DataPDF Available
ESM Annex 4 – Species pairs that are not reliably identified through
DNA barcoding
Carcharodus flocciferus (Zeller, 1847) – Carcharodus orientalis Reverdin, 1913
Fig. S1. Wing vouchers of representative
barcoded Carcharodus flocciferus and C.
orientalis. a, b. males of C. flocciferus. c, d.
males of C. orientalis. Scale bar is 10 mm.
Fig. S2. Lateral view of representative male genitalia of C.
orientalis indicating the elements measured for linear
morphometry. Cpw – cuiller proximal width; Cdw – cuiller
distal width. Scale bar is 0.5 mm.
Wing morphology: Does not allow for a reliable separation between the two species. No
feature of the wing pattern is constant enough to be used as discriminative character (fig.
Fig. S3. Lateral view of representative male genitalia of Carcharodus flocciferus and C. orientalis. a, b. C.
flocciferus (phallus removed). c. phallus of C. flocciferus. d, e. C. orientalis (phallus removed). f. phallus of
C. orientalis. Scale bar is 1 mm.
Male genitalia: It can be used for the separation of the two species. The most useful
character is the tip of the cuiller, which is broad in C. flocciferus and comparatively
narrower in C. orientalis (Higgins 1975; Slamka 2004) (figs S2, S3a-f). The validity of this
character was tested by employing both linear (fig. S2) and geometric morphometry (fig. 3
of the main text).
For linear morphometry, because the tip of the cuiller does not possess any constant
landmarks, we measured the cuiller distal width (Cdw) at 0.06 mm from the tip of the
cuiller and the cuiller proximal width (Cpw) at 0.35 mm (fig. S2). Based on our samples,
these variables form two non-overlapping morphotypes that can be attributed to C.
flocciferus and C. orientalis in a bivariate scatter plot (fig. S4).
For geometric morphometry, we defined two landmarks and 22 semi-landmarks. The two
landmarks (marked as empty circles in fig. 3a of the main text) were defined as the
intersection points with the margins of the cuiller generated by a transversal line crossing
the cuiller at 0.35 mm distance from its tip (fig. 3a main text). The RWs explaining more
than 1% of the variance were 15. A scatter plot of RW1 and RW2 (explaining respectively
37.01 and 19.63% of variance) revealed the presence of two discrete clusters of valva
shape (fig. 3a main text) equivalent to those obtained with the linear morphometric
analysis. By maximizing and minimizing inter-cluster and intra-cluster differences
respectively, K-Means identified two clusters. RW1 revealed to be significantly different
among the two clusters thus representing the variable responsible for the discrimination
(table S1). When compared with the scatter plot of fig. 3a (main text), K-Means confirmed
the validity of the two clusters.
Fig. S4. Male specimen bivariate scatter plot
using the cuiller proximal width (Cpw) and the
cuiller distal width (Cdw) as discriminative
characters. Circles Carcharodus orientalis;
triangles – C. flocciferus.
Table. S1. ANOVA table for K-Means clustering. In
bold the variable showing significant differences between
the two clusters.
Cluster Error
warp Mean
Square df Mean
Square df F Sig.
RW1 0.0635453 1 0.0009129 14 69.607679 8.371E-07
RW2 5.7327E-05 1 0.000751331 14 0.076300631 0.786410559
RW3 0.000157239 1 0.000597702 14 0.263072691 0.616014176
RW4 1.93113E-05 1 0.000172418 14 0.112002754 0.742836619
RW5 8.89821E-05 1 0.000114223 14 0.779017833 0.392344999
RW6 6.59647E-06 1 8.45746E-05 14 0.077995941 0.78411518
RW7 3.43123E-06 1 2.52897E-05 14 0.135677036 0. 718126957
RW8 6.35191E-08 1 1.95141E-05 14 0.003255046 0. 955309261
RW9 1.88692E-06 1 1.23221E-05 14 0.15313348 0.701449826
RW10 1.88742E-07 1 9.45028E-06 14 0.019972072 0. 889628511
RW11 3.23497E-06 1 7.24223E-06 14 0.4466808 0.514784216
RW12 1.3563E-07 1 6.90462E-06 14 0.019643416 0.890534002
RW13 2.45177E-07 1 3.32574E-06 14 0.073721062 0. 789956718
RW14 5.72565E-07 1 2.26356E-06 14 0.252949119 0. 622825312
RW15 4.10348E-10 1 1. 933E-06 14 0.000212286 0.988580812
DNA barcoding: Carcharodus flocciferus is paraphyletic with respect to C. orientalis (fig.
3b main text). The minimum interspecific distance between the two taxa is 0.46%. Since
no barcodes are shared, species-level identifications seem possible given the current data,
but more sampling is needed to test the robustness of these results.
Comments: Although described originally as a species, C. orientalis has for a long time
been treated as a subspecies of C. flocciferus (e.g. Higgins 1975). However, karyological
data (Lesse 1960) and further taxonomical studies (Jong 1974) suggested that C. orientalis
is a distinct species so that this taxon is currently treated accordingly by most authors.
Cohabitation between the two species has been reported from Greece (Lafranchis 2003)
and it is strongly suspected in south-eastern Romania (Dincă et al. 2009).
In Romania, C. flocciferus is relatively widespread (except for Dobrogea where it appears
to be very local) (Rákosy et al. 2003; Dincă et al. 2009). Carcharodus orientalis is
currently known only from the extreme east and south-east of the country (Dobrogea –
relatively widespread, and a handful of sites in Moldavia) (Rákosy & Varga 2001; Székely
2008; Dincă et al. 2009).
Colias crocea (Fourcroy, 1785) – Colias erate (Esper, 1805)
Wing morphology: The two species (but especially C. erate) display impressive
intraspecific variability. Their separation is possible for more or less typical specimens (fig.
S5a,e,f). However, given the large number of specimens bearing intermediate characters
between the two species (fig. S5b-d), reliable identifications based on wing morphology
alone can sometimes be unreliable. In fact, C. erate is known to display a large number of
forms in Romania (Popescu-Gorj 1978), some of them closely resembling C. crocea. It is
worth mentioning that the presence of an oval androconial patch at the base of the
hindwing upperside is not necessarily a diagnostic sign for C. crocea, as some forms of C.
erate may also have it (Popescu-Gorj 1978). Moreover, certain specimens with wing
morphology closer to C. crocea than C. erate may lack this patch (e.g. fig. S5b).
Fig. S5. Wing vouchers and male genitalia (lateral view) of representative barcoded C. crocea (a), C. erate (e,
f) and three possible hybrids (b-d). g. Neighbour-joining tree of COI barcodes of Romanian C. crocea and C.
erate with bootstrap values >50% indicated.
Male genitalia: Typical specimens of both species (fig. S5a,d-f) can be reliably
differentiated due to differences in the posterior border of the valvae (evenly rounded in C.
crocea and strongly angled in C. erate) (e.g. Niculescu 1963; Popescu-Gorj 1978; Higgins
1975; Slamka 2004). Nevertheless, based on our material, we found that this character is
also subject to individual variability to the extent that it becomes difficult to assign certain
specimens to any species (e.g. fig. S5e,f).
DNA barcoding: The two species share barcodes (fig. S5g). A similar situation was
reported for Central Asia (Lukhtanov et al. 2009). This phenomenon is likely to reflect the
presence of hybrids, or to be the consequence of historical introgression, as natural hybrids
have been recorded (Alberti 1943; Tolman & Lewington 2008; Descimon & Mallet 2009).
Pieris napi (Linnaeus, 1758) – Pieris bryoniae (Hübner, 1806)
Wing morphology: For the Romanian specimens, identification is reliable for females,
which are much darker in P. bryoniae than in P. napi (fig. S6d,h,l), but can be difficult for
males. This is due to possible confusions between P. bryoniae and certain specimens of the
spring brood of P. napi that have the dark veins on the underside of the hindwings well
developed (e.g. fig. S6c). The summer brood of P. napi is easier to distinguish from P.
bryoniae (fig. S6e-h).
Male genitalia: It does not allow for the reliable separation of the two species (fig. S7a-d)
(Higgins 1975, Gorbunov 2001).
DNA barcoding: 27 of the 29 specimens examined formed two clades with a minimum
interspecific divergence of 1.85% (fig. S8). However, two specimens identified as P. napi,
which could actually represent hybrids, (fig. S6a,h) clustered with P. bryoniae. Although P.
bryoniae has not been recorded from the area where they were collected (the base of the
Domogled Mountain, Pecinișca), it is known to occur between ca. 900-1700 m in the
mountains situated about 50 km to the north (Retezat) (Rákosy 1997). However, it could
occur much nearer as the whole area is connected by peaks of more than 1000 m. Even the
area of the Domogled Mountain, which reaches 1000 m, hosts several mountainous species
such as Aricia artaxerxes (Fabricius, 1793), Erebia ligea (Linnaeus, 1758), Erebia euryale
(Esper, 1805), Erebia melas (Herbst, 1796).
Comments: Pieris bryoniae is a taxon with controversial status. Recently, it is most often
treated as a distinct species, but also as a subspecies of P. napi (e.g. Higgins et al. 1991;
Gorbunov 2001). Studies based on enzyme electrophoretic approaches pointed out the high
similarity between the two taxa (e.g. Geiger & Scholl 1985; Geiger 1990). The two taxa
are known to hybridize regularly where their ranges contact (Geiger & Shapiro 1992;
Porter & Geiger 1995; Porter 1997; Descimon & Mallet 2009).
Fig. S6. Wing vouchers of representative barcoded Pieris napi and P. bryoniae. a-c. P. napi (males of the
spring brood). d. P. napi (female of the spring brood). e-g. P. napi (males of the summer brood). h. P. napi
(female of the summer brood). i-k. males of P. bryoniae. l. female of P. bryoniae. Scale bar is 10 mm.
Fig. S7. Lateral view of representative male genitalia of Pieris napi (a, b) and P. bryoniae (c, d). Scale bar is
0.5 mm.
Fig. S8. Neighbour-joining tree of COI barcodes of Romanian Pieris napi and P. bryoniae with bootstrap
values >50% indicated.
Coenonympha tullia (Müller, 1764) – Coenonympha rhodopensis Elwes, 1900
Wing morphology: Romanian specimens of C. rhodopensis lack (or have very little
developed) ocelli on the hindwing underside (fig. S9c,d) and can be distinguished from C.
tullia where these ocelli are more developed (fig. S9a,b) (Rákosy 1993).
Male genitalia: The material examined by us displayed differences in the shape of the
valva of the male genitalia, which is more slender in the terminal part in C. rhodopensis
compared to C. tullia (fig. S10a-c). Differences in the male genitalia were also reported by
Rákosy (1993).
Fig. S9. Wing vouchers of representative barcoded specimens of Coenonympha tullia (a, b) and C.
rhodopensis (c, d). Scale bar is 10 mm.
Fig. S10. Lateral view of representative male genitalia of C. rhodopensis (a, b) and C. tullia (c). Scale bar is
0.5 mm.
DNA barcoding: Coenonympha rhodopensis is recovered as paraphyletic with respect to
C. tullia (fig. S11). The minimum interspecific distance is only 0.3%, but no barcodes are
shared between the two taxa. Although species-level identifications seem possible based on
unique haplotypes for each species, more sampling is needed to test the robustness of these
Fig. S11. Neighbour-joining tree of COI barcodes of Romanian Coenonympha rhodopensis and C. tullia with
bootstrap values >50% indicated.
Erebia euryale (Esper, 1805) – Erebia ligea (Linnaeus, 1758)
Fig. S12. Wing vouchers of representative barcoded males of Erebia euryale and E. ligea. The detail of the
forewing under strong transmitted light to the right of each specimen illustrates the absence/presence of
androconial patches. a, c. E. euryale. b, d. E. ligea. Scale bars represent 10 mm.
Wing morphology: The two species can be separated through wing morphology. Males of
E. ligea can be easily identified by the presence of androconial patches on the forewings
(these are well visible in strong light) (fig. S12a-d). Such patches lack in E. euryale
(Sonderegger 2005; Slamka 2004; Tolman & Lewington 2008). The females are especially
distinct in the pattern on the underside of the hindwings.
Fig. S13. Lateral view of representative valvae of male genitalia of Erebia euryale (a, c, e) and E. ligea (b, d, f).
Scale bar is 1 mm.
Male genitalia: It is usually reliable for identification. Although the specimens examined
by us were found to display considerable variability in the male genitalia, Erebia ligea has
larger and more irregular teeth on the valva while in E. euryale these teeth are usually
smaller and distributed in a partial double series (Higgins 1975). In E. ligea, the terminal
part of the valva tapers in a more pronounced way than in E. euryale (Sonderegger 2005)
(fig. S13a-f).
DNA barcoding: The NJ tree displays a complex pattern involving polyphyly (fig. S14).
Although no barcodes are shared between the two species, the minimum interspecific
distance is very low (0.15%). This situation suggests either incomplete lineage sorting or
local hybridization events.
Fig. S14. Bootstrap neighbour-joining tree of COI barcodes of Romanian Erebia ligea and E. euryale with
bootstrap values >50% indicated.
Hipparchia semele (Linnaeus, 1758) Hipparchia volgensis (Mazochin-Porshnjakov,
Fig. S15. Wing vouchers of representative barcoded Hipparchia semele and H. volgensis. a, b. males of H.
semele. c. male of H. volgensis. Scale bar is 10 mm.
Wing morphology: These two species are indistinguishable based on wing morphology
(fig. S15a-c) (Kudrna 1977; Lafranchis 2004; Tolman & Lewington 2008; Pamperis 2009).
Fig. S16. Lateral view of male genitalia of representative Hipparchia semele and H. volgensis. a, b. male
genitalia of H. semele. c. male genitalia of H. volgensis. d, e. phallus of H. semele. f. phallus of H. volgensis.
Scale bar is 1 mm.
Fig. S17. Male specimen bivariate scatter
plot using the dorsal process of valva and the
uncus length as discriminative characters.
Fig. S18. Neighbour-joining tree of COI barcodes of
Romanian Hipparchia semele and H. volgensis with
bootstrap values >50% indicated.
Male genitalia: By using linear morphometry, we found that the dorsal process of the
male valva (fig. S16b) is considerably less pronounced in our specimen of H. volgensis
compared to the analyzed specimens of H. semele (fig. S16a-c, S17). This feature has been
mentioned as one of the main diagnostic characters of H. volgensis (Kudrna 1977).
DNA barcoding: Our single specimen of H. volgensis appears to have identical barcode
with several specimens of H. semele (fig. S18). Surprisingly, it is one specimen with
genitalia of H. semele type that displays a rather high maximum intraspecific divergence of
3.5%. The correct interpretation of this pattern, especially taking into account the
uncertainties on the taxonomic status of H. volgensis, is very difficult without additional
material and further studies are needed.
Comments: Hipparchia volgensis is a taxon that still has a controversial status. According
to Kudrna (1977), in the Balkans occurs H. v. delattini Kudrna, 1975. However, the taxon
delattini was sometimes raised to species rank by some authors (e.g. Wakeham-Dawson et
al. 2004; Pamperis 2009). Morphometrical analyses of the male genitalia involving H.
semele, H. volgensis and H. muelleri showed rather inconclusive results (Wakeham-
Dawson et al. 2003, 2004). In Romania, H. v. delattini has been recorded only recently
(Rákosy 1998). The specimen barcoded here was collected in the same area from where it
has been first recorded in Romania (Măcin Mountains). The locality where it was collected,
plus the small dorsal process of the valva in the male genitalia, led us to assign it to H. v.
delattini. The lack of genetic divergence in DNA barcodes is not in favour of the specific
distinctness of the taxon delattini but it is also not sufficient to allow definitive conclusions.
More material and additional studies are needed in order to correctly evaluate the
relationship between H. semele and H. v. delattini.
Hipparchia fagi (Scopoli, 1763) – Hipparchia syriaca (Staudinger, 1871)
Fig. S19. Wing vouchers of representative barcoded Hipparchia fagi (a, b) and H. syriaca (c, d). Scale bar is
10 mm.
Fig. S20. Detail of the underside of the hindwings for representative barcoded specimens of Hipparchia fagi
(a-d) and H. syriaca (e-h). A – indicates the dark base of s4; B – indicates the dark base of s3.
Wing morphology: It does not allow for a reliable separation between the two species (fig.
S19a-d). Pamperis (2009), based on data from Hesselbarth et al. (1995), mentioned that the
dark base of s4 (area A) and s3 (area B) on the underside hindwings (fig. S20a) are useful
for identification: if A > B - H. fagi and if A < B - H. syriaca. We tested this feature on our
barcoded specimens and found that it is accurate only in some cases (fig. S20a-h). For
example, the differences between samples EZROM629-08 (H. fagi) and EZROM1016-08
(H. syriaca) (fig. S20c,f) are small and, without examination of the genitalia, it would be
very difficult to tell to which species these specimens belong to. Since this overlap already
appeared in a relatively small sample, it is likely that such cases are not rare so that
identifications based on this character should be regarded with reserves.
Fig. S21. Jullien organ of the male genitalia of representative Hipparchia fagi and H. syriaca. a. right lamella
of the Jullien organ of H. fagi bearing three batons. b. Jullien organ of H. fagi bearing four batons. c, d. right
lamella of the Jullien organ of H. fagi bearing five batons. e. Jullien organ of H. syriaca. Scale bar is 1 mm.
Male genitalia: It provides reliable characters for identification. On of the most obvious is
related to the Jullien organ (Higgins 1975): lateral lamellae are narrow, each bearing three
to five long slender batons, in H. fagi (fig. S21a-d), while lamellae are wider and close
together, each bearing eight thick batons, in H. syriaca (fig. S21e).
Fig. S22. Neighbour-joining tree of COI barcodes of Romanian Hipparchia fagi and H. syriaca with
bootstrap values >50% indicated.
DNA barcoding: Hipparchia fagi is recovered as paraphyletic with respect to H. syriaca
(fig. S22). Although no barcodes are shared, the minimum interspecific distance is very
low (0.15%). Although species-level identifications seem possible given the current data,
more sampling is needed to test the robustness of these results.
Apatura ilia ([Denis & Schiffermüller], 1775) – Apatura metis Freyer, 1829
Fig. S23. Wing vouchers of representative barcoded Apatura ilia and A. metis. a, b. males of A. ilia f. clytie.
c. spring brood of A. metis (male). d. summer brood of A. metis (male). Scale bar is 10 mm.
Wing morphology: The form clytie of A. ilia is very similar in wing morphology to A.
metis and discrimination is possible based on subtle characters only (fig. S23a-d). The
most often mentioned discriminative features are (Higgins et al. 1991; Slamka 2004,
Lafranchis 2004; Tolman & Lewington 2008; Pamperis 2009):
The size of the black ocellus in s2 of the forewings upperside, larger in A. ilia than
in A. metis.
The discal orange band on the hindwing upperside, which in A. metis has a marked
discontinuity at v4.
The spot in space 2 of the hindwing underside is small or often absent in A. metis.
For the Romanian specimens, these characters are usually enough to allow the separation
between the two taxa. However, some specimens of A. ilia f. clytie may closely resemble A.
metis as it can be seen in fig. S22b, where the black ocellus in s2 is quite small (forewings
and hindwings) and there is a rather pronounced discontinuity at v4 (hindwings). The
adults of A. metis tend to be smaller than those of A. ilia.
Fig. S24. Lateral view of the male genitalia of representative Apatura ilia and A. metis. a, c. A. ilia (phallus
removed). b, d. A. metis (phallus removed). e-f. phallus of A. ilia. g. phallus of A. metis. Scale bar is 1 mm.
Male genitalia: Based on the material examined, we could find no clear discriminative
character in the male genitalia (fig. S24a-g). We noticed however that the genitalia of A.
metis are smaller compared to A. ilia, but this may be related to the smaller size of the
adults of A. metis. Higgins (1975) also reported that the genitalia of A. ilia are slightly
larger than those of A. metis. Studies based on more comparative material are needed in
order to test if the differences in genitalia size can be considered as species-specific or they
only reflect individual variability.
DNA barcoding: Apatura ilia is paraphyletic with respect to A. metis (fig. S25). The
minimum interspecific distance is very low (0.3%), but no barcodes are shared between the
two taxa and species-level identification is possible given the current data. However,
because of the relatively small sample size, the ability of DNA barcoding of discriminating
between the two species should be tested by adding more material.
Comments: The taxonomic status of A. metis has been subject to much debate (e.g.
Niculescu 1977, 1980; Varga 1978) as it has for a long time been considered as a
subspecies of A. ilia. However, currently most authors treat A. metis as a distinct species.
Fig. S25. Neighbour-joining tree of COI barcodes of Romanian Apatura ilia and A. metis with bootstrap
values >50% indicated.
Polyommatus bellargus (Rottemburg, 1775) – Polyommatus coridon (Poda, 1761)
Fig. S26. Wing vouchers of representative barcoded Polyommatus bellargus and P. coridon. a. male of P.
bellargus. b. male of P. coridon. Scale bar is 10 mm.
Fig. S27. Elements of the male genitalia of representative Polyommatus bellargus and P. coridon. a.
labides and falces of P. bellargus. b. labides and falces of P. coridon. c. phallus of P. coridon. d, e.
phallus of P. bellargus. f. labides and falces of P. coridon. g. phallus of P. coridon. Scale bar is 0.5 mm.
Wing morphology: These two species can be reliably separated based on wing characters
(fig. S26a,b). The different blue colour on the upperside of the wings and the differences in
the dark marginal border on the upperside of the wings (very narrow in P. bellargus and
much thicker in P. coridon) are some of the most obvious characters that allow for their
separation in the case of males. For females (not shown), differences are less evident, but
they are generally enough for safe determination.
Male genitalia: Although rather subtle, certain elements of the male genitalia are useful to
discriminate between the two species: the falces have a more robust appearance and are
more strongly hooked in L. coridon by comparison to P. bellargus (fig. S27, a,b,f). The
terminal part of the phallus is also slightly different (fig. S27c-e,g).
Fig. S28. Neighbour-joining tree of COI barcodes of Romanian Polyommatus coridon and P. bellargus with
bootstrap values >50% indicated.
DNA barcoding: The NJ tree displays a complex pattern involving polyphyly (fig. S28).
Although no barcodes are shared between the two species, the minimum interspecific
distance is very low (0.3%). The relatively high maximum intraspecific distance within L.
bellargus (2.3%) is most likely not an indicator of cryptic taxa. Instead, it may indicate a
case of introgression from L. coridon.
Comments: These two species are known to produce natural hybrids such as Polyommatus
x polonus (Zeller, 1845) (Lafranchis 2004; Tolman & Lewington 2008; Descimon &
Mallet 2009).
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The butterfly fauna of Russia is completely reviewed, with keys compiled for the 472 species in 140 genera occurring in the country. Surveys of their taxonomy and brief remarks on distribution and ecology are provided. The male genitalia and some other structures of all these taxa are depicted in more than 700 figures arranged in 41 plates, 472 distribution maps, and 119 colour photos of the butterflies in nature.
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Complete detailed colour guide of butterflies of Central Europe - eastern part (Czech republic, Slovakia, Poland, Hungary, Carpathiansukraine, Austria (partially). Determination of species, Habitats and Bionomy, Distribution, Threatened species. Unser Atlas ist nicht nur auf das Gebiet der Slowakei beschränkt, sondern umfasst auch die Fauna von Böhmen, Mähren, Polen, Ungarn und Karpatenukraine. Auch die Verbreitung der angeführten Arten in Österreich findet Erwähnung. Der Leser erhält in die Hände eine vollständige Fauna der Tagfalter dieser Länder einschließlich der Arten, welche dort verzeichnet, aber später nicht bestätigt wurden oder ausgestorben sind. Im Atlas werden somit mehr als 200 Schmetterlingsarten erwähnt, von welchen 195 eingehender bearbeitet wurden. Außer den Informationen über die Lebensweise (Bionomie) und die geographischen Verbreitung werden die Unterscheidungsmerkmale der ähnlichen Arten, ferner die Bedrohung und der Schutz der Falter, die jetzt recht aktuell sind, betont. Die Texte werden mit Federzeichnungen der Unterscheidungsmerkmale und mit Abbildungen der Schmetterlinge aus der Natur vervollständigt. Am Schluss findet man auf Farbtafeln alle erwähnten Arten abgebildet. 12, 3 x 17 cm, 288 pp.; Hardcover; 195 species; more than 400 (193 species) images of butterflies in natural, lifelike poses; 104 line drawings of genitalia and habitus; 60 colour plates with more than 1050 images of butterflies from collections.
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Two closely related Hipparchia taxa, Hipparchia delattini Kudrna, 1975 and H. semele muelleri Kudrna, 1975 have been described from the Balkans based on differences in male genital structure, compared to each other and to another nominal European taxon (H. semele Linnaeus, 1758). Subsequently, Kudrna (1977) synonymised both H. delattini and H. muelleri with H. volgensis (Mazochin-Porshnjakov, 1952). Application of multivariate statistical techniques on male genital data indicates a cline in several aspects of genital morphology linking these three taxa across Europe. Although clusters are repeatedly found that correspond with the three taxa, it is not possible to ascribe every individual specimen to one of the three Hipparchia taxa. Hipparchia muelleri is shown to occupy an intermediate position between H. semele and H. delattini. Generally, H. delattini is present in an area covering part of northern Greece and the central Balkans. H. semele is present in western Europe, the Balkans and down the western side of Greece. However, individual specimens that classify to H. delattini in the current analyses may occur much further west, where historically only H. semele has been, and there appears to be a correlation between putative taxa and altitude with H. delattini occurring at higher altitudes. It is suggested that genetic differentiation between these taxa has been maintained and enhanced during glacial-interglacial cycles. The results of this study are discussed in relation to other morphological characters and biogeography and require further testing with molecular data.
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DNA barcoding employs short, standardized gene regions (5' segment of mitochondrial cytochrome oxidase subunit I for animals) as an internal tag to enable species identification. Prior studies have indicated that it performs this task well, because interspecific variation at cytochrome oxidase subunit I is typically much greater than intraspecific variation. However, most previous studies have focused on local faunas only, and critics have suggested two reasons why barcoding should be less effective in species identification when the geographical coverage is expanded. They suggested that many recently diverged taxa will be excluded from local analyses because they are allopatric. Second, intraspecific variation may be seriously underestimated by local studies, because geographical variation in the barcode region is not considered. In this paper, we analyse how adding a geographical dimension affects barcode resolution, examining 353 butterfly species from Central Asia. Despite predictions, we found that geographically separated and recently diverged allopatric species did not show, on average, less sequence differentiation than recently diverged sympatric taxa. Although expanded geographical coverage did substantially increase intraspecific variation reducing the barcoding gap between species, this did not decrease species identification using neighbour-joining clustering. The inclusion of additional populations increased the number of paraphyletic entities, but did not impede species-level identification, because paraphyletic species were separated from their monophyletic relatives by substantial sequence divergence. Thus, this study demonstrates that DNA barcoding remains an effective identification tool even when taxa are sampled from a large geographical area.
The genetic relationships of populations of the holarctic Pieris napi-group are analyzed by means of enzyme electrophoretic methods in an attempt to phenetically cluster the populations, define major radiations and examine the resultant sublineages in detail in order to reconstruct the evolutionary history of the group as a whole. The data confirm previous observations that European napi is specifically distinct from all North American taxa investigated so far and show that the Nearctic taxa are split into at least five different species: angelika Eitschberger, marginalis Scudder, meckyae Eitschberger, oleracea (Harris), and virginiensis Edwards. There are also clear indications for an interruption of gene-flow among the east Asian taxa melete Mén. and nesis Fruhst. and japonica Shirozu. With respect to the evolution of the group the data demonstrate an early (Tertiary) split into Eurasian and North American sublineages. There is evidence for much subspeciation in both sublineages in Quaternary times.
1. The butterflies Pieris napi and Pieris bryoniae hybridize in a narrow zone at ≈ 1200 m in the Alps and Carpathians of Europe. They feed as larvae on a variety of hosts in the Brassicaceae, and few host species occur on both sides of the hybrid zone. 2. Females were captured on either side of the hybrid zone at Pont de Nant, Switzerland, eggs obtained, and larvae were offered plants from nineteen species of Brassicaceae and Reseda lutea (Resedaceae). 3. Nine of the hosts were found to have eggs or larvae already on them. Only Capsella bursa-pastoris and R. lutea were unsuitable. 4. Significant survivorship differences among suitable hosts were found. There was no interaction between butterfly species and host plant, which would indicate adaptation by these taxa to their respective suites of hosts. 5. Among suitable hosts, larvae of both taxa had higher mortality on plants with hairy leaves and on older plants beginning to senesce. 6. Differential selection on host use, if it occurs at all, is likely to be a very minor factor in the dynamics of the Pieris napi/bryoniae hybrid zone.
We used hierarchical and pairwise F-statistics to describe genetic differentiation and infer gene flow (M) on local and regional scales within and among parapatric European butterfly taxa in the Pieris napi (L.) group. Within-population allozyme variability is consistently high, and local effective population sizes are inferred to be in the thousands of individuals. The pairwise analysis yields an average neighbourhood area of radius 3.5 km. Among populations within most regions, differentiation is low and M > 2 effective individuals population-1generation-1. Pairwise comparisons within the brilannica group show a disjunction indicating that it is out of equilibrium, perhaps as a result of secondary contact between highland and lowland groups. Comparison between meridionalis groups on mainland Italy and Corsica yields M > 12; this is surely loo high and lack of equilibrium resulting from initial colonization is suspected. The hierarchical analysis indicates that 23 ≤0020M≤ 88 among the taxa napi, bryoniae and meridionalis that meet in hybrid zones; no effective gene flow barrier exists among them. This high estimate could also result from recent primary contact, but such a genetic barrier should produce the ‘edge effects' seen in population genetic simulations, and no evidence of this was found among geographically close samples of napi and bryoniae populations from Switzerland. Studies of gene flow among geographic regions are greatly limited by the equilibrium assumption, though studies of local differentiation are much less so. Population studies of gene flow on local scales at regional boundaries provide limited means of testing the equilibrium assumption, and both regional and local analyses provide testable predictions about local population structure. When the equilibrium assumption is not upheld, local patterns at regional boundaries can provide historical information about primary vs. secondary contact.
Enzyme electrophoretic data show a remarkably high degree of genetic similarity within the European group ofnapi s.l. whereas genetic differences exist at several loci between the European and the North American taxa ofnapi s.l. It is concluded that the European taxa did not differentiate to the species level and form a phylogenetically young group. The North American taxa included in this study are specifically distinct from Europeannapi and separated much earlier. Within these North American taxamarginalis, oleracea andvirginiensis did undergo speciation. The data show a splitting of the genusPieris into three species groups, each genetically differentiated to the same level. The splitting ofPieris into two genera, as suggested by earlier investigators, is not supported here.
The butterflies of Romania / Fluturii de zi din România
  • L Székely
Székely, L. 2008 The butterflies of Romania / Fluturii de zi din România. Brașov: Brastar Print.