but it has been completed with 12 specimens from
Iberia that were identied based on nuclear DNA data (Supp Table
S1 [online only]).
A standard genital preparation method was followed. The abdo-
mens were removed and heated in 15% KOH solution in 80°C for
30min. Next, genitalia were cleaned and dehydrated in ethanol and
mounted in euparal. Genitalia slides were digitalized using a stereo-
microscope and a digitalcamera.
TpsDig2 was used to record nine xed landmarks at the tips and
origin of the main processus (Tóth and Varga 2011). The raw coord-
inates were transformed using Procrustes generalized least squares
using geomorph R package (Adams etal. 2021).
All further analysis was performed based on the transformed
coordinates. We used linear discriminant analysis (LDA) to deter-
mine the morphological relationships between the studied taxa
using MASS R package (Venables and Ripley 2002). Leave-one-out
cross-validation classication was also used to quantify the classi-
cation success. The signicance of the visible pattern was analyzed
by pairwise permutational MANOVA using Bonferroni-corrected
signicance levels using RVAideMemoire R package (Hervé, 2021).
Average landmark coordinates of the processus posterior were calcu-
lated for all the studied species, then a PCA was performed on these
mean shapes.
Flight Time Data (Navarre, NorthernSpain)
Adult records were retrieved from the data of two transects located
at Taxoare (Aranguren, Navarre). In this locality, only orange-
headed caterpillars were found, and DNA results classied all the
sequenced specimens as M.pseudornata. Adults were counted fol-
lowing a standardized methodology that consists of sampling every
two weeks, from the rst week of April to the last week of September,
along a walked transect at distances of 2.5 m on both the sides and
5 m ahead of the recorder (Pollard and Yates 1993).
Nomenclature
This paper has been registered in Zoobank (www.zoobank.org), the
ofcial register of the International Commission on Zoological
Nomenclature. The LSID (Life Science Identier) number of the
publication is: urn:lsid:zoobank.org:pub:D1410808-7450-4190-
BA1A-C57BA477AD46
Fig. 1. Iberian sampling sites of Melitaea pseudornata stat. nov. and Melitaea phoebe. The relationship between flight time and altitude is depicted. All
identifications are based, at least, on the sequencing of the wg gene.
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4Insect Systematics and Diversity, 2022, Vol. 6, No. 2
Results
Phylogenetic Inference and Species Delimitation
The haplotype network based on the barcode region of the COI (Fig.
2A,B) showed that M.ornata and M. pseudornata shared barcode
haplogroups with M. phoebe individuals. However, M. ornata
from the Balkan Peninsula and eastwards maintained very distinct
barcodes. Some individuals of M.pseudornata displayed exclusive
haplotypes within Haplogroup 2, although separated from those of
M.phoebe by only one or two substitutions.
Nuclear markers retrieved all the taxa as monophyletic with a
posterior probability (PP) of 1 (Figs 2B, Supp Fig. S2 [online only])
and a bootstrap value (BS) > 70, except for M.phoebe in the ML
inference, which had a lower support (Supp Fig. S3 [online only]).
Melitaea pseudornata was recovered sister to M. ornata in the
Bayesian and ML inferences. Interestingly, the Bayesian phylogeny
retrieved two clades within M. phoebe: a clade formed by all the
Iberian specimens and a French individual (PP=0.82), and a clade
with the rest of individuals (PP=0.77).
Both BPP runs selected the seven-species hypothesis as the most
likely (PP=1). All the taxa of the M.phoebe species group and the
Iberian taxon were separated as distinct species.
Genitalia
Despite interindividual variability was present in this group
of taxa, the processus posterior of M. pseudornata showed on
average a unique shape, distinct to M. ornata and M. phoebe
(Fig. 3B–D). The average shape of the processus posterior was
very similar to M.ornata as the inner process have the similar
length. On the other hand, it showed some similarities to M.abbas
and M.punica as the outer process pointed upward. On the LDA
scatterplots, the centroid of M. pseudornata was positioned at
a distance from all the other groups but close to M.ornata and
M.phoebe considering the rst three axes, which explain 74.81
% of the variance between groups (Fig. 3). The rst axis ex-
plained 38.17%, the second 20.53%, and the third 16.11% of
variance between groups. Leave-one-out cross-validation classi-
cation assigned 77.78% of the individuals correctly in the case of
M.pseudornata (Supp Table S4 [online only]). This success was
very similar to the overall precision of the classication in the
group, which was78.34%.
The pairwise permutational MANOVA indicated statistically
signicant differences (P < 0.05 in all cases) in the processus pos-
terior shapes among the taxa analyzed (Supp Table S5 [online only]).
Fig. 2. A. Haplotype network of the Melitaea phoebe species group based on the barcode fragment of the COI gene. The area of the circles is proportional to
the number of sequences they represent. B.European distribution of the three haplogroups of Melitaea pseudornata stat. nov., M.phoebe and Melitaea ornata.
C.Bayesian inference phylogeny obtained using a partitioned alignment of the nuclear genes EF-1ɑ, MDH, wg and RPS5. Posterior probabilities at species-level
and for higher relationships are indicated, and scale units are presented in substitutions per site.
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5Insect Systematics and Diversity, 2022, Vol. 6, No. 2
M. pseudornata showed a similar morphological differentiation
compared to the closely related species (Supp Table S6 [online only]).
Notes About Morphology and Ecology of
M.pseudornata
Larval Morphology. Black with white dots and black head until the
last stage (L7), when it turns orange; nevertheless, locally some in-
dividuals retain the black head in the last stage (Sánchez Mesa and
Muñoz Sariot 2017). In south-eastern Iberia (Baetic System), caterpil-
lars have orange scoli (Sánchez Mesa and Muñoz Sariot 2017, Muñoz
Sariot and Sánchez Mesa 2019a). In northern Iberia, larvae have black
scoli and present an orange lateral stripe, similar to the Iberian speci-
mens of M.phoebe (Fig. 4), although sometimes very diffuse.
Adult Morphology. Wings were very variable and similar to
M.phoebe (Supp Fig. S4 [online only]); after a visual inspection of the
wing traits partially diagnostic between M.ornata and M.phoebe—
mentioned in Russell and Tennent (2016)—no clear differential
patterns were found between M. pseudornata and M. phoebe.
Regarding the tip of the antennae, stubbier shapes are more frequent
in M. pseudornata compared to M. phoebe, although it does not
seem a fully diagnostic trait (Sánchez Mesa and Muñoz Sariot 2017).
Pictures of the wings of individuals used in this study have been de-
posited in gshare (DOI: 10.6084/m9.gshare0.16832830)
FlightTime. In the Baetic System, only one generation was recorded
(Sánchez Mesa and Muñoz Sariot 2017). Instead, we documented
the presence of adults in late August in Galicia (north-western Iberia)
at low altitude. In Navarre (north-central Iberia), in a locality where
only M.pseudornata has been recorded, biweekly adult counts con-
ducted for three consecutive years showed a consistent bi-modal
shape, with peaks at end of May/beginning of June, and at end of
July/beginning of August, consistent with the existence of two gen-
erations (Supp Fig. S5 [online only]). Thus, this taxon seems to be
uni- or bivoltine depending on the locality, which is possibly related
to the desiccation of the host plant during summer or, in high alti-
tude areas, to a shorter summer period.
Fig. 3. A. Landmarks on the processus posterior of a Melitaea phoebe male genitalia. B.Typical processus posterior shapes of Melitaea pseudornata stat. nov.,
M.phoebe and Melitaea ornata. C.PCA built using the mean shapes of all the studied species. Species mean shapes are shown. D.Linear discriminant analysis
(LDA) scatterplot for the studied taxa. Group centroids are shown. All the groups had equal prior probability independently from the sample size of the group.
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6Insect Systematics and Diversity, 2022, Vol. 6, No. 2
Habitat. Similar to M. phoebe, but typically inhabiting mid-
mountain biotopes, between 500 m and 1500 m (Fig. 1). Present
at sea-level in Galicia. It occupies the Atlantic and Mediterranean
biogeographic regions.
Host Plants. Baetic System: eggs and/or L1 larvae on Carduus
platypus subsp. granatensis (Willk.) Nyman (Asteraceae),
Carduncellus hispanicus Boiss. ex DC. (Asteraceae), Cirsium arvense
(L.) Scop. (Asteraceae), Cirsium vulgare (Savi) Ten. (Asteraceae),
Cirsium pyrenaicum (Jacq.) All. (Asteraceae), Cirsium acaulon subsp.
gregarium (Boiss. ex DC.) Talavera (Asteraceae); caterpillars in the
last instar were found in the previously cited host plants and on
Onopordum acanthium L. (Asteraceae) and Onopordum illyricum
L. (Asteraceae). Navarre: L1 caterpillars on Centaurea jacea subsp.
angustifolia (DC.) Gremli (Asteraceae).
Parasitoids. Baetic System: Cotesia melitaearum (Wilkinson,
1937) (Hymenoptera: Braconidae). Navarre: Dolichogenidea
sp. Viereck, 1911 (Hymenoptera: Braconidae), which is a novel
parasitic relationship in the genus Melitaea, and Cynipoidea
(Hymenoptera).
Discussion
The Iberian Taxon, a NewSpecies
The results here presented suggest that the Iberian taxon should
be elevated to the species status. First, molecular evidence (nuclear
markers) retrieved the Iberian individuals as a monophyletic clade,
well-diverged from M.ornata, although sister to it (Figs. 2C, Supp
Figs. S2–S3 [online only]); species delimitation analyses supported
the specic status for this clade. Second, differences in the genitalia
between the Iberian taxon and M.ornata were comparable to those
found interspecically among other species of the group (Fig. 3B–D;
Supp Tables S4–S6 [online only]). Third, their phenology is distinct
since M. ornata has apparently only one generation each year—
second generations have been obtained only in captivity (Russell and
Pateman 2013, Russell et al. 2014)—while the Iberian taxon has
two generations in a signicant part of the distribution range. Worth
mentioning, there is no evidence of a close relationship of the Iberian
taxon with the northAfrican M.punica, a hypothesis that was sug-
gested due to similarities present in the caterpillars (Sánchez Mesa
and Muñoz Sariot 2017, Muñoz Sariot and Sánchez Mesa 2019a).
The denomination of this novel species, however, is not straight-
forward. Russell et al. (2020) attributed several taxa to the Iberian
Fig. 4. Pictures of the northern and southern forms of Melitaea pseudornata stat. nov. and of Melitaea phoebe. An approximate distribution of M.pseudornata
based on the sequenced specimens is given; populations in eastern and southern Iberia are apparently more scattered and restricted to mountain ranges.
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7Insect Systematics and Diversity, 2022, Vol. 6, No. 2
M.ornata-like taxon. They based their proposals on the external morph-
ology of the adults, but in these traits the Iberian taxon cannot be reli-
ably differentiated from M.phoebe. Several of our specimens showed
typical ornata-like characteristics but, based on nuDNA, they proved to
be M.phoebe, and vice versa. The thickness of the tip of the antennae
seems not to be a dening trait either because thick tips are found in
both taxa (see Sánchez Mesa and Muñoz Sariot 2017, Muñoz Sariot
and Sánchez Mesa 2019a), although thicker shapes are more common
in M.pseudornata than in the Iberian M.phoebe. Overall, the evidence
available points that this taxon can only be reliably distinguished by
nuDNA data and by the reddish head of the last (L7) instar caterpil-
lars (Sánchez Mesa and Muñoz Sariot 2017)—distinct to M.phoebe,
with invariably black head, and to M.ornata, with reddish head from
L4 to the last instar (Russell and Tennent 2016). Given the absence of
these data in the taxonomic proposals made by Russell et al. (2020),
we think that further analyses of the type specimens are required in
order to conrm the identications. In consequence, here we used the
name of the rst taxon whose identication was based on the color of
the head of the caterpillars, which is pseudornata (Muñoz Sariot and
Sánchez Mesa 2019a, b). Thus, we tentatively name the novel species as
M.pseudornata Muñoz Sariot & Sánchez Mesa, 2019, stat. nov.
The Distribution Range of M.pseudornata
Identications based on the wg gene (Supp Fig. S1 [online only])
conrmed the presence of M.pseudornata across most of the Iberian
Peninsula, apparently restricted to areas of oceanic inuence and/or
mountain ranges. So far, it has been only found in Spain. This distri-
bution is similar to the prediction made by Tóth etal. (2013, 2017),
who showed through ecological niche modelling analyses that vir-
tually all the Iberian Peninsula represents a climatically suitable
habitat for the sibling species M.ornata. Although further explor-
ation is required, M.pseudornata was the sole species found in a vast
area of north-western Spain. In contrast, only M.phoebe was found
in the south-west of the Iberian Peninsula (although sampling in this
region is low) and in Catalonia (except in the southern mountains
of Els Ports, where it is replaced by M. pseudornata). Both species
are locally sympatric in some mountain ranges in the Baetic System
(south-eastern Iberia), but this seems not to be usual and the pattern
documented agrees with a situation of parapatry, in which contact
zones may reect some kind of competition or incompatibility.
The ranges of the species pair M.pseudornata and M.ornata match
with a distribution pattern typically produced by glacial cycles, even if
initial divergence predates them (Ebdon et al. 2021). Glacial periods
caused the isolation of populations in the southern peninsulas, which
promoted allopatric differentiation and, sometimes, speciation (Hewitt,
2000). In the Iberian Peninsula, there are about twenty buttery species
that have a sibling widespread through Europe (Dincă et al. 2015).
Many of them establish contact zones around the Pyrenees—typically
in the Ebro River valley, the Pyrenees themselves, or in S.France—such
as the pairs Iphiclides feisthamelii (Duponchel, 1832) (Papilionidae)–
Iphiclides podalirius (Linnaeus, 1758) (Gaunet etal. 2019)or Aricia
cramera (Eschscholtz, 1821) (Lycaenidae)–Aricia agestis ([Denis &
Schiffermüller], 1775) (Vodă et al. 2015). In our case, the existence
of a contact zone cannot be determined since it is unknown whether
M. pseudornata is present in France; meanwhile, the closest area
where M. ornata has been reported is Provence. Hence, as far as we
know, M.pseudornata is allopatric with respect to M.ornata.
M.pseudornata and M.phoebe, Two
InteractingSpecies
Mitochondrial DNA (Figs. 2A,B) showed that M. pseudornata
shared two haplogroups (here, groups of haplotypes linked by two
or fewer mutations) with M.phoebe. One of the shared haplogroups,
Haplogroup 1, is exclusive to Iberia. The second shared haplogroup,
Haplogroup 2, was also found in M. phoebe from all Europe
(including Iberia) and in M. ornata. Considering that M. ornata
conserves a well diverged COI lineage in the Balkan Peninsula and
eastwards (Haplogroup 3), the fact that in other parts of Europe
this species is clustered in the same haplogroup with M.phoebe and
M.pseudornata while nuclear markers differentiate them suggests
mitochondrial introgression. Thus, M.ornata would have partially
lost its original mtDNA in favor of an introgressed mtDNA pre-
sumably coming from M.phoebe, a scenario already proposed by
Tóth etal. (2017). The same situation may apply to M.pseudornata,
whose mtDNA could have been completely erased after the intro-
gression events with M.phoebe—as occurred in other Iberian spe-
cies such as Iphiclides feisthamelii (Gaunet etal. 2019). Overall, we
cannot determine from these data how common hybridization be-
tween M.phoebe and M.pseudornata might be at present but, given
that they share two well-differentiated haplogroups, introgressive
hybridization seems to have occurred at least twice in thepast.
Past hybridization between M.phoebe and M.pseudornata could
have had an impact on the morphology of the larvae and the adults.
Asign of this can be the presence, in populations of northern Iberia,
of the orange lateral stripe in the caterpillars, very similar to those
present in the Iberian M. phoebe (Fig. 4); these stripes are absent
in M.ornata (Russell and Tennent 2016). Regarding the adults, a
combination of traits of the wing underside such as the premarginal
markings and color tone of the hindwings and are considered to be
relatively useful to distinguish between M. ornata and M. phoebe
(Russell and Tennent 2016). However, between the Iberian M.phoebe
and M.pseudornata, these traits are regularly shared (Supp Fig. S4
[online only]). Furthermore, no other external traits seem to unam-
biguously differentiate the adults of M.pseudornata and M.phoebe.
M.pseudornata populations may also be affected by ecological
character displacement regarding the larval host plant. In the Baetic
System, M.pseudornata females are known to oviposit (or L1 larvae
were found) on Carduncellus Adans, Carduus L., and Cirsium Mill.,
whereas M.phoebe oviposits on Centaurea L. Contrastingly, in cen-
tral Navarre, an area where only M.pseudornata has been found,
we only observed larvae (including L1) feeding on Centaurea jacea.
This behavior could be inuenced by a potential competitive pres-
sure caused by the more generalist M.phoebe, a hypothesis already
suggested by Tóth etal. (2015) for M.ornata.
SupplementaryData
Supplementary data are available at Insect Systematics and
Diversityonline.
Specimen Collection Statement
Insect Systematics and Diversity supports compliance with the Nagoya Proto-
col. The authors attest that all legal and regulatory requirements, including
export and import collection permits, have been followed for the collection
of specimens from source populations at any international, national, regional,
or other geographic level for all relevant eld specimens collected as part of
this study.
Acknowledgments
We thank Vladimir Žikić for the identications of the parasitoids, Amador
Prieto, Santi Patino and Javi Valencia for the identication of the host plants
from Navarre and Zsolt Bálint for providing the equipment of the Hungarian
Natural History Museum to digitalize the genitalia preparata. We also acknow-
ledge V.Dincă, E.García-Barros, J.Hernández-Roldán, S.Montagud, A.Sendra,
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8Insect Systematics and Diversity, 2022, Vol. 6, No. 2
F. González, M. Munguira, R. Requejo, L. Dapporto, S. Viader, R. Vodă,
M.Tarrier, H. Romo, P.Escuer, L.Parmentier, S.Cuvelier, A. Mir, A.Iglesias,
L. Kaminski and M. Menchetti for providing samples used in this study. We
are grateful to the Aranguren City Council (Navarre) for the support for the
monitoring and to the volunteers who have collected observations of the phen-
ology of this species. Financial support for this research was provided by pro-
jects PID2019-107078GB-I00 funded by Ministerio de Ciencia e Innovación
(MCIN)/Agencia Estatal de Investigación (AEI)/ 10.13039/501100011033 and
2017-SGR-991 funded by Generalitat de Catalunya to Roger Vila and by grant
BES-2017-080641, funded by MCIN/AEI/10.13039/501100011033 and by
‘European Social Fund (ESF) Investing in your future’ to Joan C.Hinojosa.
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