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ORIGINAL PAPER
Journal of Insect Conservation (2024) 28:1107–1119
https://doi.org/10.1007/s10841-024-00602-2
Introduction
Over the past ∼ 150 years, our planet’s biodiversity has
declined at such a staggering rate that some are talking of
sixth mass extinction (Wake and Vredenburg 2008; McCal-
lum 2015). The ultimate goal of nature conservation is to
preserve the natural biodiversity on Earth. There are many
approaches employed to reach this goal, from protecting
ecosystems and particular habitats to focusing on endan-
gered taxa. Identifying operational conservation units is the
conservation. The basic concept is to identify evolutionary
-
cally unique entities, Ryder 1986; Moritz 1994). While
Kornél Takáts
kornel.takats@ttk.elte.hu
1
2
3
4
5
Opole 45-052, Poland
Abstract
Parnassius apollo
subspecies rank taxa described from the Carpatho-Pannonian region (Central Europe), often based on old, one-by-one
-
transsylvanicus and ssp. rosenius) and the rest of the populations (including our out-
P. apollo
our results support a single conservation unit in the region. We suggest that (i) extensive monitoring is needed to reveal
studies on Central European P. apollo
strategies. We emphasize that modern integrative taxonomy is not only important for clarifying taxonomical issues, but
also for providing basis for sound conservation management.
Keywords Parnassius · Wing
shape
Received: 30 January 2024 / Accepted: 22 May 2024 / Published online: 3 August 2024
© The Author(s) 2024
From 20 to 2? Landmark-based geometric morphometrics reveal
negligible wing-shape divergence between 20 subspecies of the
Apollo buttery, Parnassius apollo (Lepidoptera, Papilionidae), in the
Carpatho-Pannonian region
KornélTakáts1,2,3 · SándorCsősz2,3 · GergelySzövényi3· GergelyKatona4· Paweł J.Domagała5·
GáborHerczeg2,3
1 3
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Journal of Insect Conservation (2024) 28:1107–1119
2001
be prioritized based on their unique heritable characteristics
2004).
approaches can be seen as “reductionistically mistaken”
(Casacci et al. 2014). Further, even though studying ‘his-
-
ous collections is more and more promising recently, such
for every potential study object (Anderung et al. 2008;
Ellis 20082011; Call et al. 2021
2021
use to infer species boundaries and to describe biodiver-
2009; Yazdi
et al. 2012
highest heritability among various trait types (e.g. Mous-
taxonomy can be used as a surrogate for genetic analyses,
the conservation aspect, the distinction and management of
-
2006
not only serves the purpose of classifying individuals, but
The general biodiversity decline can be detected in but-
Parnassius
apollo
1999). It is
-
-
2021
and Warren 1999 -
2010). Maes et
al. (2019
occurring in Europe mainly in the 500–2500 m above sea
1935; Glassl 2005; Möhn 2005). Its
species in open mountain habitats, and P. apollo is consid-
-
ous populations inhabiting spatially more or less separate
2012
2022). In the case of P. apollo, 290 subspecies have been
described altogether (Möhn 2005), including 26 subspecies
rank taxa from the Carpatho-Pannonian region (for a map
of the region, see Fig. 1; for detailed compilation of traits
used in original descriptions and subsequent taxonomical
Table 1
(almost) all of the described subspecies in the Carpatho-
Pannonian region (Table 1
in a good agreement (Möhn 2005; Glassl 20052011).
-
ogy of the dozens of subspecies is lacking, even though it
In the present study, our goal is to provide an extensive
1) of
the described subspecies occurring in the Carpatho-Panno-
nian region based on available specimens from museum and
university collections. We aim (i) to provide taxonomically
relevant information and (ii) to equip conservation manage-
only the old, one-by-one subspecies descriptions available.
Methods
Sampling
sample sizes that are relevant for us (N > 10 for each sex
material. About 2/3 of the Carpatho-Pannonian specimens
used for our analyses can be found in the collection of the
=
al. 2016
from other museums and university collections (Table 1; for
2
P. a . rhaeticus
to a separate lineage (Todisco et al. 2010). The starting
-
1
-
-
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Journal of Insect Conservation (2024) 28:1107–1119
1952;
1988
Dragomirescu 1991; Glassl 2005; Möhn 2005).
number of specimens to our analyses (P. a. ruthenicus and
P. a. vistulicus) or specimens of uncertain provenance (P. a.
cominius and P. a. artemidor, and doubtful specimens from
distribution area of P. a. albus is outside of the Carpatho-
-
2
analysed 20 Carpatho-Pannonian subspecies and one out-
1).
The subspecies included in our study belongs to six
Fig. 1 Map depicting the former (early 20th century) distribution of the putative Parnassus apollo
×
1988
Dragomirescu 199120022003; Popov and Plushtch 20042008; Kalivoda 20082011). Dark red
2018) and the thick
×
the scheme for 10 ×
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Journal of Insect Conservation (2024) 28:1107–1119
2008
shape variation is more likely to describe adaptive variation.
-
-
P. apollo and the photographing setups, camera-dependent
the museum specimen (see Fig. 2).
Landmarks
1991; Rohlf and Marcus 19932012
Carpatho-Pannonian area (Fig. 12). The geo-
-
Romanian Western Carpathians including one subspecies,
-
P. a. rhae-
ticus subspecies.
Photographing
Due to the high museum value of the specimens, removal
Table 1
+
Abbr. GEO N m N f N t
P. apollo serpentinicus , 1925 20 11 31 -
P. a. antiquus 10 10 20 +
P. a. braniskoi 12 19 31 -
P. a. candidus , 1911 16 53 -
P. a. carpathicus ., 1893 CARP 169 82 251 +
P. a. djumbirensis , 1939 152 40 192 +
P. a. frankenbergeri 21 12 33 +
P. a. interversus , 1922 29 18 +
P. a. liptauensis , 1932 12 10 22 +
P. a. niesiolowskii , 1963 21 38 +
P. a. nitriensis , 1952 36 24 60 -
P. a. oravensis , 1969 ORAV 25 14 39 +
P. a. rosnaviensis , 1952 120 46 166 +
P. a. strambergensis , 1912 12 10 22 -
P. a. sztrecsnoensis , 1915 55 +
P. a. zelnyi 28 10 38 +
P. a. jaraensis , 1922 JARA WCARP 60 30 90 -
P. a. rosenius , 1923 ECARP 13 13 26
P. a. transsylvanicus , 1912 ECARP 36 -
P. a. timacus TIMA 10 11 21
P. a. rhaeticus , 1906 40 +
949 1426
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Journal of Insect Conservation (2024) 28:1107–1119
the ordinating Principal Component Analysis (PCA) by
continuous morphometric data and displays patterns graphi-
cally, aiming to cover the maximum variation in the data,
but has no estimation of the ideal number of clusters, and
-
via a Gap statistics partitioning algorithm (Tibshirani et
al. 2001) ran on the PC scores, using the package “clus-
2013
= 5, (maximum number of clusters)
= 1000 (bootstrap iterations), ref.gen = “PC” (reference
principal components of the data). This method estimates
the optimal number of clusters based on statistic thresholds
and automatically assigns objects into partitions.
-
mum variation in the data revealed no pattern (see Results),
-
-
ley 2002
-
to the model equally. Again, this approach did not support
= -
age) via 2006) on the
subspecies and geo-regions. The method turned out to be
et al. 2013; Cespedes et al. 20152018).
=-
starting and termination points of veins (Fig. 2). Additional
the upper (3 semilandmarks) and inner (2 semilandmarks)
margins (Fig. 2), via the ‘Append tps curve to landmarks’
2021). To generate the
-
2013)
slid to minimize the Procrustes distance) and gathering the
1991
only.
Statistical analyses
-
-
rately for the sexes using “lme4” and “lmerTest” packages
2015; Kuznetsova et al. -
a priori groupings via
Fig. 2
Parnassus apollo1935
ax1 and ax2axillaris 1 and 2 veins, cu1 and cu2cubitus 1 and 2 veins,
m1, m2 and m3media veins, r1, r2, r3, r4 and r5
the radius P.
apollo, this is marked by “r2 + 3”, scsubcosta. For the explanation of
(the colour version is available online as open access)
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Journal of Insect Conservation (2024) 28:1107–1119
rest in both females and males (Fig. 5
rejected the separation of the subspecies in both sex from
3
Carpathian subspecies in females.
-
indistinguishable (Table 2). The agglomerative hierarchi-
-
ter including the rest of Carpatho-Pannonian populations
(Fig. 5
dendrograms (Fig. 5
(Eastern Carpathians vs. the rest) remained supported in
>
1).
Discussion
The original descriptions of the studied 20 Carpatho-Pan-
nonian P. apollo subspecies are based on the ‘triad’ of size
-
analysis results in a dendrogram providing approximately
> 95. Validity of
have been done in R (R Core Team 2020), version 4.0.2.
1982; Klingenberg 2013) to compare the shape for the dif-
ferent groups supported by our analyses.
Results
-
20,928 = 18.583; P < 0.001;
20,456 = 11.92; P < 0.001). We found that apart
to be smaller than ssp. antiquus (Fig. 3
5,943
= 42.035; P < = 24.144; P < 0.001),
(Fig. 3).
-
= =
=
no recognizably separated entities in the extensive set of
males and females (Fig. 4a, c). Gap statistics estimated a
single cluster in both sexes (Fig. 4b, d) based on the PC
scores, supporting the (lack of) pattern returned by the PCA.
Table 2 1
Male ECARP WCARP NRC
36 1 0 3 0 0 40 0.90
0 0 2 0 1 20 0.85
ECARP 0 0 80 4 0 0 84 0.95
25 8 13 66
1 0 0 0 9 0 10 0.90
WCARP 0 0 2 6 0 52 60
Female ECARP WCARP NRC
20 1 0 5 1 0
1 6 0 4 0 0 11 0.54
ECARP 0 0 49 0 0 0 49 1
13 20 0 291 6 19 349 0.83
1 0 0 0 8 2 11
WCARP 1 0 0 3 1 25 30 0.83
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Journal of Insect Conservation (2024) 28:1107–1119
to a genetically distinct lineage (Todisco et al. 2010
clearly smaller than the Carpatho-Pannonian subspecies,
P. a. antiquus
subspecies held almost negligible information. In males, P.
a. strambergensis and P. a. antiquus
Considering the high end of the size range, none of the sam-
ples could be separated. Our results suggest that size rela-
incorrectly used in a discriminatory context in some of the
labels as “medium” (e.g. P. a. oravensis
1969 and P. a. antiquus) or “large”
(e.g. P. a. nitriensis Issekutz 1952 and P. a rosnaviensis
Issekutz 1952
at the pattern on the biologically relevant geo-region level,
-
-
at least one of its elements mentioned as a separating trait
1
In descriptions of P. apollo subspecies (as in other but-
via
P. a. rhaeticus, belonging
Fig. 3 Centroid size variation among the studied Parnassus apollo subspecies. Means +
1
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Journal of Insect Conservation (2024) 28:1107–1119
(ii) comprises a set of nonindependent, often directly con-
founding variables and (iii) mixing information of size and
shape. Further, a set of linear measures contains no infor-
mation about the spatial relationships of the endpoints
used for the measurements, making the visualisation of the
morphometry using Procrustes superposition, on the other
hand, covers all shape information independently from size
and any a priori 2010;
2020; Viacava et al. 2023). Our PCA (+ GAP
+ 1) sub-
each other and the size divergence is not considerable. This
of local adaptation vs. phenotypic plasticity (see. e.g. West-
Eberhard 2003) in this highly plastic life history trait cannot
be separated.
based geometric morphometrics instead of analysing an a
priori set of linear measurements. This approach is superior
to the latter, because any set of a priori selected variables
is (i) biased, potentially leaving out relevant information,
Fig. 4
of subspecies and geo-regions, see Table 1
version is available online as open access)
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Journal of Insect Conservation (2024) 28:1107–1119
(ECARP) and the rest of the Carpatho-Pannonian popula-
taxonomic value. Agglomerative hierarchical clustering also
separate genetic lineage (Todisco et al. 2010) is clustering
Heliconius species has
been revealed (Montejo-Kovachevich et al. 2021). As the
-
sonable to assume that this change in shape is also heritable.
-
2012
as a distinct subspecies. We note that the COI sequence
-
Carpathian subspecies (P. a. transsylvanicus; P. a. rosenius)
other. Again, the taxonomy of 20 (+ 1) subspecies received
zero support and thus they may not meet subspecies criteria
2012 2020
alternative explanations. First, one might consider that fore-
P. apollo taxonomy.
al. 2021; Viertler et al. 2022), including lepidopterans (e.g.
20162020) and P. apollo
Table 1
of Carpatho-Pannonian P. apollo, based on the often old,
as subspecies (Glassl 2005; Möhn 2005 2011) needs
reconsideration.
the biologically relevant geo-regions revealed a clear
Fig. 5 -
viations of subspecies and geo-regions, see Table 1P
an asterisk (the colour version is available online as open access)
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Journal of Insect Conservation (2024) 28:1107–1119
ago (most of) these areas might have been interconnected
and the recent fragmentation might be a result of recent
-
2011) and the potential for consequent genetic
-
on P. apollo
-
tive and professional training mechanisms, and legislation
(Thomson ).
Taken together, despite the numerous disperse popula-
P.
apollo subspecies in the Carpatho-Pannonian region. Fur-
from the Eastern Carpathians, has high probability of being
already extinct. Our results bring attention to the potential
problems of “old” taxonomy, based on one-by-one subspe-
cies descriptions lacking the methodology and rigour of
modern taxonomy. We must note that some putative subspe-
biologically minor morphological shifts, phenotypic plas-
ticity alone as a driver cannot be unequivocally excluded,
-
cal conclusion, integrative taxonomy should be employed,
scale genetic analyses.
Supplementary Information The online version contains
supplementary material available at
024-00602-2.
Acknowledgements -
porting the digitalization of the Parnassius apollo
We are highly indebted for numerous people for their inevitable help
2
-
Author contributions
2002) and the sequence from an Eastern
-
-
2011; Takáts and Mølgaard 2016
al. 2021
-
-
bian Carpathians (putative P. a. timacus
separation from ROCP and obviously, from ECARP, in the
From the conservation aspect, our results suggest the
+
one could consider based on the recent taxonomic treat-
ments of Carpatho-Pannonian P. apollo (Glassl 2005; Möhn
2005-
has been already foreseen in the national conservation strat-
-
ing that further studies are needed to clarify the question
have been no published observations of P. apollo in the
(Vizauer 2010; Moldovan 2016), apart from a single obser-
morphologically unique P. apollo populations have indeed
gone extinct.
Pannonian region. Given that proven (in the sense that indi-
conservational consequences of our results might be the
most prominent in this geo-region. The 15 putative subspe-
cies (not counting P. a. cominius
P. a. vistulicus and P. a. ruthenicus
conclusion is strengthened by Todisco et al. (2010) report-
-
ern Carpathian putative subspecies (P. a. antiquus, P. a.
interversus, P. a. niesiolowskii and P. a. liptauensis). The
1 3
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Journal of Insect Conservation (2024) 28:1107–1119
-
. Accessed (2020-06-22)
Parnassius apollo -
continental patterns of mitochondrial genetic diversity. Commun
Papilio machaon
Parnassius Apollo
Parnassius apollo
-
-
-
org/10.1046/j.0962-1083.2001.01411.x
Parnassius apollo
Möhrendorf, p 214
-
-
-
Parnassius
apollo serpentinicus
Parnassius apollo
-
-
-
evo.12114
Parnassius apollo
-
parnassius_apollo
Funding statement -
Data availability -
Declarations
Competing interests
Open Access This article is licensed under a Creative Commons
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