ArticlePDF Available

Unexpected layers of cryptic diversity in Wood White Leptidea butterflies

Authors:

Abstract and Figures

Uncovering cryptic biodiversity is essential for understanding evolutionary processes and patterns of ecosystem functioning, as well as for nature conservation. As European butterflies are arguably the best-studied group of invertebrates in the world, the discovery of a cryptic species, twenty years ago, within the common wood white Leptidea sinapis was a significant event, and these butterflies have become a model to study speciation. Here we show that the so-called 'sibling' Leptidea actually consist of three species. The new species can be discriminated on the basis of either DNA or karyological data. Such an unexpected discovery challenges our current knowledge on biodiversity, exemplifying how a widespread species can remain unnoticed even within an intensely studied natural model system for speciation.
Content may be subject to copyright.
ARTICLE
NATURE COMMUNICATIONS | 2:324 | DOI: 10.1038/ncomms1329 | www.nature.com/naturecommunications
© 2011 Macmillan Publishers Limited. All rights reserved.
Received 12 Jan 2011 | Accepted 28 Apr 2011 | Published 24 May 2011 DOI: 10.1038/ncomms1329
Uncovering cryptic biodiversity is essential for understanding evolutionary processes and
patterns of ecosystem functioning, as well as for nature conservation. As European butterflies are
arguably the best-studied group of invertebrates in the world, the discovery of a cryptic species,
twenty years ago, within the common wood white Leptidea sinapis was a significant event, and
these butterflies have become a model to study speciation. Here we show that the so-called
‘sibling’ Leptidea actually consist of three species. The new species can be discriminated on the
basis of either DNA or karyological data. Such an unexpected discovery challenges our current
knowledge on biodiversity, exemplifying how a widespread species can remain unnoticed even
within an intensely studied natural model system for speciation.
1 Institut de Biologia Evolutiva (CSIC-UPF), Passeig Marítim de la Barceloneta 37–49, 08003 Barcelona, Spain. 2 Departament de Genètica i Microbiologia,
Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain. 3 Department of Karyosystematics, Zoological Institute of Russian Academy
of Science, Universitetskaya nab. 1, 199034 St Petersburg, Russia. 4 Department of Entomology, St Petersburg State University, Universitetskaya nab.
7/9, 199034 St Petersburg, Russia. 5 Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, 08010 Barcelona, Spain.
Correspondence and requests for materials should be addressed to R.V. (email: roger.vila@ibe.upf-csic.es).
Unexpected layers of cryptic diversity in wood
white Leptidea butterflies
Vlad Dincaˇ1,2, Vladimir A. Lukhtanov3,4, Gerard Talavera1,2 & Roger Vila1,5
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1329
NATURE COMMUNICATIONS | 2:324 | DOI: 10.1038/ncomms1329 | www.nature.com/naturecommunications
© 2011 Macmillan Publishers Limited. All rights reserved.
Given the global biodiversity crisis1–3, cataloguing the earth’s
species has become a race against time. Several studies have
highlighted the presence and importance of cryptic biodi-
versity, which might represent a substantial proportion of Earths
natural capital. An estimate of cryptic species diversity is important
to better understand evolutionary processes and patterns of eco-
system functioning, while also having deep implications for nature
conservation4,5. e recent increase in the number of reported
cryptic species is, in large part, owing to an increasing amount of
studies incorporating DNA-based techniques, including large-scale
approaches such as DNA barcoding6, which oen provide resolu-
tion beyond the boundaries of morphological information. How-
ever, documenting cryptic diversity based on DNA data alone is
generally not sucient, prompting calls for integrative morphologi-
cal, ecological and molecular approaches7,8. Recent estimates on the
distribution of cryptic diversity are contradictory, and are based
on a thin empirical foundation4,9. In any case, it is to be expected
that the highest number of yet-to-be-discovered cryptic species
is concentrated in the most biodiverse and least explored regions
of our planet (that is, tropical areas). In temperate regions such as
Europe, it is assumed that the level of unrecognized diversity is low,
not only because of lower species richness, but also because taxo-
nomic research has been intense for many groups of organisms.
Such a case is represented by butteries, probably the best-studied
group of invertebrates, which have become a agship for insect
conservation eorts in Europe10,11.
e discovery of a new European species of wood white (Leptidea
sp.) at the end of the twentieth century was an important event in
buttery systematics. Leptidea sinapis (Linnaeus, 1758), a common
buttery with Palaearctic distribution was found to ‘hide’ a cryptic
species, Leptidea reali (Reissinger, 1989)12,13. Aer the two species
were shown to be separable based on their genitalia—but not on
their wing morphology13—several studies revealed that L. reali is
oen sympatric with L. sinapis and that its distribution is almost
as wide as that of L. sinapis14,15. Molecular data (allozyme markers
and mitochondrial DNA) also supports the specic distinctness of
L. reali16. Moreover, much attention has been paid to behavioural
and ecological aspects of the species pair L. sinapis L. reali, to the
point that they have become a model for studying speciation in
cryptic species. Such studies revealed that: a premating reproductive
barrier exists (females only accept conspecic males)17,18; the two
species display only limited ecological dierentiation (larval food
plant preference and performance)17,19; niche separation between
the two species (forests or meadows) is not caused by xed between-
species dierences20; dierences in phenology and voltinism are
mostly the result of environmentally induced pleiotropic eects21;
larval diapause is determined by information from the host plant22;
and behavioural polyphenism has been documented in female
propensity to mate23.
In this paper, we integrate molecular (mitochondrial and nuclear
DNA markers), cytological (chromosome number) and morpho-
logical data (male genitalia morphometry) to study the species pair
L. sinapisL. reali. We found an unexpected pattern showing that
L. reali actually comprises two synmorphic, yet genetically and
karyotypically distinct, groups, with the new cryptic entity being
sister to L. sinapis + L. reali, producing what may be called nested
cryptic species. erefore, the so-called ‘twin speciesL. sinapis
L. reali are actually a triplet of cryptic species, a result that asks for a
reconsideration of previous knowledge and exemplies the advan-
tages of an integrative approach when studying closely related taxa.
Results
Analysis of mitochondrial and nuclear DNA markers. e
Bayesian and maximum likelihood gene genealogies estimated for
each of the mitochondrial (cytochrome oxidase subunit I (COI),
NADH dehydrogenase subunit 1 (ND1)) and nuclear loci (internal
transcribed spacer 2 (ITS2), wingless (Wg), carbamoyl-phosphate
synthetase 2/aspartate transcarbamylase/dihydroorotase (CAD))
gave largely congruent results for the species pair L. sinapis and
L. reali. Depending on their degree of variability, the markers had
dierent resolving power, but all suggested that specimens that are
morphologically attributable to L. reali (based on their genitalia) are
not monophyletic. Moreover, the more variable genes COI, ND1 and
ITS2 showed that L. reali formed two clades and was paraphyletic
with respect to L. sinapis (Supplementary Figs S1–S5).
e topology of the partitioned Bayesian, maximum likelihood
and maximum parsimony multi-gene trees revealed three major
well-resolved clades within the L. sinapisL. reali group (Fig. 1).
Whereas L. sinapis was recovered as monophyletic, specimens
morphologically attributable to L. reali (based on their genitalia)
formed two strongly supported clades and were paraphyletic with
respect to L. sinapis. One of these clades was sister to L. sinapis and
included all specimens from Spain and Italy, as well as several from
southern France (Fig. 1). is clade is certainly attributable to gen-
uine L. reali, as the type locality of this species lies in the French
Pyrenees12. e other clade of specimens with reali-like morphol-
ogy consisted of samples from several countries ranging from
Ireland and France in the west, to eastern Kazakhstan in the east,
and was recovered as sister to L. sinapis plus genuine L. reali with
good support (Fig. 1). is pattern was recovered by the Bayesian
coalescent-based species tree estimation as well, conrming the top-
ological relationships of the three lineages (Fig. 2). e species-tree
approach is less prone to misleading results than combining data by
partitions, because it incorporates uncertainty associated with gene
trees (probability of unsorted ancestral polymorphism), nucleotide
substitution model parameters, and the coalescent process. ese
results, together with the karyotypic data, strongly suggest that the
non-Mediterranean clade of L. reali represents a dierent species.
e oldest available name that we could assign to the new species is
juvernica (Williams, 1946), described as a subspecies for Irish popu-
lations with reali-like morphology14. erefore, in accordance with
the International Code of Zoological Nomenclature, we hereaer
refer to the new species as Leptidea juvernica stat. nov.
Our sampling revealed that L. reali and L. juvernica stat. nov. dis-
play non-overlapping geographical distributions, but some popula-
tions are parapatric —at least in southeastern France, where they are
separated by only 87 kilometres (Fig. 3, Supplementary Table S1). It is
worth noting that we did not nd any case of introgression between
these two species in the parapatry zone or elsewhere.
Karyotype analysis. Diploid chromosome numbers 2n = 52, 2n = 53
and 2n = 54 were found in L. reali. e individuals with 2n = 52 and
2n = 54 presented 26 and 27 bivalents during rst meiotic division
(MI), and 26 and 27 chromosomes during second meiotic division
(MII), respectively. Individuals with 2n = 53 were heterozygous for
one chromosomal fusion/ssion and demonstrated 25 bivalents and
one trivalent in MI (Supplementary Note 1). us, we established
the chromosome number of L. reali is not xed and ranges between
2n = 52–54.
Leptidea juvernica stat. nov. displayed clearly higher chromo-
some numbers and, at the same time, a higher level of chromosome
number variation than L. reali. We have found in mitotic cells, or
have reconstructed based on meiotic cells, the following numbers:
2n = 80, 2n = 82 and 2n = 84, 2n = ca. 81–84, 2n = ca. 83–85. Some of
the individuals studied were chromosomal heterozygotes display-
ing up to six multivalents in metaphase I of meiosis (Supplementary
Note 1). Given the karyotypes observed in MI and MII cells and tak-
ing into account all possible combinations of gametes, we concluded
that chromosome numbers ranging from 2n = 76 to 2n = 88 are
expected to be found in L. juvernica stat. nov. Our results show that
L. reali and L. juvernica stat. nov. are dierentiated by at least 11
chromosomal fusions/ssions.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1329
NATURE COMMUNICATIONS | 2:324 | DOI: 10.1038/ncomms1329 | www.nature.com/naturecommunications
© 2011 Macmillan Publishers Limited. All rights reserved.
Morphological analysis. e Shapiro-Wilk test supported normal dis-
tributions for the ve measured variables (phallus length (PL), saccus
length (SL), vinculum width (VW), genital capsule length and uncus
length (UL)) (P > 0.05). For the discriminant analysis, the variables
included in the prediction equation with the stepwise method and
using Wilks’ Lambda were PL, VW and SL. e rst two canonical
discriminant functions explained 100% of the variance and were used
in the analysis. e rst function alone accounted for 99.4% of the
variance displaying a strong canonical correlation of 0.951 and a highly
signicant Wilks’ Lambda (0.091, P < 0.001). e second function
explained 0.6% of the variance, displayed a canonical correlation of
0.227 and had a signicant Wilks’ Lambda (0.949, P = 0.032). e struc-
ture matrix that was obtained (Supplementary Table S2) showed the
canonical weight of each variable which is an indicator of its discrimi-
natory power.
All specimens attributed to L. sinapis in the molecular analy-
sis were correctly classied by the discriminant analysis, sup-
porting previous results indicating that male genitalia allow for
0.01
RVcoll.09-X181 (IE)
RVcoll.07-E253 (FR)
RVcoll.07-Z236 (KZ)
RVcoll.160410QT77 (CH)
RVcoll.07-E217 (IT)
RVcoll.10-C194 L. lactea
RVcoll.10-A742 (EE)
RVcoll.10-C189 L. lactea
RVcoll.08-L090 (ES)
RVcoll.10-C195 L. lactea
RVcoll.10-A411 (FR)
RVcoll.10-B385 (BG)
RVcoll.Nz075 L. amurensis
RVcoll.LR08-D679 (SI)
RVcoll.08-H275 (ES)
RVcoll.10-A412 (FR)
RVcoll.03-H535 (ES)
RVcoll.07-Z083 L. morsei
RVcoll.09-V207 L. duponcheli
RVcoll.07-E140 (IT)
RVcoll.07-Z210 (KZ)
RVcoll.08-J396 (ES)
RVcoll.LR08-D680 (SI)
RVcoll.10-A262 (FR)
RVcoll.08-Y010 (RU)
RVcoll.07-Z081 (KZ)
RVcoll.08-H281 (ES)
RVcoll.09-T250 (FR)
RVcoll.07-Z124 L. morsei
RVcoll.07-E237 (IT)
RVcoll.09-V713 (ES)
RVcoll.08-Y007 (RU)
RVcoll.07-E083 (IT)
RVcoll.07-D500 (RO)
RVcoll.10-C244 (CZ)
RVcoll.09-X268 (FR)
RVcoll.09-X183 (IE)
RVcoll.07-E082 (IT)
RVcoll.06-H636 (KZ)
RVcoll.07-E081 (IT)
RVcoll.10-C186 L. amurensis
RVcoll.10-C243 L. duponcheli
RVcoll.06-H638 (KZ)
RVcoll.10-A259 (FR)
RVcoll.09-T245 (FR)
RVcoll.08-M325 (RO)
RVcoll.06-H639 (KZ)
RVcoll.07-C470 (ES)
RVcoll.Nz091 L. amurensis
RVcoll.07-E254 (FR)
RVcoll.10-B453 (HR)
RVcoll.07-D962 (RO)
RVcoll.07-E553 (RO)
RVcoll.08-M498 L. morsei
RVcoll.07-Z082 (KZ)
RVcoll.06-K559 (RO)
RVcoll.07-E080 (IT)
RVcoll.08-M322 (RO)
RVcoll.08-Y012 (RU)
RVcoll.09-X180 (IE)
Leptidea sinapis
Leptidea reali
Leptidea juvernica
97/56/52
53/–/–
98/52/–
99/57/56
81/59/66
67/–/–
100/99/99
97/–/53
97/–/–
98/–/79
100/95/98
66/73/–
93/–/–
99/64/69
100/100/99
100/84/76
83/–/–
100/93/98
77/59/–
91/–/72
100/96/99
54/–/–
95/–/86
61/–/–
100/100/99
100/74/– 100/97/97
100/99/99
100/95/85
100/100/99
100/100/99
100/100/99
Figure 1 | Leptidea molecular phylogeny. Bayesian ultrametric tree based on the combined analysis of COI, ND1, ITS2, Wg and CAD. Leptidea juvernica stat.
nov. is monophyletic and sister to L. sinapis + L. reali. Bayesian posterior probabilities, maximum likelihood and maximum parsimony bootstrap
values ( > 50%) are shown above recovered branches. IE, Ireland; ES, Spain; FR, France; IT, Italy; CH, Switzerland; SI, Slovenia; HR, Croatia; RO, Romania;
BG, Bulgaria; EE, Estonia; RU, Russia; KZ, Kazakhstan.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1329
NATURE COMMUNICATIONS | 2:324 | DOI: 10.1038/ncomms1329 | www.nature.com/naturecommunications
© 2011 Macmillan Publishers Limited. All rights reserved.
the separation of L. sinapis and L. reali sensu lato. On the other
hand, classication was much less accurate for L. reali and L.
juvernica stat. nov. (61.5% for L. reali and 62.5% for L. juvernica
with 53.8% and 58.3%, respectively, aer cross validation) (Fig. 4,
Supplementary Table S3), indicating that they cannot be reliably
identied based on the parameters involved. To further test these
results, another discriminant analysis was run including only L.
reali and L. juvernica stat. nov. Two variables were introduced in
the prediction equation: SL and genital capsule length (GL). e
rst function explained 100% of the variance and was used in the
analysis. is function displayed a moderate canonical correla-
tion of 0.357 and a signicant Wilks’ Lambda of 0.873 (P = 0.003).
Classication results were similar to the previous analysis, with
56.4% of L. reali and 64.6% of L. juvernica stat. nov. correctly iden-
tied (53.8% and 62.5%, respectively, aer cross-validation). is
conrmed that, although there seemed to be a slight tendency of
larger genitalia for L. reali specimens (Fig. 4), identication was
unreliable based on male genitalia.
Female genitalia of a few specimens corresponding to L. reali and
L. juvernica stat. nov. were also examined. Although our sample was
too small to permit statistical analyses, we did not notice any appar-
ent dierence in the length of the ductus bursae, the most useful
character to discriminate females of L. sinapis from L. reali13,24.
Discussion
is study presents strong evidence for the existence of a previously
unnoticed, widespread species of Leptidea. is is clearly supported by
our combined molecular phylogeny based on two mitochondrial and
three nuclear markers, as well as by the coalescent-based species tree
reconstruction, which showed that the new species L. juvernica stat.
nov. is sister to the species pair L. sinapis and L. reali. No topological
discordance in the relationships among the three species was detected
in the single-gene trees (Fig. 2), except for CAD and Wg, which mixed
specimens of L. sinapis and L. reali. e slower mutation rate and/or
coalescent process of these two nuclear markers is most probably the
cause, but it is worth noting that they recovered the new species as a dif-
L. reali
L. reali
L. sinapis
L. sinapis
L. juvernica
L. juvernica
COI ND1 ITS2
Wg CAD Species tree
99
Figure 2 | Co-estimation of five gene trees embedded in a shared species
tree. Two mitochondrial (COI and ND1) and three nuclear loci (ITS2, Wg
and CAD) were used to estimate a species tree (in black) for L. sinapis,
L. reali and L. juvernica stat. nov. using *BEAST. The consensus is represented
in white within the species tree and the posterior probability for the L. reali
L. sinapis clade is shown. Trees are figured with DensiTree55 displaying all
trees of the Markov chain Monte Carlo chain with a burn-in of 5,000 trees.
Higher levels of uncertainty are represented by lower densities.
Leptidea juvernica
2n=54
2n=81
(2n=76–86) (2n=76–84)
30
40
60
70
80
W
S
0 1,000 km2,000
N
E
20 30 40 50 60 70 8010 0 10
2n=52, 53
(2n=52–54)
2n=80
2n=80, 82, 84
Leptidea reali
(2n=76–88)
90
50
a b c d
Longitude (deg.)
Latitude (deg.)
Figure 3 | Chromosome number results and sampling localities. Leptidea reali (red dots), L. juvernica stat. nov. (blue dots) and L. sinapis (empty circles).
Although L. reali and L. juvernica display non-overlapping distributions, they come into close contact in southeastern France and are differentiated by at
least 11 fixed chromosome fusions/fissions. (a) Karyotype of L. reali, Spain, RVcoll.03-H535: MI cell demonstrating 25 bivalents and one multivalent
(most likely a trivalent, indicated by an arrow). (b) L. reali, Spain, RVcoll.07-F514: MII cell demonstrating 27 chromosomes. (c) L. juvernica, Russia,
RVcoll.08-Y012: MI cell demonstrating 42 bivalents. (d) L. juvernica, Russia, RVcoll.08-Y010: MII cell demonstrating 41 chromosomes. Scale bar
corresponds to 10 µm in all figures.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1329
NATURE COMMUNICATIONS | 2:324 | DOI: 10.1038/ncomms1329 | www.nature.com/naturecommunications
© 2011 Macmillan Publishers Limited. All rights reserved.
ferent entity. When combining the three nuclear loci, the monophyly
of all species was strongly supported (Supplementary Fig. S6).
Our conclusions based on molecular data were also supported by
karyotype analyses, which revealed dierent chromosome numbers
between L. reali (2n = 52–54) and L. juvernica stat. nov. (2n = 80–
84). Although the chromosome number is not xed in these species,
intraspecic variation is limited and the interspecic gap is pro-
nounced (at least 11 chromosomal rearrangements). It is relevant
that karyotype characteristics (chromosome numbers and level of
chromosome number variability) are nearly identical in geographi-
cally distant populations within each species, whereas L. reali from
Italy and L. juvernica stat. nov. from Slovenia are drastically dier-
ent, despite geographical proximity.
Mating between species with dierent karyotypes is known to
produce hybrids that are heterozygous for chromosomal rearrange-
ments xed between parental species. ese hybrids typically have
reduced fertility due to partial missegregation of homologous chro-
mosomes during the MI25. Although dierent kinds of chromosomal
rearrangements have various eects on the fertility of heterozygous
hybrids26, hybrid fertility is generally negatively correlated with
the extent of karyotypic divergence between parental taxa27,28, and
multiple chromosome fusion/ssions, such as those we detected in
L. reali and L. juvernica stat. nov., can strongly contribute to postzygotic
reproductive isolation. Although we have no direct data on the degree
of postzygotic isolation, the chromosomal dierentiation between
L. reali and L. juvernica stat. nov. is high and can be considered as
additional independent evidence that there are two distinct species.
Morphometry results showed that specimens of L. reali and
L. juvernica cannot be reliably distinguished, whereas L. sinapis
was clearly dierentiated based on genitalic measurements. Wing
and preimaginal stage morphology did not appear to be useful for
identication either, as already shown by several studies comparing
L. sinapis and L. reali sensu lato13. erefore, L. reali and L. juver-
nica stat. nov. seem to represent the plesiomorphic state and to have
remained in morphological stasis, wheras L. sinapis evolved genitalic
dierences. e fact that the new cryptic species reported here is appar-
ently fully synmorphic to L. reali explains why it remained unnoticed
for such a long time despite intensive research. We propose the
common name ‘cryptic wood white’ for L. juvernica stat. nov.
e relationships between the three studied species suggest
that the common ancestor of the triplet of species (ancestor A)
(Fig. 5a,b) probably originated in central or western Asia and sub-
sequently spread over western Europe. e hypothesis of an eastern
origin is also supported by the exclusively eastern distribution of the
closest relatives (L. amurensis, L. morsei and L. lactea) to the triplet
of cryptic species (Fig. 1). About 270,000 years ago (Supplementary
Table S4), probably in southwestern Europe, ancestor A speciated
producing the common ancestor of L. sinapis and L. reali (ancestor
B), and the L. juvernica stat. nov. lineage that established across tem-
perate Europe and Asia (Fig. 5c). About 120,000 years ago, ancestor
B diverged into L. sinapis and L. reali (Fig. 5d). Later on (ca. 27,000
years ago), L. sinapis expanded north and east into the territory of
L. juvernica stat. nov. (Fig. 5e). On the basis of our sampling, L. reali
and L. juvernica stat. nov. are most likely parapatric, with L. reali
conned to southwestern Europe and L. juvernica stat. nov. spread
across temperate Europe and Asia. is provides a totally new view
on L. reali which is actually a west Mediterranean species and not a
widely distributed taxon as concluded before. Our sampling suggests
a potential contact zone in southeastern France, where populations
of the two species are separated by less than 90 kilometres (Fig. 3).
To know the causes behind the apparent inability of L. reali and
L. juvernica stat. nov. to coexist will require further studies. It has
been shown that, besides dierences in the genitalia, behavioural
aspects related to mate choice maintain reproductive isolation
between L. sinapis and L. reali sensu lato17,18. Previous data on the
biology and ecology of L. reali sensu lato, as well as our view of the
speciation processes undergone by Leptidea, need to be extensively
revised in light of these results17–23,29. Our observations revealed that
both L. reali and L. juvernica stat. nov. can use Lathyrus pratensis as
a larval food plant (oviposition observed in Spain for L. reali and in
Romania for L. juvernica stat. nov. and adults obtained from these
eggs by rearing in the laboratory). It is thus to be expected that
ecological dierentiation between the two species is minimal.
In this study, we show that assessing cryptic diversity is a chal-
lenging task even in well-studied groups of organisms. What has
been formerly called the cryptic species pair, L. sinapis L. reali
comprises a triplet of species, and new research is needed to clarify
their distribution, ecology and conservation status. Our ndings
exemplify that cryptic biodiversity may consist of nely nested
layers and highlight the importance of using an array of techniques
when dealing with closely related species.
Methods
Specimen sequencing. e mitochondrial marker COI was sequenced in 166
specimens, the mitochondrial ND1 in 85 specimens, the nuclear ITS2 in 91
specimens, the nuclear wingless (Wg) in 67 specimens and the nuclear CAD in
43 specimens.
irteen GenBank COI sequences of L. sinapis from Spain30, France31, Slov-
enia32, Greece21 and Kazakhstan33, seven sequences of L. juvernica stat. nov. from
Slovenia32, three sequences of L. amurensis33 from Russia and two sequences of
L. morsei33 from Kazakhstan were also added to the dataset. Additionally, one
sequence of L. sinapis from Austria and three sequences of L. juvernica
stat. nov. from Germany were included from the publicly available project ‘Fauna
Bavarica—Lepidopera Rhopalocera’ included in the Barcode of Life Data System at
http:\\www.barcodinglife.org. Four ND1 Leptidea GenBank sequences (two L. reali
and two L. sinapis)16 were also added to the dataset. All novel sequences obtained
in this study have been deposited to GenBank under accession codes JF512569 to
JF513007 (for details see Supplementary Table S1).
Total genomic DNA was extracted using Chelex 100 resin, 100–200 mesh,
sodium form (Bio-rad), under the following protocol: one leg was removed and
introduced into 100 µl of Chelex 10% and 5 µl of Proteinase K (20 mg ml1) were
added. e samples were incubated overnight at 55 °C and were subsequently incu-
bated at 100 °C for 15 min. Aerwards they were centrifuged for 10 s at 3,000 r.p.m.
5.02.50.0–5.0 –2.5
Function 1
5.0
2.5
0.0
–2.5
Function 2
PL
SL
UL
VW
GL
Figure 4 | Discriminant analysis based on male genitalia morphometry.
L. sinapis (black) is identifiable based on male genitalia, but there is broad
overlap between L. reali (red) and L. juvernica stat. nov. (blue). Circles
represent individuals and squares represent centroids for each species.
Elements of the male genitalia measured were PL, SL, VW, GL and UL. The
discriminant variables were PL and SL for function 1 and SL and VW for
function 2. The upper left corner image indicates the variables measured
for Leptidea male genitalia.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1329
NATURE COMMUNICATIONS | 2:324 | DOI: 10.1038/ncomms1329 | www.nature.com/naturecommunications
© 2011 Macmillan Publishers Limited. All rights reserved.
e primers used were: for COI (676 bp) LCO 1490 (5-GGTCAACAAATCATAA
AGATATTGG-3)34 and Nancy (5-CCCGGTAAAATTAAAATATAAACTTC-3)35, or
(658 bp) LepF1 (5-ATTCAACCAATCATAAAGATATTGG-3) and LepR1
(5-TAAACTTCTGGATGTCCAAAAAATCA-3)36; for ND1 (790–794 bp)
5- CTGTTCGATCATTAAAATCTTAC-3 (forward)37 and 5-ATCAAAAG
GAGCTCGATTAGTTTC-3 (reverse)38; for ITS2 (684 bp) ITS3 (5-GCATCGAT
GAAGAACGCAGC-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3)39; for Wg
(403 bp) Wg1 (5-GARTGYAARTGYCAYGGYATGTCTGG-3) and Wg2
(5-ACTICGCRCACCARTGGAATGTRCA-3)40; for CAD (571 bp) CADFa
(5-GDATGGTYGATGAAAATGTTAA-3) and CADRa (5-CTCATRTCGTAAT
CYGTRCT-3).
Double-stranded DNA was amplied in 25 µl volume reactions: 13.22 µl ultra
pure (HPLC quality) water, 2.5 µl 10× buer, 4.5 µl 25 mM MgCl2, 0.25 µl 100 mM
dNTP, 1.2 µl of each primer (10 mM), 0.13 µl Taq DNA Gold Polymerase (Qiagen)
and 2 µl of extracted DNA. e typical thermal cycling prole for COI was 95 °C
for 60 s, 44 °C for 60 s and 72 °C for 90 s, for 40 cycles. e annealing temperature
varied according to marker: 48 for ND1, 47 for ITS2, 51 for Wg, and 48 for CAD.
PCR products were puried and sequenced by Macrogen Inc. All the samples
are stored in the Institut de Biologia Evolutiva collection in Barcelona, Spain, and
are available upon request.
Phylogenetic analyses and species tree estimation. COI, ND1, ITS2, Wg and
CAD sequences were edited and aligned using Geneious Pro 4.7.541. ese resulted
in ve alignments of 676 bp and 195 specimens for COI, 794 bp and 89 specimens
for ND1, 715 bp and 91 specimens for ITS2, 403 bp and 67 specimens for Wg,
and 571 bp and 43 specimens for CAD. For COI, duplicate haplotypes (excluding
outgroups) were removed using TCS 1.2142.
Individual Bayesian and ML phylogenetic trees were inferred using COI, ND1,
ITS2, Wg, and CAD with BEAST 1.6.043 and GARLI 1.044. Relationships based on
the combined dataset were estimated using partitioned Bayesian and ML analyses
using BEAST 1.6.0 and GARLI-PART v. 0.9744 with substitution models by mark-
ers according to the suggestions of jModeltest 0.145. e models employed for the
partitioned ML analysis were TPMuf + I + G for COI, HKY + I for CAD and ND1,
TVM for ITS2 and TPM2 for Wg. For the partitioned BI, GTR + I + G was used for
COI, HKY + I for CAD and ND1, GTR for ITS2 and HKY for Wg.
Branch support was assessed by 100 bootstrap replicates for maximum
likelihood, and Markov chain Monte Carlo convergence was checked aer two
independent runs of 10 million generations each (with a pre-run burn in of 100,000
generations) for Bayesian inference. A multilocus coalescent-based Bayesian spe-
cies tree was estimated with *BEAST46. L. sinapis, L. reali and L. juvernica stat. nov.
specimens were dened as three taxonomic units in accordance with clades previ-
ously inferred by single-gene and ve-loci combined trees. A relaxed clock with
uncorrelated lognormal distribution47 and a Yule speciation process as tree prior
were used. e length of the Markov chain Monte Carlo chain was set at 50 million
generations sampling every 1,000 runs with a burn-in set to the rst 500,000 genera-
tions. A maximum parsimony tree based on the ve markers combined was inferred
with MEGA448 and branch supports were assessed by 100 bootstrap replicates.
Dating divergence events. Node ages were inferred with BEAST 1.6.043 using the
COI haplotype dataset under a coalescent model with constant population size. We
calibrated the phylogeny at two selected nodes: the L. sinapis common ancestor
node as an example of a very recent clade supposedly under a coalescent process,
and the root of the tree as a clearly coalesced node. For the age of the root node, we
used a normally distributed prior ranging between 2.2 and 4 MYA based on slow
and fast published invertebrate mitochondrial rates of 1.3 and 2.3% uncorrected
pairwise distance per million years49,50. e prior range assumed for the common
ancestor of L. sinapis was a normal distribution between 8,500–31,000 years, as
previously inferred51. e dataset was analysed using the GTR + I + G model and
applying an uncorrelated lognormal relaxed molecular clock47 along the branches.
Base frequencies were estimated, six gamma rate categories were selected and
a randomly generated initial tree was used. Parameters were estimated using
two independent runs of 10 million generations each (with a pre-run burn in of
100,000 generations) to ensure convergence, and were checked with the program
Tracer v1.5.
A
B
b c
d e
L. sinapis
L. reali
L. juvernica
27,000 years
46,000 years
73,000 years
121,000 years
269,000 years
A
B
a
L. juvernica
L. juvernica
L. reali L. reali
L. juvernica
L. sinapis
L. sinapis
Figure 5 | Phylogenetic relationships and proposed speciation scenario. (a) L. juvernica stat. nov. is sister to L. sinapis + L. reali. Age estimations are
indicated for each node. (b) The common ancestor of the whole group (ancestor A) probably originated in central or western Asia and subsequently
colonized western Europe. (c) Ancestor A split into L. juvernica in temperate Europe and Asia and the common ancestor of L. sinapis and L. reali (ancestor
B) in southwestern Europe (d) Ancestor B speciated into L. sinapis and L. reali. (e) Subsequently, L. sinapis rapidly spread north and east into the territory of
L. juvernica.
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1329
NATURE COMMUNICATIONS | 2:324 | DOI: 10.1038/ncomms1329 | www.nature.com/naturecommunications
© 2011 Macmillan Publishers Limited. All rights reserved.
Karyotype analyses. Gonads were stored in Carnoy xative (ethanol and glacial
acetic acid, 3:1) for 2–6 months at 4 °C and then stained with 2% acetic orcein for
30 days at 20 °C. Cytogenetic analysis was conducted using a two-phase method of
chromosome analysis52.
In our study, we have counted the diploid chromosome numbers (2n) in
mitotic spermatogonial cells and the haploid chromosome numbers (n) in met-
aphase II of male meiosis. We also counted the number of chromosomal elements
(n) (bivalents + multivalents) in metaphase I of male meiosis. In the last case, the
number of chromosomal elements was equal to the haploid number (n), if all the
elements were represented by bivalents, or less if some elements were represented
by multivalents. To distinguish between bivalents and multivalents, we used a
special method53. Briey, by varying the pressure on the coverslip, we were able to
manipulate chromosomes, for example, change their position and orientation in
intact (not squashed) spermatocyte cells, and consequently to analyse the structure
of the bivalents and multivalents.
In total, preparations from 68 males were analysed. As cell divisions are
extremely rare in Leptidea during imago stage54, metaphase plates were observed
in only 14 individuals (Supplementary Table S1). ese individuals have also been
used for morphological and molecular analysis.
Genitalia preparation and morphometrics. Male genitalia were prepared
according to the following protocol: maceration for 15 min at 95 °C in 10% potas-
sium hydroxide, dissection and cleaning under a stereomicroscope and storage in
tubes with glycerine.
Genitalia were photographed in a thin layer of distilled water (without being
pressed under a cover slip) under a Carl Zeiss Stemi 2000-C stereomicroscope
equipped with a DeltaPix Invenio 3S digital camera. Measurements were per-
formed based on the digital photographs by using AxioVision soware. A total
of 39 specimens of L. reali, 48 of L. juvernica stat. nov. and 48 of L. sinapis were
included in the morphometrical analyses (Supplementary Table S5). Five elements
of the male genitalia were measured: PL, SL, VW, GL (measured from the ventral
edge of the vinculum to the uncus apex) and UL. e rst three elements combined
were reported to be the best to discriminate between L. sinapis and L. reali13,16,24.
Statistical analyses were carried out using the soware SPSS 14.0 for Windows.
e rst batch of analyses was run by including three groups: Leptidea reali,
L. juvernica stat. nov. and L. sinapis. Subsequently the analyses were repeated
including only L. reali and L. juvernica stat. nov. A Shapiro-Wilk normality test was
employed. Subsequently, a discriminant analysis was performed by employing the
stepwise method. In order to test the obtained classication, a cross validation was
carried out (‘leave-one-out’ method).
References
1. Gaston, K. J. & Fuller, R. A. Biodiversity and extinction: losing the common
and the widespread. Prog, Phys, Geogr. 31, 213–225 (2007).
2. omas, J. A. et al. Comparative losses of British butteries, birds, and plants
and the global extinction crisis. Science 303, 1879–1881 (2004).
3. Brooks, T. M. et al. Global biodiversity conservation priorities. Science 313,
58–61 (2006).
4. Bickford, D. et al. Cryptic species as a window on diversity and conservation.
Trends Ecol. Evol. 22, 148–155 (2006).
5. Esteban, G. F. & Finlay, B. J. Conservation work is incomplete without cryptic
biodiversity. Nature 463, 293 (2010).
6. Hebert, P. D. N., Cywinska, A., Ball, S. L. & deWaard, J. R. Biological
identications through DNA barcodes. Proc. R. Soc. B 270, 313–321 (2003).
7. Schlick-Steiner, B. C., Seifert, B., Stauer, C., Christian, E., Crozier, R. H. &
Steiner, F. M. Without morphology, cryptic species stay in taxonomic crypsis
following discovery. Trends Ecol. Evol. 22, 391–392 (2007).
8. Beheregaray, L. B. & Caccone, A. Cryptic biodiversity in a changing world.
J. Biol. 6, 9 (2007).
9. Pfenninger, M. & Schwenk, K. Cryptic animal species are homogeneously
distributed among taxa and biogeographical regions. BMC Evol. Biol. 7, 121 (2007).
10. New, T. R., Pyle, R. M., omas, J. A., omas, C. D. & Hammond, P. C.
Buttery conservation management. Annu. Rev. Entomol. 40, 57–83 (1995).
11. omas, J. A. in Ecology and Conservation of Butteries (ed. Pullin, A. S.)
180–197 (Chapman & Hall, 1995).
12. Réal, P. Lépidoptères nouveaux principalement jurassiens. Mém. Comité de
Liaison Rech. Ecofaunist. Jura 4, 1–28 (1988).
13. Lorković, Z. Leptidea reali Reissinger, 1989 (=lorkovicii Real 1988), a new
European species (Lepid., Pieridae). Nat. Croatica 2, 1–26 (1993).
14. Mazel, R. Leptidea sinapis L., 1758—L. reali Reissinger, 1989, le point de la
situation (Lepidoptea: Pieridae, Dismorphiinae). Linneana Belgica 18, 199–202
(2001).
15. Mazel, R. & Eitschberger, U. Répartition géographique de Leptidea sinapis
(L., 1758) et L. reali Reissinger, 1989 au nord de l’Europe, en Russie et dans
quelques pays d’Asie (Lepidoptera: Pieridae, Dismorphiinae). Linneana Belgica
18, 373–376 (2002).
16. Martin, J., Gilles, A. & Descimon, H. in Butteries: Ecology and Evolution
Taking Flight (eds Boggs, C. L., Watt, W. B. & Ehrlich, P. R.) 459–476
(Chicago University Press, 2003).
17. Freese, A. & Fiedler, K. Experimental evidence for specic distinctness of the
two wood white buttery taxa, Leptidea sinapis and L. reali (Pieridae). Nota
Lepid. 25, 39–59 (2002).
18. Friberg, M., Vongvanich, N., Borg-Karlson, A.- K., Kemp, D. J., Merilaita, S. &
Wiklund, C. Female mate choice determines reproductive isolation between
sympatric butteries. Behav. Ecol. Sociobiol. 62, 873–886 (2008).
19. Friberg, M. & Wiklund, C. Host plant preference and performance of the
sibling species of butteries Leptidea sinapis and Leptidea reali: a test of
the trade-o hypothesis for food specialisation. Oecologia 159, 127–137
(2009).
20. Friberg, M., Olofsson, M., Berger, D., Karlsson, B. & Wiklund, C. Habitat
choice precedes host plant choice—niche separation in a species pair of a
generalist and a specialist buttery. Oikos 117, 1337–1344 (2008).
21. Friberg, M., Bergman, M., Kullberg, J., Wahlberg, N. & Wiklund, C. Niche
separation in space and time between two sympatric sister species—a case of
ecological pleiotropy. Evol. Ecol. 22, 1–18 (2008).
22. Friberg, M. & Wiklund, C. Host-plant-induced larval decision-making in a
habitat/host-plant generalist buttery. Ecology 91, 15–21 (2010).
23. Friberg, M. & Wiklund, C. Generation dependent female choice: behavioral
polyphenism in a bivoltine buttery. Behav. Ecol. 18, 758–763 (2007).
24. Fumi, M. Distinguishing between Leptidea sinapis and L. reali (Lepidoptera:
Pieridae) using a morphometric approach: impact of measurement error on the
discriminative characters. Zootaxa 1819, 40–54 (2008).
25. Kandul, N. P., Lukhtanov, V. A. & Pierce, N. E. Karyotypic diversity and
speciation in Agrodiaetus butteries. Evolution 61, 546–559 (2007).
26. King, M. Species Evolution (Cambridge University Press, 1993).
27. White, M. J. D. Animal Cytology and Evolution (Cambridge University Press,
1973).
28. Gropp, A. H., Winking, H. & Redi, C. in Genetic Control of Gamete Production
and Function (eds Crosignani, P. G., Rubin, B. L. & Franccaro, M.) 115–134
(Academic Press, 1982).
29. Beneš, J., Konvicˇka, M., Vrabec, V. & Zámecˇník, J. Do the sibling species of
small whites, Leptidea sinapis and L. reali (Lepidoptera, Pieridae) dier in
habitat preferences? Biologia 58, 943–951 (2003).
30. Braby, M. F., Vila, R. & Pierce, N. E. Molecular phylogeny and systematics
of the Pieridae (Lepidoptera: Papilionoidea): higher classication and
biogeography. Zool J. Linn. Soc. 147, 239–275 (2006).
31. Mutanen, M., Wahlberg, N. & Kaila, L. Comprehensive gene and taxon
coverage elucidates radiation patterns in moths and butteries. Proc. R. Soc.
B 277, 2839–2848 (2010).
32. Verovnik, R. & Glogovcˇan, P. Morphological and molecular evidence of a
possible hybrid zone of Leptidea sinapis and L. reali (Lepidoptera: Pieridae).
Eur. J. Entomol. 104, 667–674 (2007).
33. Lukhtanov, V. A., Sourakov, A., Zakharov, E. V. & Hebert, P. D. N. DNA
barcoding Central Asian butteries: increasing geographical dimension does
not signicantly reduce the success of species identication. Mol. Ecol. Resour.
9, 1302–1310 (2009).
34. Folmer, O. et al. DNA primers for amplication of mitochondrial Cytochrome
C oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol.
Biotech. 3, 294–299 (1994).
35. Simon, C. et al. Evolution, weighting, and phylogenetic utility of mitochondrial
gene sequences and a compilation of conserved polymerase chain reaction
primers. Ann. Entomol. Soc. Am. 87, 651–701 (1994).
36. Hebert, P. D. N. et al. Ten species in one: DNA barcoding reveals cryptic species
in the neotropical skipper buttery Astraptes fulgerator. Proc. Natl Acad. Sci.
USA 101, 14812–14817 (2004).
37. Aubert, J., Barascud, B., Descimon, H. & Michel, F. Ecology and genetics of
interspecic hybridization in the swallowtails, Papilio hospiton Gene and
P. machaon L., in Corsica (Lepidoptera: Papilionidae). Biol. J. Linn. Soc. 60,
467–492 (1997).
38. Aubert, J., Barascud, B., Descimon, H. & Michel, F. Systematique moleculaire
des Argynnes (Lepidoptera: Nymphalidae). Comptes rendus de l’Academie des
Sciences. Serie 3. Sciences de la Vie 319, 647–651 (1996).
39. White, T. J. et al. in PCR Protocols: A Guide to Methods and Applications
(eds Innis, M. A. et al.) 315–322 (Academic Press, 1990).
40. Brower, A. V. Z. & DeSalle, R. Mitochondrial vs. nuclear DNA sequence
evolution among nymphalid butteries: the utility of Wingless as a source of
characters for phylogenetic inference. Insect Mol. Biol. 7, 1–10 (1998).
41. Drummond, A. J. et al. Geneious v4.7. Available from http://www.geneious.
com/ (2009).
42. Clement, M., Posada, D. & Crandall, K. Tcs: a computer program to estimate
gene genealogies. Mol. Ecol. 9, 1657–1660 (2000).
43. Drummond, A. J. & Rambaut, A. BEAST: Bayesian evolutionary analysis by
sampling trees. BMC Evol. Biol. 7, 214 (2007).
44. Zwickl, D. J. Genetic Algorithm Approaches for the Phylogenetic Analysis of Large
Biological Sequence Datasets Under the Maximum Likelihood Criterion. PhD
dissertation, e University of Texas at Austin (2006). Available from: https://
www.nescent.org/wg_garli/Main_Page.
45. Posada, D. jModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25,
1253–1256 (2008).
ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1329
NATURE COMMUNICATIONS | 2:324 | DOI: 10.1038/ncomms1329 | www.nature.com/naturecommunications
© 2011 Macmillan Publishers Limited. All rights reserved.
46. Heled, J. & Drummond, A. J. Bayesian inference of species trees from
multilocus data. Mol. Biol. Evol. 27, 570–580 (2010).
47. Drummond, A. J., Ho, S. Y. W., Phillips, M. J. & Rambaut, A. Relaxed
phylogenetics and dating with condence. PLoS Biol. 4, e88 (2006).
48. Tamura, K., Dudley, J., Nei, M. & Kumar, S. MEGA4: Molecular Evolutionary
Genetics Analysis (MEGA) soware version 4.0. Mol. Biol. Evol. 24, 1596–1599
(2007).
49. Quek, S. P. et al. Codiversication in an ant-plant mutualism: stem texture
and the evolution of host use in Crematogaster (Formicidae: Myrmicinae)
inhabitants of Macaranga (Euphorbiaceae). Evolution 58, 554–570 (2004).
50. Brower, A. V. Z. Rapid morphological radiation and convergence among races
of the buttery Heliconius erato inferred from patterns of mitochondrial-DNA
evolution. Proc. Natl Acad. Sci. USA 91, 6491–6495 (1994).
51. Lukhtanov, V. A., Dincă, V., Talavera, G. & Vila, R. Unprecedented within-
species chromosome number cline in the Wood White buttery and its
signicance for karyotype evolution and speciation. BMC Evol. Biol. 11, 109
(2011).
52. Lukhtanov, V. A., Vila, R. & Kandul, N. P. Rearrangement of the Agrodiaetus
dolus species group (Lepidoptera, Lycaenidae) using a new cytological
approach and molecular data. Insect Syst. Evol. 37, 325–334 (2006).
53. Lukhtanov, V. A. & Dantchenko, A. V. Principles of highly ordered metaphase
I bivalent arrangement in spermatocytes of Agrodiaetus (Lepidoptera).
Chromosome Res. 10, 5–20 (2002).
54. Lorković, Z. in Butteries of Europe, Vol 2 (ed. Kudrna, O.) 332–396 (Aula, 1990).
55. Bouckaert, R. R. DensiTree: making sense of sets of phylogenetic trees.
Bioinformatics 26, 1372–1373 (2010).
Acknowledgments
We are grateful to B. Aldwell, K. Bond, S. Cuvelier, M. Fumi, J. Harding, J. Haslett, J.
Hernández-Roldán, M. Hughes, G. Jecoate, M. Marín, X. Merit, S. Montagud,
E. Regan, I. Rippey, V. Tshikolovets, N. Shapoval, S. Viader and M. Warren for providing
samples. Special thanks to B. Nelson for organizing a specimen loan from National
Museums Northern Ireland, and to G. Lamas for comments on the manuscript. Support
for this research was provided by the Russian Foundation for Basic Research (grants
RFFI 09-04-01234, 11-04-00734 and 11-04-00076), by grant NSH-3332.2010.4 (Leading
Scientic Schools) and by the programs of the Presidium of Russian Academy of Science
‘Gene Pools and Genetic diversity’ and ‘Origin of biosphere and evolution of geo-
biological systems’ to V.A.L.; by the Spanish MICINN (projects CGL2007-60516/BOS
and CGL2010-21226/BOS to V.D., G.T. and R.V., and predoctoral fellowship BES-2008-
002054 to G.T.); and by a predoctoral fellowship from UAB to V.D.
Author contributions
All authors conceived and designed the research. G.T. and V.D. performed phylogenetic
analyses. V.D. performed morphometrical analyses. V.A.L. performed karyological
analyses. V.D. and R.V. wrote the manuscript, and all authors discussed the results and
commented on the manuscript.
Additional information
Accession Codes: All novel sequences obtained in this study have been deposited to
GenBank under accession codes JF512569 to JF513007.
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing nancial interests: e authors declare no competing nancial interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
How to cite this article: Dincă, V. et al. Unexpected layers of cryptic diversity in wood
white Leptidea butteries. Nat. Commun. 2:324 doi: 10.1038/ncomms1329 (2011).
... mnemosyne sp.2 from the Middle East and P. mnemosyne sp.3 from Southern Europe (Condamine 2018;Condamine et al. 2018). In respect to the high levels of genetic divergence and weak morphological differences, the P. mnemosyne species complex is similar to a group of cryptic taxa discovered within the genus Leptidea Billberg, 1820 (Pieridae) (Dincă et al. 2011). The latter complex contains three species: Leptidea sinapis (Linnaeus, 1758), L. reali (Reissinger, 1989), and L. juvernica (Williams, 1946). ...
... The latter complex contains three species: Leptidea sinapis (Linnaeus, 1758), L. reali (Reissinger, 1989), and L. juvernica (Williams, 1946). These morphologically cryptic white-wood butterfly taxa can reliably be identified by means of either DNA sequences or karyological data (Dincă et al. 2011;Lehtonen et al. 2017;Talla et al. 2019 (2018) and Condamine et al. (2018) belongs to P. nebrodensis. It is a distinct species and can be distinguished from P. mnemosyne s. str. ...
Article
Full-text available
Recent multi-locus phylogenetic studies repeatedly showed that what was thought to be the Clouded Apollo butterfly Parnassius mnemosyne (Linnaeus, 1758) represents a cryptic species complex. This complex contains at least three distant species-level phylogenetic lineages. Here, we compile a set of morphology- and DNA-based evidences supporting the distinctiveness of two species in this group, i.e. P. mnemosyne s. str. and P. nebrodensis Turati, 1907 stat. rev. These species can be distinguished from each other based on a combination of diagnostic characters in the male genitalia structure, wing scale patterns, and the forewing venation. The species status of P. nebrodensis is supported based on unique nucleotide substitutions in the mitochondrial (COI, ND1, and ND5) and nuclear (Wg and EF-1a) genes. P. nebrodensis is endemic to the Western Mediterranean Region. This species shares a disjunctive range through the Pyrenees, Western and Central Alps, Apennines, and the Nebrodi and Madonie mountains on Sicily. Altogether 38 nominal taxa initially described as P. mnemosyne subspecies are considered here to be junior synonyms of P. nebrodensis. At first glance, P. nebrodensis can be assessed as an endangered species due to its restricted distribution, narrow range of habitats, and ongoing population decline. Isolated populations of this species scattered through mountain ranges need special management and conservation efforts.
... The use of discriminant analysis in other groups of Lepidoptera using measurements of parts of the wings and structures of the male genitalia has been previously used to delimit species (Kolev 2005;Hernández-Roldán and Munguira 2008;Prieto et al. 2009;Núñez et al. 2021) and even to confirm cryptic species (Dincâ et al. 2011). According to the discriminant analyses in which linear measurements were used in wing characters (Fig. 3A, B), the results were consistent with the Bayesian tree. ...
Article
Our research focuses on demonstrating the existence of cryptic species named under Biblis aganisa Boisduval. We used COI sequences to delimit Biblis species for Mexico using species delimitation analyses and examined phylogenetic relationships with sequences from Mexico, Costa Rica, Argentina, USA, and Guana Island using a Bayesian inference tree. We performed a discriminant analysis with quantitative traits using female and male wing and genitalia, and a tree of maximum parsimony based on 39 qualitative characters of wings, head, and male genitalia. The results were congruent in the three analyses. Three groups were formed based on DNA, ECO 01 + DHJ02, ECO 02 + DHJ01, and ECO 03. The characters that contributed over 50% separation were for wings: wing length, anal margin length, and distance from the band to the outer margin; for male genitalia, angle of the integument, uncus, and the length of the hypandrium, while for females, it was the angle of the anteapophysis and the length of the abdomen. For the analysis of qualitative characters, a tree of maximum parsimony was obtained where 20 characters were informative. We confirmed the existence of three cryptic Biblis species in Mexico, two not yet described, and one corresponding to B. aganisa (ECO 02), which is sympatric in Oaxaca and Sinaloa (ECO 03) and in the Yucatan Peninsula (ECO 01).
... In particular, this is true for the pair of sibling species Leptidea sinapis -L. juvernica (Dincǎ et al., 2011). ...
Article
The hundred-year dynamics of gamma diversity and butterflies’ abundance have been assessed for the first time in Russia for the Nizhny Novgorod Region. According to analyzed data, we forecast that no significant change in the number of 104 species of butterflies is expected, the number of 33 species in the near future is likely to increase, and the number of 11 species will continue to decline in the near future. A decrease in abundance of 23 steppe and arcto-boreal species is also expected. The reasons for the decrease in the number of species are considered, supported by the long-term monitoring dataset. Extrapolation of our conclusions for Russian regions with similar conditions (Kirov, Ivanovsk, Vladimir, Kostroma, Penza, and Ulyanovsk regions and the Republics of Mari El, Chuvash, and Mordovia), taking into account the natural conditions of these areas, seems applicable and promising.
... Although populations may have diversified enough to become separate species with distinct vocalizations, their plumage patterns can be conserved and are hence not useful for species identification. Differences in song may help uncover these cryptic species [13]. Many birds are "high canopy birds", making them difficult to trap to allow useful morphological measurements such as weight, bill length and tail or wing length, or the gathering of genetic material. ...
... as of mid-October 2020; Ratnasingham and Hebert, 2007). Many European Lepidoptera species show deeply divergent mitochondrial intraspecific lineages raising the possibility of high levels of cryptic diversity (Dincȃ et al., 2011(Dincȃ et al., , 2013Hausmann et al., 2013;Mutanen et al., 2013Mutanen et al., , 2016Vodȃ et al., 2015;Hernández-Roldán et al., 2016). Little is known about the extent to which these barcode splits reflect biological species. ...
Article
Full-text available
Gracillariidae is the most species-rich leaf-mining moth family with over 2,000 described species worldwide. In Europe, there are 263 valid named species recognized, many of which are difficult to identify using morphology only. Here we explore the use of DNA barcodes as a tool for identification and species discovery in European gracillariids. We present a barcode library including 6,791 COI sequences representing 242 of the 263 (92%) resident species. Our results indicate high congruence between morphology and barcodes with 91.3% (221/242) of European species forming monophyletic clades that can be identified accurately using barcodes alone. The remaining 8.7% represent cases of non-monophyly making their identification uncertain using barcodes. Species discrimination based on the Barcode Index Number system (BIN) was successful for 93% of species with 7% of species sharing BINs. We discovered as many as 21 undescribed candidate species, of which six were confirmed from an integrative approach; the other 15 require additional material and study to confirm preliminary evidence. Most of these new candidate species are found in mountainous regions of Mediterranean countries, the South-Eastern Alps and the Balkans, with nine candidate species found only on islands. In addition, 13 species were classified as deep conspecific lineages, comprising a total of 27 BINs with no intraspecific morphological differences found, and no known ecological differentiation. Double-digest restriction-site associated DNA sequencing (ddRAD) analysis showed strong mitonuclear discrepancy in four out of five species studied. This discordance is not explained by Wolbachia-mediated genetic sweeps. Finally, 26 species were classified as “unassessed species splits” containing 71 BINs and some involving geographical isolation or ecological specialization that will require further study to test whether they represent new cryptic species.
Article
Full-text available
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.
Article
Full-text available
1. The description of how biological information is compiled over time is essential to detect temporal biases in biodiversity data that could directly influence the utility, comparability, and reliability of ecological and biogeographical studies. 2. We explore trends in species recording over time using one of the most spatially and temporally comprehensive country-level databases for any group of insects in the world – the database of butterfly occurrences from Great Britain. 3. Firstly, we used two crucial milestones (the year in which the taxonomic inventory is complete, i.e., when the last species was recorded, the year in which all species are recorded together for the first time) to delimit three main phases in the process of biodiversity recording (taxonomic, faunistic and exhaustive phases). Secondly, we aimed to quantify how far species features (attractiveness and detectability) influence the process of recording through time. 4. During the first stage of biodiversity compilation, when the main aim is to complete the taxonomic inventory (taxonomic period), entomologists tend to record attractive species more frequently. However, once the inventory is complete, particularly in the period during which more spatially and temporally comprehensive information about species distribution is amassed (the exhaustive period), the recording pattern clearly changes to more detectable species. 5. Common, highly detectable species are undersampled in the first phase of biodiversity data compilation and oversampled in the final stages. Awareness of such temporal patterns in recording is necessary in order to correctly interpret and address bias in insect biodiversity trends.
Article
Full-text available
Recombination reshuffles the alleles of a population through crossover and gene conversion. These mechanisms have considerable consequences on the evolution and maintenance of genetic diversity. Crossover, for example, can increase genetic diversity by breaking the linkage between selected and nearby neutral variants. Bias in favor of G or C alleles during gene conversion may instead promote the fixation of one allele over the other, thus decreasing diversity. Mutation bias from G or C to A and T opposes GC-biased gene conversion (gBGC). Less recognized is that these two processes may -when balanced- promote genetic diversity. Here we investigate how gBGC and mutation bias shape genetic diversity patterns in wood white butterflies (Leptidea sp.). This constitutes the first in-depth investigation of gBGC in butterflies. Using 60 re-sequenced genomes from six populations of three species, we find substantial variation in the strength of gBGC across lineages. When modeling the balance of gBGC and mutation bias and comparing analytical results with empirical data, we reject gBGC as the main determinant of genetic diversity in these butterfly species. As alternatives, we consider linked selection and GC content. We find evidence that high values of both reduce diversity. We also show that the joint effects of gBGC and mutation bias can give rise to a diversity pattern which resembles the signature of linked selection. Consequently, gBGC should be considered when interpreting the effects of linked selection on levels of genetic diversity.
Article
Full-text available
The study of global biodiversity will greatly benefit from access to comprehensive DNA barcode libraries at continental scale, but such datasets are still very rare. Here, we assemble the first high-resolution reference library for European butterflies that provides 97% taxon coverage (459 species) and 22,306 COI sequences. We estimate that we captured 62% of the total haplotype diversity and show that most species possess a few very common haplotypes and many rare ones. Specimens in the dataset have an average 95.3% probability of being correctly identified. Mitochondrial diversity displayed elevated haplotype richness in southern European refugia, establishing the generality of this key biogeographic pattern for an entire taxonomic group. Fifteen percent of the species are involved in barcode sharing, but two thirds of these cases may reflect the need for further taxonomic research. This dataset provides a unique resource for conservation and for studying evolutionary processes, cryptic species, phylogeography, and ecology.
Book
Full-text available
The study summarizes the literature data and our own knowledge about the occurrence of diurnal butterflies (Lepidoptera, Papilionoidea) in the Ondavská vrchovina highlands orographic unit (Western Carpathians, Slovakia). In the past, little attention was paid to the study of butterflies in this area. Therefore, intensive research on butterfly biodiversity is currently being carried out, mainly in the northern part of the territory by the author of this study. It will serve as the basis for further research in this area., i.e., it will confirm, refute or supplement the species list with new knowledge. Several faunistic data from this area were listed in the “Lepidoptera Prodrome of Slovakia” (HRUBÝ 1964) and its supplements (REIPRICH 1977; REIPRICH & OKÁLI 1988; 1989a, b), mainly due to several authors (e.g., PANIGAJ Ľ, JÁSZAY T., REIPRICH A., PETRAŠOVIČ J.). A survey of butterflies was conducted in the vicinity of 34 villages during the years 2008 – 2020. This study builds on the results obtained in the study area of north-eastern Slovakia and complement faunistic data which may be the basis for further ecological evaluation. A total of 17, 793 individuals were evaluated and 88 species belonging to 6 families were identified. The number of species found according to the habitat preferences of the butterflies was as follows: 15 ubiquitous species, 39 mesophilic species, 29 xerothermophilic species, 4 hygrophilous and 1 tyrphophilous species. Similarly, the studied sites represent a set of several microhabitats that creates favourable conditions for the survival of several species. According to the faunal element of butterfly distribution, 8 different types were confirmed, dominated by Palaearctic species dominated (59.0%). Among these, Palaearctic species (44.3%) and West Palearctic species (14.7%) significantly predominated. Likewise, Eurosiberian species were represented quite often (20.4%), while other types were represented by only a smaller number of species. The occurrence of these species was in accordance with the location of the territory within Europe. Thermophilic butterfly species belonging to the Ponto-Mediterranean (4.5%) and Mediterranean (2.3%) faunistic elements were also significant, which further points to the thermophilic character of the lepidopteran fauna of the Ondavská vrchovina highlands. According to the mobility of butterflies, the occurrence of mainly sedentary species (77.3%) with lesser or greater affinity for the environment was confirmed. The occurrence of sedentary species, especially extremely to very sedentary ones, is important from a conservation point of view, as these species are strongly associated with habitats. They represent an important indicator of the quality of lepidopterocenoses, because their number significantly decrease with changes in the quality of the environment. According to the type of vulnerability and endangerment of butterflies, the occurrence of mainly five categories was confirmed, up to 69 species (79.3%) of which belonged to the category of least concerned species (LC). In addition, 17 species with a different status of threatened or of European and National importance were also recorded (Parnassius mnemosyne L., Iphiclides podalirius L., Carcharodus flocciferus Zell., Aporia crataegi L., Lycaena dispar Haw., L. alcipron Rott., Satyrium w-album Knoch, Pseudophilotes vicrama Moore, Phengaris arion L., Polyommatus daphnis Den. et Schiff., Polyommatus bellargus Rott., Argynnis laodice Pall., Brenthis ino Rott., Melitaea phoebe Den. et Schiff., M. diamina Lang, M. aurelia Nick. and M. britomartis Assm.) as well as Nymphalis xanthomelas Den. et Schiff., which has insufficient data on Slovakia. In conclusion, analyses of published and unpublished data showed that 114 species of butterflies belonging to 55 genera from 6 families were confirmed in the monitored area as follows by family: Papilionidae (3 species of 3 genera), Hesperiidae (11 species of 7 genera), Pieridae (14 species of 7 genera), Riodinidae (1 species of 1 genus), Lycaenidae (35 species of 13 genera) and Nymphalidae (50 species from 24 genera). However, by comparison it was found that out of the total number of species recorded, 91 species were found for both periods, i.e., older (until 1990) and new (after 1990). Thus, in total, 8 new species were recorded for the fauna (Carcharodus flocciferus Zell., Colias erate Esp., Polyommatus amandus Schn., Nymphalis xanthomelas Den. et Schiff., Neptis rivularis Sc., Euphydryas maturna L., Melitaea didyma Esp. and Brintesia circe F.). In contrast, 15 species were not captured in the present (Pyrgus serratulae Ram., P. alveus Hb., Favonius quercus L., Satyrium ilicis Esp., Cupido minimus Fssl., Glaucopsyche alexis Poda, Polyommatus dorylas Den. et Schiff., P. thersites Cant., P. coridon Poda, Argynnis pandora Den. et Schiff., Melitaea trivia Den. et Schiff., Lasiommata maera L., Coenonympha tullia Müll., Hyponephele lycaon Kühn and Chazara briseis L.). At the same time, for 13 species (Carcharodus alcae Esp., C. flocciferus Zell., Aporia crataegi L., Pieris bryoniae Hb., Satyrium w-album Knoch, Cupido alcetas Hffmsg., Aricia eumedon Esp., Polyommatus amandus Schn., Limenitis populi L., L. camilla L., Neptis rivularis Sc., Euphydryas maturna L. and Erebia ligea L.) their occurrence was limited to a few localities or even to a single site, so their future occurrence is highly endangered and questionable and they will thus require increased attention. The occurrence of Colias alfacariensis Rib. in the north-east of Slovakia is also questionable and requires further investigation with respect to problematic determination.
Article
Full-text available
Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.
Article
Full-text available
Overlapping measurements in the length of the genitalia of Leptidea sinapis/reali collected in Slovenia triggered an investigation of a possible natural hybridization between these two well known sibling species of butterflies. Random polymorphic: DNA (RAPD) was used to generate species specific markers and sequences of the cytochrome oxidase subunit one gene for determination of the progeny. RAPD's clustering and mitochondrial DNA (mtDNA) phylogeny were congruent with the taxonomic placement of specimens of both species, but slightly incongruent with the results of the analysis of genital morphology. Two specimens with L reali genitalia measurements, but genetically belonging to L. sinapis, had species specific RAPD markers of both species indicating probable hybrid origin. All the specimens with genitalia of intermediate length were also genetically assigned to L. sinapis indicating a possible one way introgression as predicted from their genitalia morphology. Leptidea sinapis was found predominantly in xerothermic habitats in Slovenia, whereas L. reali was more of a generalist except in the sub-Mediterranean region where it is limited to humid meadows.
Article
Full-text available
Do the sibling species of small whites, Leptidea sinapis and L. reali (Lepidoptera, Pieridae) differ in habitat preferences? Biologia, Bratislava, 58: 943—951, 2003; ISSN 0006-3088. We analysed the habitat preferences of two cryptic siblings of Leptidea butter-flies, L. sinapis and L. reali, using (i) data from the Czech distribution atlas (1,400 identified specimens from 200 grid squares), (ii) data on the habitat association of 359 specimens from 111 sites in the Czech Republic and Slo-vakia, and (iii) an ordination analysis of the two butterflies in 21 limestone quarries in Moravia (Czech Republic). The distribution atlas data revealed that L. sinapis is significantly rarer in the Czech Republic than L. reali and is restricted to warm areas of low elevations, while L. reali is widespread. The two species tend not to co-occur and there is a clear latitudinal gradient in distribution of L. sinapis. Regarding habitat preferences, L. sinapis was overrepresented in xerothermic habitats and absent in hygrophilous habitats, while L. reali occurred in all habitat types. The pattern of occurrence of the species in the limestone quarries suggested that L. sinapis is a xerophilous specialist associated with early-successional vegetation, whereas L. reali is a generalist in Central Europe. Given the decline of xerothermic habitats due to successional changes, L. sinapis should be, at least in the Czech Republic, included in the list of endangered species.
Article
After analysis of the original description of Leptidea reali as a new species from the E Pyrenees, it is now characterised in more detail, including notes on oviposition, food plants, habitat and sexual isolation. It is found not only in the Pyrenees but also in Croatia and some adjoining countries, as well as in Sweden, Poland, and Ukraine, where it occurs simpatrically with sinapis, predominantly in lowlands, with Lathyrus pratensis as the main host plant. -Author
Article
The phylogenetic relationships of 19 European species of the subfamily Argynninae were studied using electrophoresis at 12 presumptive enzyme loci and by sequencing of a segment spanning 445 bp at the mtDNA ND1 locus. Both types of markers show that the genera defined by systematics using morphological characters correspond to monophyletic assemblages. At the upper classification level of tribes, mtDNA appears to be a better evolutionary marker than enzymes. Even though the molecular method employed satisfactorily defined the phyletic relations among Boloriidi, they failed to clearly unravel them within the Argynnidi, which have probably undergone an ancient and rapid evolutionary radiation. The genus Issoria, which is attributed to the Argynnidi using morphological characters, is assigned to the Boloriidi by molecular data.
Article
A morphometric approach was used to test the possibility of discriminating between L. sinapis and L. reali by taking into account some new genitalic characters in addition to those used in previous surveys. Principal component analysis, performed on the size-and-shape data sets and on the size-adjusted data sets, has allowed two completely separate morphotypes to be detected, both in males and in females. Discriminant analysis has confirmed the separation of previously detected morphotypes and has correctly classified 100% of the specimens in both sexes with six discriminative characters being identified in males and two in females. However, some of these discriminative characters were not considered reliable enough because of the high associated measurement error and the scarce discriminative power. Reliable discriminative characters were: vinculum (≈ valve) width, length of phallus (≈ aedoeagus ≈ aedeagus), saccus and uncus in males and ductus bursae length in females. The main topics discussed are: a comparison of the discriminative characters with previous studies, the sources of measurement error and the devices used to reduce it, as well as the between and within-species variability of the characters.
Chapter
Five of the world’s six species of Maculinea butterfly live in Europe. They are among the few insects for which specific conservation measures have been taken, and are regarded as ‘flagship’ species by many Western conservationists (Anon., 1993a). Thus Maculinea butterflies are regularly used for logos and on stamps (Anon., 1981, 1993b), have been the subject of numerous radio and television broadcasts and have frequently featured in magazines as diverse as the National Geographic and The Economist and in newspapers ranging from the European to the Sun.
Article
Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. C SIMON, F FRATI, A BECKENBACH, B CRESPI, H LIU, P