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From 20 to 2? Landmark-based geometric morphometrics reveal negligible wing-shape divergence between 20 subspecies of the Apollo butterfly, Parnassius apollo (Lepidoptera, Papilionidae), in the Carpatho-Pannonian region

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For effective conservation management of endangered taxa, it is important to define operational units for conservation. In the absence of detailed genetic analyses, morphology-based taxonomy is often used as a surrogate. The Apollo butterfly, Parnassius apollo, is one of the most endangered butterfly species in Europe (considered as a flagship species) with 26 subspecies rank taxa described from the Carpatho-Pannonian region (Central Europe), often based on old, one-by-one descriptions. We applied landmark-based geometric morphometrics on wing shape to determine the number of morphologically distinguishable groups in the region, based on 949 males and 477 females from 20 Carpatho-Pannonian putative subspecies (both extant and potentially extinct). We found a single division between the Eastern Carpathian populations (described as two subspecies: ssp. transsylvanicus and ssp. rosenius) and the rest of the populations (including our outgroup from the Swiss Alps). Since P. apollo was not observed in the Eastern Carpathians in the last two decades, and the currently known extant populations in the Carpatho-Pannonian region are all located in the Northern Carpathians, our results support a single conservation unit in the region. We suggest that (i) extensive monitoring is needed to reveal whether the unique Eastern Carpathian populations have really gone extinct and (ii) more taxonomical/phylogenetic studies on Central European P. apollo are needed for establishing the taxonomy of the species and efficient conservation strategies. We emphasize that modern integrative taxonomy is not only important for clarifying taxonomical issues, but also for providing basis for sound conservation management.
Map depicting the former (early 20th century) distribution of the putative Parnassus apollo subspecies (area boundaries drawn by brown lines, brown dashed lines indicate areas or parts of areas that were not included in our analysis) and the geo-regions (black dashed lines) that were inhabited by the species in the Carpatho-Pannonian region. NCARP: Northern Carpathians including 15 examined subspecies, ECARP: Eastern Carpathians including two subspecies, WCARP: Romanian Western Carpathians including one subspecies, BURGL: Burgenland (foothills of Eastern Alps) including one subspecies, SERB: Serbian Carpathians including one subspecies, ALPS: Swiss Alps (our outgroup) including one subspecies. Different shapes (circles or triangles, no specific meaning) with darker colours indicate localities (10 × 10 km UTM quadrats) for which photographs of specimens have been collected, lighter colours indicate additional literature data of occurrence (Jakšić 1988; Ruşti and Dragomirescu 1991; Konvička and Fric 2002; Höttinger 2003; Popov and Plushtch 2004; Dabrowski 2008; Kalivoda 2008; Kříž 2011). Dark red lines mark the boundaries of Pannonian Basin and the Carpathians (including its foothills areas) according to Kocsis et al. (2018) and the thick blue lines indicate the country borders (AUT – Austria, CZE – Czech Republic, HRV – Croatia, HUN – Hungary, POL – Poland, ROM – Romania, SRB – Serbia, UKR – Ukraine). Some 100 × 100 km UTM quadrats are marked with two-letter abbreviations (capital black letters), and we provide the scheme for 10 × 10 km UTM resolution in the WL quadrat for reference (the colour version is available online as open access)
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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 buttery, Parnassius apollo (Lepidoptera, Papilionidae), in the
Carpatho-Pannonian region
KornélTakáts1,2,3 · SándorCsősz2,3 · GergelySzövényi3· GergelyKatona4· Paweł J.Domagała5·
GáborHerczeg2,3
1 3
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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 20082011; 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 20052011).
-
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 

-
-

1 3
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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 199120022003; Popov and Plushtch 20042008; Kalivoda 20082011). Dark red
2018) and the thick

×
the scheme for 10 ×
1 3
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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 19932012

     
Carpatho-Pannonian area (Fig. 12). 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
1 3
1110
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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. 20152018).
=-

    
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 apollo1935
ax1 and ax2axillaris 1 and 2 veins, cu1 and cu2cubitus 1 and 2 veins,
m1, m2 and m3media veins, r1, r2, r3, r4 and r5
the radius   P.
apollo, this is marked by “r2 + 3”, scsubcosta. For the explanation of
 
(the colour version is available online as open access)
1 3
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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
1 3
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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
1 3
1113
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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.
20162020) 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 1P
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
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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 -

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
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       
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References

        

Parnassius apollo
      


  

  -



-
  

        -
   
 


      



unit concept and its applicability in biological conservation. Ital

40



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Journal of Insect Conservation (2024) 28:1107–1119
       -
 Parnassius apollo       
and perspectives of conservation. Funct Ecosyst Communities

         -
        
     
sagmb-2013-0016
   
(2020) Comparative phylogenetics of Papilio  

    
sysbio/syaa029
         
-
tion of Erebia pronoe
    
zootaxa.4441.2.5
-
       

        


   
        

doi.org/10.3389/fevo.2021.660546
         


        

-
  



     Parnassius apollo
-

       
       

    
       
       

-
        



 
taxonomy is essential to link molecular research to nomenclature.

          -
      

Takáts K, Mølgaard M (2016) Partial mtCOI-sequences of balkanic
species of Pseudochazara

  -

         -


       Parnassius apollo


   -


Parnassius apollo
  

-

26. 
          
      -
        
   


-

      
 




  
-

 
red lists for prioritising conservation actions for European but-


  
     

-
  -


Parnassius apollo
 
23), Goecke and Evers, Keltern, 33 pp
         






       
     

   
      

         
        
     
       
 

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Journal of Insect Conservation (2024) 28:1107–1119
   

        
      

West-Eberhard MJ (2003) Developmental plasticity and evolution.
     

-
Myrmica ants by geometric morphomet-

 -



morphometrics for biologists. Elsevier Academic, Amsterdam, p
488
   






       
 Parnassius apollo   
  

Publisher’s Note -

    


    -
phy of Parnassius apollo  
    

-
  

   

         -

         


        -
mance of geometric morphometrics and linear-based methods in
the taxonomic resolution of a mammalian species complex. Ecol

   
     -
       

        
     
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     
pnas.0801921105
         
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