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Genus Chersotis Boisduval, 1840 (Lepidoptera: Noctuidae) in Croatia with some notes on the other Balkan countries: DNA barcoding, distribution and new records

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  • National Museum of Natural History, Bulgarian Academy of Sciences

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An updated distribution of the genus Chersotis Boisduval, 1840 in Croatia, Albania and Bulgaria is presented. A total of seven species have been recorded in Croatia so far, with Chersotis laeta leonhardi (Rebel, 1904) being recorded for the first time during this survey. Chersotis laeta achaiana Thurner, 1967 is reported as new for Albania. The record of Chersotis fimbriola (Esper, [1803]) is marked as questionable for Croatia and Bulgaria as no exact locality is given nor is the specimen available for inspection for Croatia and the origin of the Bulgarian specimen is a case of doubt. For all other six species from Croatia an update in their distribution in the country is given. Additionally, all the species of the genus Chersotis occurring in Croatia have been barcoded and the results concur to the species identification based on the external and internal characteristics. Three taxa, Chersotis elegans (Eversmann, 1837) and Ch. laeta leonhardi from Croatia and Ch. laeta achaiana Thurner, 1967 from Albania have been DNA barcoded for the first time while for Chersotis laeta macini Rákosy, Stangelmaier & Wieser, 1996 a partial DNA barcode sequence was obtained.
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Ecologica Montenegrina, 48, 2021, 86-108
Genus Chersotis Boisduval, 1840 (Lepidoptera: Noctuidae) in Croatia
with some notes on the other Balkan countries: DNA barcoding,
distribution and new records
TONI KOREN1,7*, MARTINA PODNAR2, ANA MRNJAVČIĆ VOJVODA3,
STOYAN BESHKOV4, IVA MIHOCI5 & MLADEN KUČINIĆ6,8
1 Association Hyla, Zagreb, Croatia, Association Hyla, Zagreb, Croatia
2 Croatian Natural History Museum, Demetrova 1, 10000, Zagreb, Croatia
3 Croatian Agency for Agriculture and Food, Gorice 68b, Zagreb, Croatia
4 National Museum of Natural History, Bulgarian Academy of Sciences, Sofia, Bulgaria
5 Croatian Natural History Museum, Demetrova 1, 10000, Zagreb, Croatia
6 Department of Biology, Faculty of Science, University of Zagreb, Zagreb, Croatia
7E-mail: koren.toni1@gmail.com
8E-mail: kucinic@zg.biol.pmf.hr
* Corresponding author
Received 24 October 2021 Accepted by V. Pešić: 28 November 2021 Published online 16 December 2021.
Abstract
An updated distribution of the genus Chersotis Boisduval, 1840 in Croatia, Albania and Bulgaria is presented. A total of
seven species have been recorded in Croatia so far, with Chersotis laeta leonhardi (Rebel, 1904) being recorded for the
first time during this survey. Chersotis laeta achaiana Thurner, 1967 is reported as new for Albania. The record of
Chersotis fimbriola (Esper, [1803]) is marked as questionable for Croatia and Bulgaria as no exact locality is given nor
is the specimen available for inspection for Croatia and the origin of the Bulgarian specimen is a case of doubt. For all
other six species from Croatia an update in their distribution in the country is given. Additionally, all the species of the
genus Chersotis occurring in Croatia have been barcoded and the results concur to the species identification based on
the external and internal characteristics. Three taxa, Chersotis elegans (Eversmann, 1837) and Ch. laeta leonhardi from
Croatia and Ch. laeta achaiana Thurner, 1967 from Albania have been DNA barcoded for the first time while for
Chersotis laeta macini Rákosy, Stangelmaier & Wieser, 1996 a partial DNA barcode sequence was obtained.
Key words: Faunistic, south-east Europe, mtDNA, BOLD, distribution maps, genitalia.
Introduction
The DNA barcoding based on the sequencing of the partial cytochrome c oxidase subunit 1 mitochondrial
gene (mtCOI), along with the Barcode of Life Data Systems (BOLD) database (Hebert et al., 2003a, 2003b;
Ratnasingham & Hebert, 2007) added global biodiversity knowledge.
Ecologica Montenegrina 48: 86-108 (2021)
This journal is available online at: www.biotaxa.org/em
http://dx.doi.org/10.37828/em.2021.48.12
KOREN ET AL.
Ecologica Montenegrina, 48, 2021, 86-108 87
The method has proved to be useful in studies of the taxonomy, phylogenesis, phylogeography and
biodiversity of different groups of organisms (for example Amora et al., 2015; Brehm et al., 2019; Cárdenas
et al., 2013; De Barros et al., 2017; Dela Cruz et al., 2016; Elías-Gutiérrez et al., 2008; Guo et al., 2016;
Huemer et al., 2020; Kučinić et al., 2019a, 2019b).
Lepidoptera are one of the four major insect Orders, with described number of species being greater
than 155 000 (Nieukerken et al., 2011). Also, there is a great number of new species not yet described,
especially from the tropic regions. Classification of Lepidoptera based on morphological characteristics,
especial that of the adult were found to be confusing and inconsistent. The use of molecular methods in such
a diverse group is useful for identification and classification of species.
At the global scale, Lepidoptera, one of the most popular insect groups in terms of their ecology,
systematic and taxonomy have been studied in detail by the DNA barcoding method. The use of molecular
methods has brought new insights into the relationships between Lepidopteran taxa changing the phylogeny
based on the morphological features (Mitchell et al., 2006; Zahiri et al., 2011; Zahiri et al., 2012; Zahiri et
al., 2013a; Zahiri et al., 2013b). Lepidoptera have seen more intensive DNA barcode analysis than any other
insect order with nearly half the world’s known species included in the library (Nieukerken et al., 2011). The
barcoding of Lepidoptera has been systematically done on national and regional scales, e.g. Austria, Finland,
Germany, Norway and Switzerland (Huemer et al., 2014) as well in the continental scale (Brehm et al.,
2019; Zahiri et al., 2017). Recently, 97.3% of the currently recognized Noctuoidea species in North America
have been barcoded, of which 92.8% possess a diagnostic array of DNA barcodes (Zahiri et al., 2017).
In Europe, Germany is one of the countries that leads the barcoding of its Lepidoptera fauna. Ten
years ago, 1264 of the 1338 species of butterflies and larger moths (Papilionoidea and Macroheterocera) of
Germany were barcoded and added to the DNA barcode library, of which it was found that 99 % species
possess diagnostic barcode sequences (Hausmann et al., 2011).
Another good example of an extensive DNA barcoding project is the one done on the Lepidoptera of
Austria where in the last 10 years more than 3000 species of Lepidoptera (Huemer & Hebert, 2016; Huemer
et al., 2019) were barcoded. Only recently results of the DNA barcoding of the Noctuoidea superfamily with
671 species has been published (Huemer et al., 2019). The results were more than promising with the
diagnostic DNA barcodes obtained for 658 species and only 13 species could not be reliably identified from
their DNA barcode (Huemer et al., 2019). This showed that indeed also in Noctuidae, the usage of barcoding
may be a good complementary technique for the correct species identification.
Barcoding of Lepidoptera proved to be useful also on a larger scale, in the comparison of the 1000
Lepidoptera species between Austria and Finland (Huemer et al., 2014). The results showed that for 98.8%
of the taxa DNA barcode library possessed diagnostic COI sequences and that the barcode libraries based on
regional sampling can often be effective for a much larger area (Huemer et al., 2014).
In Croatia the DNA barcoding has been successfully used to study several insect groups, including
Trichoptera (e.g. Ćukušić et al., 2017; Kučinić et al., 2019a, 2019b, 2020), Diptera (Bušić et al., 2021),
Hemiptera (fam. Aphididae) (Franjević et al., 2016) and Coleoptera (Mičetić Stanković et al., 2018). In the
Lepidoptera surveys of Croatia, DNA barcoding has been in larger scale used only for PhD thesis (e.g.
Geometridae - Mihoci, 2012) while this lacks in other works.
Faunistic studies of Noctuoidea of Croatia has a very long tradition, and indeed, this family can be
regarded as one of best studied insect groups in the country, with the first checklists being created more than
20 years ago (Kučinić, 1997). Afterwards, many previously unrecorded species have been recorded
increasing the known diversity of this group (e.g. Mrnjavčić Vojvoda et al., 2014; Koren & Gomboc, 2015).
With the expansion of invasive plants new species are also arriving in the country, e.g. Acontia candefacta
(Hübner, [1831]) (Koren, 2019). Also, with the rising temperatures due to the climatic changes, new waves
of southern species being recorded in the country are also expected in the future. Still, many interesting
habitats in the country, especially high mountains, remain with only limited amount of surveys, opening the
possibility for interesting records.
What is also interesting to note that almost all the surveys carried in the country dealt with only the
external or in recent times also the internal morphology (see Mrnjavčić Vojvoda et al., 2014; Gomboc &
Koren, 2015). This gives only a limited insight into distribution of the species with their ecology, genetic or
their conservation status being unknown.
The genus Chersotis Boisduval, 1840 is one of the largest Noctuinae genera, consists of 117 species
group taxa representing 75 species with exclusively Palaearctic distribution (Varga et al., 2013), of which 20
can be found in Europe (Fibiger, 1990; Fibiger, 1993; Witt & Ronkay, 2011; Fibiger & Skule, 2013; Varga
GENUS CHERSOTIS IN CROATIA
88
et al., 2013). The name Chersotis originates from the ancient Greek ‘Chersos’ meaning ‘Dry land’ (Varga et
al., 2013). The species of this genus can usually be found in the mountainous or meso-montane
xerothermophilic habitats. They are generally rare but can sometimes be very abundant, especially in the
mountains. Adults are active during the night but can also be observed during the day feeding or resting on
different meadow flowers. They hibernate mostly as larvae. The food plants include various species from the
genera Vicia, Trifolium, Plantago, Galium, Asperula, Cirsium etc. (Kučinić & Lorković, 1998).
In this paper we provide (1) an updated distribution of the genus Chersotis in Croatia with some
additions and corrections for Albania and Bulgaria; (2) the results of the DNA barcoding of the six species of
the genus currently confirmed in Croatia
Materials and methods
Field surveys
In Croatia specimens were collected using two different methods. One was heath moth traps that were
powered by a 12 V battery and left on the locality during the whole night and then collected in the morning.
The second were light tents consisting of a white sheet and a 230 W UV lamp connected to a chargeable 12
V battery. Depending on the researcher, one to 8 light tents were used per locality. Voucher specimens are
deposited in the Croatian Natural History Museum (CNHM) under the collections Mihoci, Vajdić and
Kučinić as well in the private Lepidopterological collection Koren. On the other Balkan’s countries, except
for Romania, collecting methodology involved 2 or 3 portable light traps with an 8 W 368 nm “Blacklight”
white and 8 W “Blacklight” black tubes, both powered by 12 V 9Ah batteries, as well as a Finnish “tent trap”
with a 160 W MV bulb at the top of the pole and a 20 watt (368 nm) black light bulb over the catching pot
below. An additional 20 W (368 nm) light bulb was also positioned about 70 m from the tent trap. The
distance between the Finnish “tent trap” and the light traps, as well as between the light traps themselves was
sometimes more than 1 km, as they were deployed in different expositions if wherever possible. All traps ran
throughout the night. Genitalia were fixed on glass slides in Euparal mountant after staining with a 2%
Merbromin solution. All genitalia slides were photographed with a Zeiss stereo microscope Stemi 2000-C
with AxioCam ERc 5s digital camera and self modificated transmitted light 4200K. Some of the everted
vesicas in 3D were photographed in alcohol before being mounted on glass. Insects were photographed with
Sony DSChX400v digital camera, scale line is 1 cm. The genitalia slides elaborated by S. Beshkov (SB) are
part of his collection in the National Museum of Natural History, Sofia (NMNHS). All trips of SB were self-
financed and undertaken in his own time.
The list of localities on which Chersotis spp. were recorded contains the relevant toponyms, a short
description of the habitat, altitude, coordinates, dates of the visits and observers. Localities are mainly
arranged in geographical order from northwest towards southeast (Appendix I). In addition, Chersotis spp.
from North Macedonia, Albania and Romania were barcoded in order to provide a reference for the missing
species in the BOLD database (Tab. 1).
DNA extraction
Total genomic DNA was extracted from a single leg by using GenElute Mammalian Genomic DNA
Miniprep Kit (Sigma-Aldrich) and eluted with 60 µl of elution buffer. The 658 bp long fragment of the
mitochondrial cytochrome c oxidase subunit 1 (COI) gene, corresponding to DNA barcode region, was
amplified via PCR reaction for 23 samples (Tab. 1) by using standard Lepidoptera PCR primers (Hebert et
al., 2004): LepF1 (5′-ATT CAA CCA ATC ATA AAG ATA TTG G-3′) and LepR1 (5′-TAA ACT TCT
GGA TGT CCA AAA AAT CA-3′). Additionally, a shorter (307 bp) 5’ fragment of the same region, was
amplified for the one degraded DNA sample (CROB719) by using LepF1 and an internal MLepR1 (5'-GCT
TTC CCA CGA ATA AAT AAT A-3; Hajibabaei et al., 2006) primers. PCR mixtures (20 µl) contained 1 x
DreamTaq Green Buffer, 200 µM of each dNTP, 0.4 μM of each primer, 0.625 U of DreamTaq DNA
Polymerase and 1 µl of DNA eluate. In both cases, the cycling conditions comprised an initial denaturation
step of 95°C for 2 min followed by 35 cycles of 30 s at 95 °C, 30 s at annealing temperature of 50°C, 60 s at
72°C and afterwards the final extension step at 72°C for 7 min. All PCR products were sequenced bi-
directionally by Macrogen Europe (Amsterdam, The Netherlands) using amplification primers. The
sequences were inspected and primers trimmed manually by using BioEdit v. 7.2.5 Sequence Alignment
Editor (Hall, 1999). All sequences are deposited in BOLD under accession numbers given in Table 1.
KOREN ET AL.
Ecologica Montenegrina, 48, 2021, 86-108 89
Table 1. List of Chersotis species discussed in this study: first column - identification according to morphological
features; followed by specimens’ locality, voucher code, DNA code and BOLD accession number. *uncomplete DNA
barcode.
Species
Locality
Specimen
voucher
DNA code
BOLD accession
number
Ch. anatolica
Albania, Tomorr Mts, above
Ujanik Village
AMV192
CROB725
CROLE001-21
Albania, Mt Tomorr, Tomorr peak
AMV193
CROB726
CROLE002-21
Ch. cuprea
Croatia, Poštak, Ljubina poljana
MŠ207
CROB258
CROLE003-21
Croatia, Poštak, Ljubina poljana
MŠ208
CROB259
CROLE004-21
Croatia, Sjeverni Velebit, Zavižan,
Velebit Botanical
AMV56
CROB511
CROLE005-21
Ch. elegans
Croatia, Biokovo, mountain hut
below peak Sv. Jure
AMV60
CROB515
CROLE006-21
Albania, Mt Tomorr, Tomorr peak
AMV188
CROB723
CROLE007-21
Albania, Galičica Mts, above
Bregas (=Korita) Village,
AMV186
CROB724
CROLE008-21
Ch. fimbriola
thurneri
North Macedonia, Demir Kapija
Town, near Vardar River Valley
AMV207
CROB721
CROLE009-21
Albania, Galičica Mts, above
Bregas (=Korita) Village
AMV201
CROB722
CROLE010-21
Ch. laeta achaiana
Albania, Mt Tomorr, Tomorr peak
AMV205
CROB720
CROLE011-21
Ch. laeta leonhardi
Croatia, Mt. Biokovo
AMV3
CROB447
CROLE012-21
Croatia, Mt. Biokovo
AMV119
CROB484
CROLE013-21
Serbia, Javor Mts, below Vasilijn
Vrh, near Lepojevići Village
AMV189
CROB716
CROLE014-21
Albania, Shkallë Village, Dajt Mt
AMV196
CROB718
CROLE015-21
Ch. laeta macini
Romania, Enisala
AMV194*
CROB719
CROLE016-21
Ch. margaritacea
Croatia, Velebit, Siljevača,
Visočica
AMV57
CROB512
CROLE017-21
Croatia, Sjeverni Velebit, Zavižan,
Velebit Botanical
AMV58
CROB513
CROLE018-21
Croatia, Biokovo, Silnji gozd
(beech)
AMV59
CROB514
CROLE019-21
Ch. multangula
Croatia, Lička Plješivica
MŠ209
CROB260
CROLE020-21
Croatia, Biokovo, Silnji gozd
(beech)
AMV63
CROB517
CROLE021-21
Croatia, Mali Alan, Velebit,
pastures and edge
AMV64
CROB518
CROLE022-21
Croatia, Dinara, mountain hut
Brezovac
AMV65
CROB519
CROLE023-21
Ch. rectangula
Croatia, Ivančica, military
complex at the peak
AMV66
CROB520
CROLE024-21
Phylogenetic analysis
Data set for phylogenetic analyses (data set 1) comprised all publicly available Chersotis DNA barcode
sequences (total of 178 on 4. 11. 2020.) downloaded from the Barcode of Life Data System (BOLD;
Ratnasingham & Hebert, 2007) and GenBank (www.ncbi.nlm.nih.gov/genbank/) and 24 Chersotis sequences
obtained in this study (Tab. 1).
GENUS CHERSOTIS IN CROATIA
90
Sequences of Rhyacia simulans (Hufnagel, 1766) (ABOLA072-14 and LEFIJ872-10/ JF853873)
Eugraphe sigma ([Denis & Schiffermüller], 1775) (ABOLA060-14), Noctua pronuba (Linnaeus, 1758)
(ABOLA061-14), Noctua fimbriata (Schreber, 1759) (ABOLA064-14) and Nola thymula (Millière, 1867)
(GWOTD203-12 /KX040180) were used as outgroups. Prior to analysis, 202 Chersotis sequences were
collapsed into 81 unique haplotypes using DNA collapser option of the online service FaBox 1.5 (Villesen,
2007). Otherwise, identical haplotypes differing only by length were excluded and a final alignment
comprised 46 Chersotis and six outgroup sequences.
Phylogenetic analyses of data set 1 were carried out using two different methods: Neighbor-Joining
method (NJ; Saitou & Nei, 1987) and Bayesian inference (BI) as implemented in MrBayes (version 3.2.7a,
Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck, 2003).
Neighbor-joining tree based on Kimura-2-parameter distances was calculated using MEGA version 7
(Kumar et al., 2016) phylogenetic software. Branch support was assessed via non parametric bootstrapping
(2000 pseudoreplicates). For BI analysis, the best-fit model of nucleotide substitution (GTR +I +G) was
selected by jModelTest2 (Darriba et al., 2012) based on Bayesian information criterion (BIC). Data set was
further partitioned by codon positions. Two independent runs were carried out with four Monte Carlo
Markov chains running for 10 million generations and sampling trees every 1000th generation. The first 25 %
of trees were discarded as a burn-in. The convergence of MCMC chains was checked in Tracer v. 1.7.1.
(Rambaut et al., 2018). Model selection as well as BI analysis were performed using the CIPRES Science
Gateway (Miller et al., 2011).
Species delineation
In order to sort Chersotis sequences into putative species, the molecular-based species delineation was
conducted by Automatic Barcode Gap Discovery (ABGD, Puillandre et al., 2012) approach. Input data
comprised only the whole-length (658 bp) Chersotis DNA barcode sequences (a total of 177, 23 of them
from this study, Appendix I). Analysis was performed online on the ABGD webserver
(http://www.abi.snv.jussieu.fr/public/abgd/abgdweb.html), using the default parameters (gap width X = 1.5,
prior intraspecific divergences from P = 0.001 to P = 0.1, with 20 steps) and the Kimura-2-parameter (K80)
model. Some of the sampled specimens, especially from difficult for identification species-groups or with
several subspecies, were identified by male genitalia, including everted vesicas and female genitalia as well.
Distance analysis
Intra- and interspecific uncorrected pairwise distances (p-distances) were calculated in MEGA version 7
(Kumar et al., 2016) under complete deletion option. To avoid bias due to different sequence length, only the
whole-length Chersotis DNA barcode sequences were used and data set was the same as the one described
above for the ABGD analysis. Additionally, to address the only available sample of Ch. laeta macini
(CROB719), intra- and inter- specific p-distances based on 307 bp long 5’ fragment of DNA barcode region
were also calculated for selected taxa (Ch. fimbriola and Ch. laeta).
Results
During this research six species of genus Chersotis were recorded. For all recorded species, new faunistic
findings will be given along with the results of DNA barcoding.
Faunal and DNA barcoding analysis
Chersotis cuprea ([Denis & Schiffermüller], 1775).
New records: NP Risnjak, Gerovski kraj, meadow Lazac, 12.8.2019, 5 ex., leg. Koren; NP Risnjak,
Gerovski kraj, meadow Šegine, 12.8.2019, 3 ex., leg. Koren; NP Risnjak, Guslica, 15.8.2019, 3 ex., leg.
Koren; Mt. Velebit, Zavižan, 18.8.2017, >20 ex., leg. Koren; Lička Plješevica at 1230 m, 04.09.2008, 1 ex.,
leg. Mihoci; Lička Plješevica at 1500 m, 13.08.2008. 2 ex., leg. Mihoci; Mt. Biokovo, Lađena, 27.08.1990, 1
ex., leg Kučinić.
Genetic data: Ch. cuprea specimens, CROB259 and CROB511 analyzed in this study share the
haplotype with Austrian (ABOLC043-16, ABOLD199-16) and German (FBLMX164-11, GWORZ514-10)
KOREN ET AL.
Ecologica Montenegrina, 48, 2021, 86-108 91
specimens. They are comprised within well supported monophyletic clade together with a third Croatian
sample and Norwegian (LON271-08, LON3653-16), Italian (LEATB737-13, LEATC168-13) and Finnish
(LEFIA609-10) specimens. Intraspecific distances within this taxon were in range 0.2-0.8 (Tab. 2).
Chersotis elegans (Eversmann, 1837)
New records: Mt. Biokovo, mountain hut above Sv. Jure peak, 30.7.2018, 1 ex., leg Koren; Mt. Biokovo,
Ravna Vlaška, 07.08.2007, 1 ex., leg. Mihoci.
Genetic data: no specimen of Ch. elegans existed in the BOLD database. In order to confirm the
correct identification of the specimens from Mt. Biokovo, male genitalia were checked in accordance to
Fibiger (1997). During this research we barcoded one specimen from Croatia and one from Albania. Two
COI haplotypes (p-distance = 0.5%) were obtained within three analyzed specimens of Ch. elegans. Those
sequences form a well-supported clade and represent the first published Ch. elegans DNA barcodes. For its
closest species, Ch. anatolica, analyzed samples from Albania have identical COI haplotypes and clustered
within well supported monophyletic Ch. anatolica clade together with Italian (PHLAB1175-10) and North
Macedonian (PHLAF399-11) samples of the same species. It should be noted that even though these two
species have great morphological similarity, they are not sister species (Dufay & Varga, 1995). The overall
intraspecific distance in this species is low and ranged between 0.2 and 0.3 % (Tab. 2). Both female and male
genitalia, including everted vesicas are however quite different (Fig. 3. a-e), the difference are much bigger
than between Ch. fimbriola and Ch. laeta and between the subspecies of Ch. laeta.
Chersotis fimbriola (Esper, [1803]).
Genetic data: Barcoded samples from Demir Kapija and Korita Village, Galičica Mts (CROB721 and
CROB722) clustered with Austrian (ABOLD207-16, ABOLD208-16, ABOLD210-16 and DEEUR686-16)
and Spanish (LENOA115-11) specimens. The clade is well supported by BS and moderately by BPP values
and intraspecific distances ranged from 0.2 to 0.6%.
Chersotis laeta leonhardi (Rebel, 1904).
New records: Mt. Biokovo, Podglogovnik, 19.06.2007, 1 ex., leg. Mihoci; Mt. Biokovo, meadows at 1080
m, 18.06.2007, 5 ex., leg. Mihoci; Mt. Biokovo, Ravna Vlaška, 13.07.2013, 2 ex., leg. Mihoci; Mt. Biokovo,
above Vošac, 15.07.2013, 6 ex., leg. Mihoci; Mt. Biokovo, electric substation, 18.06.2007, 3 ex., leg.
Mihoci; 02.07.2007, 5 ex., leg. Mihoci; Mt. Biokovo, below Sv. Jure, 24.07.1994, 1 ex., leg. Kučinić,
02.07.2007, 9 ex., leg. Mihoci, 16.07.2013, 5 ex., leg. Mihoci; Mt. Biokovo, Lađena, 22.07.1995, 1 ex., leg.
Vajdić, 24.07.1995. 1 ex., leg. Kučinić; Mt. Biokovo, near the chapel, 13.07.1994, 1 ex., leg. Kučinić,
14.06.1991, 1 ex., leg. Kučinić.
Genetic data: Sequences from this study represent the first published DNA barcodes for this species
and clustered within two well supported monophyletic clades, the first one containing all samples of Ch.
laeta leonhardi, and the second clade samples of Ch. laeta macini and one of Ch. laeta achaiana (Fig. 1).
The intraspecific p-distances ranged from 0,2-2.9 % (2.7-2.9 % between two Ch. laeta subspecies, Table 2).
They marginally overlapped with interspecific distances revealed between Ch. laeta and its sister species Ch.
fimbriola (2.9-3.2 %, Tab. 2). Moreover, they are in the range or even larger than interspecific distances
between several well-defined species of the genus such are: Ch. andreae vs. Ch. multangula (2.7-3-0 %), Ch.
anatolica vs. Ch. elegans (2.4-2.7%) and Ch. margaritacea cyrnea vs. Ch. margaritacea (1.7 2.4 %) (Tab.
2). Those findings are consistent with the results of the ABGD analysis which advocates distinct species
status of Ch. laeta leonhardi and Ch. laeta achaiana which are found sympatric and synchronic in Albania,
Tomorr Mt., as well as with unresolved trichotomy obtained in NJ analysis between Ch. fimbriola, Ch. laeta
achaiana/macini (Fig. 5: a-e) and Ch. laeta leonhardi (Fig. 6). All above strongly argues for the necessity of
taxonomic revision within Ch. laeta which, based on molecular data, could be split into two species.
Additional morphological analysis of the greater number of specimens from Ch. laeta subspecies from
different localities should be undertaken to support the rising of the Ch. laeta leonhardi on the species level.
Chersotis margaritacea (de Villers, 1789).
New records: Velebit, Visočica, 13.8.2015, 3 ex., leg. Koren; Mt. Velebit, Zavižan, 18.8.2017, 12 ex., leg.
Koren; Lička Plješivica (1500 m), 13.08.2008. 2 ex., leg. Mihoci; Mt. Biokovo, entrance to the nature park,
12.09.2007, 1 ex., leg. Mihoci; Mt. Biokovo, vrata Biokova, 13.09.2007, 2 ex., leg. Mihoci; 01.10.2007, 3
ex., leg. Mihoci; Mt. Biokovo, Podglogovnik, 01.10.2007, 1 ex., leg. Mihoci; Mt. Biokovo, below Sv. Jure
GENUS CHERSOTIS IN CROATIA
92
peak, 28.08.1990. 5 ex., leg. Kučinić, 07.08.2007, 1 ex. leg. Mihoci; 30.8.2018, 2 ex., leg. Koren; Mt.
Biokovo, Sv. Jure peak, 07.08.2007, 1 ex., leg. Mihoci; Mt. Biokovo, Kotišina, 30.09.1995, 1 ex., leg.
Vajdić; Mt. Biokovo, Lađena, 27.09.1995., 1 ex., leg. Vajdić; Mt. Biokovo, meadow at 1083 m, 29.08.1990,
2 ex., leg. Kučinić; Mt. Biokovo, Silnji gozd, permanent plot, 14.09.1990, 2 ex., leg. Kučinić, 30.8.2018, 3
ex., leg. Koren.
Genetic data: Croatian sample CROB513 belonging to Ch. margaritacea shares the haplotype with
Austrian (ABOLA900-15, ABOLB529-15, LEATD628-13, LEATD629-13), French (LENOA110-11,
PHLAA406-09) and Italian (LEATB740-13 LEATB741-13) individuals. Together with remaining Ch.
margaritacea samples originated from Croatia, Austria, Germany and France it clustered within clade well
supported by BS, but moderately by BPP values. The intraspecific distances ranged from 0.2 to 1.1 %.
Chersotis multangula (Hübner, [1803]).
New records: Mt. Papuk, Poljanice, 4.7.2019, 1 ex., leg. Koren; Mt. Velebit, Mali Alan, 9.7.2018, 1 ex., leg.
Koren; Lička Plješevica, Bijeli potoci, 2.7.2018, 2 ex., leg. Koren, Kozja Draga, 28.7.2015, 1 ex., leg. Koren,
Dinara, Brezovac, 24.7.2018, 2 ex., leg. Koren; Mt. Biokovo: Ravna Vlaška, 19.06.2007. 1 ex., leg. Mihoci,
07.08.2007. 3 ex., leg. Mihoci, 13.07.2013. 3 ex., leg. Mihoci; Mt. Biokovo, electric substation, 07.08.2007,
3 ex., leg. Mihoci; Mt. Biokovo, below Sv. Jure peak, 24.07.1994. 1 ex., leg. Kučinić, 26.07.1995. 1 ex., leg.
Kučinić, 07.08.2007. 5 ex., leg. Mihoci, 28.7.2018, 2 ex., leg. Koren, 30.8.2018, 3 ex., leg. Koren; Mt.
Biokovo, Sv. Jure peak, 07.08.2007. 1 ex., leg. Mihoci; Mt. Biokovo, Lađena, 24.07.1995. 1 ex. Leg.
Kučinić, 30.07.2009. 1 ex., leg. Vajdić; Mt. Biokovo, Silnji gozd, 28.7.2018, 1. ex., leg. Koren.
Genetic data: Two different haplotypes were found in Croatian Ch. multangula specimen. The one
present in individuals CROB260 and CROB517 is shared with a number of individuals from Germany
(FBLMV003-09, GBLAA2027-15, GBLAB307-13, GBLAD973-14) and individuals from Austria
(LEATB426-13, PHLAG887-12) and France (LENOA107-11). Another one shared between CROB518 and
CROB519 was also recorded in samples from Austria (ABOLA071-14), Germany (GWORZ513-10) and
Italy (LEATB599-13, LEATB600-13, PHLAB1179-10, PHLSA585-11). Remaining sample which clustered
within Ch. multangula clade originates from Germany (GWORK401-09). Intraspecific distances within this
taxon are low being in range 0.2-0.3%.
Chersotis rectangula ([Denis & Schiffermüller], 1775).
New records: Lička Plješevica at 1365 m, 05.09.2008., 1 ex., leg. Mihoci.
Genetic data: The haplotype found in a single analyzed Croatian sample of Ch. rectangula is the
same as the one previously observed in Italian sample (LEATB602-13). Along with them, the well supported
Ch. rectangula clade comprised the samples from Austria (ABOLA069-14, GWORU307-10) and Italy
(GWOTF344-12, LEATB601-13). Intraspecific genetic distances within this clade are also low and range
from 0.2 to 0.3%.
Sequence analysis
No double peaks in chromatograms or indels in the alignment what would indicate the erroneous
amplification of a nuclear mitochondrial pseudogene (numt), were detected. However, the screening for
Wolbachia infection as the potential cause for the eventual failure of DNA barcoding, was not performed.
Phylogenetic analysis
Both applied methods of phylogenetic inference revealed a similar topology of the phylogenetic tree
characterized by well-supported monophyletic species clades and a large basal polytomy (Fig. 1). These two
basic results clearly show the basic strengths and limitations of DNA barcoding: good resolution at the
species and species group level (see: cryptic species, sister species) and rather low resolution at higher
phylogenetic (cladistic) level (i.e.Hajibabaei et al., 2006; Zahiri et al., 2014; Tan et al., 2021). Species of the
genus Chersotis did not form monophyletic clade. The branching off of the Noctua clade as a sister group to
the clade comprising all remaining species was revealed only in NJ analysis but it was not well supported by
bootstrap (BS) values. Both methods revealed well supported sister-group relationships of Chersotis
margaritacea margaritacea (de Villers, 1789) to Chersotis margaritacea cyrnea (Spuler, 1907), and of Ch.
elegans (Eversmann, 1837) to Chersotis anatolica (Draudt, 1936) as well as an unresolved trichotomy
between Chersotis ocellina ([Denis & Schiffermüller], 1775), Chersotis alpestris (Boisduval, [1837]) and
Chersotis oreina Dufay, 1984 and also between Chersotis rectangula ([Denis & Schiffermüller], 1775),
KOREN ET AL.
Ecologica Montenegrina, 48, 2021, 86-108 93
Chersotis andereggii (Boisduval, 1832) and Chersotis juncta (Grote, 1878). The sister group relationship of
Chersotis multangula (Hübner, [1803]) to Chersotis andreae Dufay, 1973 was supported in NJ, but not in
Bayesian analysis.
Furthermore, in NJ analysis, the well supported Ch. fimbriola, Ch. laeta leonhardi and Ch. laeta
macini / Ch. laeta achaiana clades appear in an unresolved trichotomy, while in Bayesian analysis the sister-
group relationship of Ch. laeta leonhardi and Ch. laeta macini / Ch. laeta achaiana is well supported by
Bayesian posterior probabilities (BPP) values (0.99). Finally, the clustering of Ch. larixia as a sister taxon to
Ch. elegans Ch. anatolica clade was not well supported by either, BS or BPP values. The inclusion of
several Chersotis species that have not yet been DNA barcoded would probably shed more light into the
phylogenetic relationships within the genus.
Figure 1. Phylogram based on Bayesian analysis of DNA barcode sequences (mitochondrial COI gene). Numbers at
nodes indicate Bootstrap values for NJ analysis and Bayesian posterior probabilities (BS / BPP). For the samples from
the previous studies, only a single representative of each haplotype is shown. Samples from this study are in bold.
Putative Chersotis species as suggested by most conservative ABGD partition are depicted by rectangles on the right of
the clades. * not included in ABGD analysis.
GENUS CHERSOTIS IN CROATIA
94
Species delineation
By different values of prior maximal interspecific distances (P) ranging from 0.001 to 0.021544, ABGD
analysis revealed a total of 7 partitions comprising 17, 19 or 32 groups. 17 groups were revealed
consequently in all initial partition (the first four most conservative partitions with values of prior maximal
distance between 0.004642 and 0.021544), while the recursive run resulted in two more hypothetical species
(19) in the partitions 5 and 6 with P values between 0.001668 and 0.002783. In the last recursive partition
with very low P value (P= 0.001) as many as 32 putative species were revealed. Most conservative split (17
species) almost perfectly reflects the current taxonomy of the genus Chersotis (Fig. 1). Two additional
species revealed in the first two recursive partitions (5 and 6) account to Ch. fimbriola which was further
split into a total of 3 putative species: the first one comprising Albanian and North Macedonian Ch. fimbriola
samples, the second one containing Austrian samples, and the third one accounting to Spanish Ch. fimbriola
sample.
Distance analysis
Intra- and interspecific genetic distances among species of genus Chersotis are shown in Table 2. The
intraspecific distances ranged from 0.2 to 1.1 %. The lowest interspecific distance was 1.7% and the highest
8.2%. P-distance based on 307 pb long fragment between Ch. laeta macini and Ch. laeta achaiana samples
(CROB719 and CROB 720) amounts only 0.3%, while much higher values ranged from 3.9 - 4.2% and 2.6 -
3.6% were observed in their comparison with Ch. laeta leonhardi and Ch. fimbriola, respectively.
Discussion
On the Balkan Peninsula there are 12 species reported from the genus Chersotis (Hacker, 1989; Fibiger,
1993), one of which Chersotis andereggii (Boisduval, 1832) seems to be not surely proved. The report of
Hacker (1989) for Greece follows uncertain literature data for Peloponnesus, Chelmos Mnt., 2200 m
(Beshkov & Slivov, 2006). According to Beshkov & Slivov (2006) reports in the literature for Ch.
andereggii in Serbia and North Macedonia are incorrect. Report for Bulgaria (Beshkov & Slivov, 2006)
perhaps is also incorrect, although it is based upon correctly identified specimens. The reason for this doubt
is, that we hardly believe that these specimens are really correctly labeled and are from Bulgaria indeed.
Same is the situation with the occurrence of Chersotis fimbriola in Bulgaria; specimens from Balchik belong
to another species Ch. laeta macini and that reported from Pirin Mts is perhaps also mislabeled specimen
from the collection of Al. Slivov (Beshkov, 2000). Chersotis alpestris (Boisduval, [1837]) is perhaps another
mislabeled specimen from the collection of Al. Slivov wrongly reported by Beshkov (2000) as new for the
Balkan Peninsula and Bulgaria. About the doubt case of the data of Al. Slivov and why a lot of his records
should be treated as doubtful see in Kolev (2002) and Ignatov et al. (2013). Three of these 12 species
reported from the Balkans are known only from Greece, as one of them, Chersotis larixia (Guenée, 1852), is
found only in Crete (Fibiger, 1993), another one, Chersotis zukowskyi hellenica Boursin, 1961, is known
only from Chelmos Mts in Peloponnesus and Chersotis capnistis schnacki Fibiger & Moberg, 1993 is known
only from high mountains of South and Central Greece (Varga et al., 2013). In the intraspecies level many
species have additional subspecies that are in most cases clearly defined both with external morphology and
allopatry (Fibiger, 1993; Varga at al., 2013).
Up to now, five species of the genus Chersotis have been recorded in Croatia (Kučinić & Lorković,
1998; Koren & Gomboc, 2015). During this survey, an additional species, Ch. laeta, has been recorded in
Croatia for the first time. For each species we present their literature records in Croatia, the new records
gathered during this survey and the result of the barcoding are presented under Results.
The species Ch. cuprea was till now recorded for Croatia only for Mt Velebit (Kučinić & Lorković,
1998). This Trans-Palaearctic, the most distributed species of the genus (Varga et al., 2013) with pseudo-
boreoalpine range has a wide Euro Asiatic distribution reaching northern Turkey, the Caucasus area,
Armenia, Russia, Japan and eastern Tibet (Fibiger, 1993). Previous to this research from Croatia only known
from Mt. Velebit (Kučinić & Lorković, 1998). During a visit to Zavižan, Mt. Velebit, a numerous population
of this species was confirmed at the only historical locality (Kučinić & Lorković, 1998). Most specimens
were already very worn indicating the end of the flight season in the area. This is in accordance with the
known flight period which is from July to September (Fibiger, 1993). On Mt. Velebit we also observed two
specimens during the day, a known behavior of this and other members of genus Chersotis which regularly
visit flowers.
KOREN ET AL.
Ecologica Montenegrina, 48, 2021, 86-108 95
Table 2. Uncorrected pairwise genetic distances (p-distances, in percentages) based on DNA barcode region of the COI gene within and between Chersotis species.
cuprea
multan.
marga.
elegan.
fimbri.
l.acha.
l. leon.
anatol.
rectan.
ander.
alpestr.
andre.
larixia
oreina
cyrnea
juncta
ocell.
cuprea
0.2-0.8
multangula
6.7-7.1
0.2-0.3
margaritacea
6.7-7.4
4.7-5.5
0.2-1.1
elegans
6.4-7.0
5.6-5.8
5.2-5.6
0.5
fimbriola
6.5-7.1
6.4-7.0
5.8-6.4
6.4-7.0
0.2-0.6
laeta achaiana
7.3-7.4
6.2-6.5
5.5-5.9
6.8-7.1
2.9-3.2
n.d.
laeta leonhardi
7.1-7.4
6.5-6.8
5.9-6.5
6.2-6.5
3.8-4.1
2.7-2.9
0.2
anatolica
6.5-6.8
6.1-6.5
6.1-6.4
2.4-2.7
6.5-7.0
6.8-7.0
6.7-7.1
0.2-0.3
rectangula
6.8-7.3
5.0-5.5
6.1-6.5
6.2-6.5
6.7-7.0
6.2-6.4
6.7-7.0
6.1-6.4
0.2-0.3
andereggii
6.4-6.8
4.7-4.9
5.3-5.8
6.5-6.8
6.5-7.1
6.7-6.8
6.8-7.1
6.4-6.7
4.1-4.6
0.02
alpestris
7.4-8.1
6.2-6.8
5.3-6.2
5.8-6.4
7.4-8.2
7.1-7.6
7.1-7.6
5.5-6.1
7.1-7.9
6.8-7.4
0.3-0.5
andreae
6.1-6.4
2.7-3.0
4.6-4.9
5.2-5.5
5.9-6.2
5.9
6.1-6.2
5.5-5.6
4.6-4.7
4.9
6.1-6.5
n.d.
larixia
5.8-6.1
4.6-4.7
5.5-5.6
3.5-3.8
5.9-6.4
6.4
6.5-6.7
4.0-4.1
5.3-5.6
5.3-5.5
6.5-7.0
4.9
n.d.
oreina
6.8-7.0
5.8-6.1
5.5-5.9
5.6-5.8
6.8-7.3
7.0
6.5-7.0
5.5-5.6
6.7-7.1
6.8-7.1
3.0-3.5
5.6-5.8
5.9
0.3
cyrnea
6.5-6.8
4.4-4.6
1.7-2.3
5.6-5.8
5.6-6.1
5.9
6.1-6.2
6.4-6.5
5.8-5.9
5.0-5.2
6.1-6.5
4.0
5.3
5.9
n.d.
juncta
6.5-6.8
4.6-4.7
5.6-5.9
5.2-5.3
6.5-7.0
6.4
6.8-7.0
5.0-5.2
3.5-3.6
3.6-3.8
6.2-6.7
4.7
4.9
5.8
5.3
n.d.
ocellina
7.8-7.9
6.2-6.7
6.5-7.1
6.1-6.5
7.3-7.8
6.8-7.0
7.3-7.6
5.2-5.6
7.6-7.9
7.4-7.8
3.6-4.3
5.8-5.9
6.7-6.8
3.3-3.5
6.8-7.0
6.4-6.5
0.2
GENUS CHERSOTIS IN CROATIA
96
Figure 2. Distribution of genus Chersotis in Croatia. a) Chersotis cuprea, b) Chersotis elegans, c) Chersotis laeta. d)
Chersotis margaritacea, e) Chersotis multangula, f) Chersotis rectangula. Black circles: new records, white circles:
literature records.
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Ecologica Montenegrina, 48, 2021, 86-108 97
Additionally, we recorded Ch. cuprea at three additional areas; National Park Risnjak, Lička
Plješivica and Biokovo. Accordingly, Ch. cuprea is now known from five distinct mountain areas (Fig. 2) in
the Country, and has accordingly a much wider distribution than was previously known (see Kučinić &
Lorković, 1998). The habitats in Croatia are in accordance with the habitats known for this species in the
literature. In central and southern Europe, it inhabits open bushy or woody areas in the subalpine zone while
in the northern Europe it is usually found in the lowlands (Fibiger, 1993).
Ch. elegans has a Mediterranean-Asiatic distribution and is in many parts of its range sympatric with
its sister species, Chersotis anatolica (Draudt, 1936), from which it can be reliably distinguished only on
basis of the genitals (Fibiger, 1993). It flies between July and September and recently was found as a new for
Bulgaria, Serbia (Beshkov & Nahirnić, 2017) and Albania (Beshkov & Nahirnić, 2017).
Previous to this research only two historical records were known for this species, both from Mt.
Učka (Rebel, 1912; Stauder, 1925). On Mt. Biokovo two specimens were collected so far, despite numerous
light trappings on the same localities during the last 30 years. This may indeed indicate that the population of
this species in the area is quite scarce, or that the main habitat of the species was not explored so far. The
habitats of Ch. elegans are open subalpine zone of mountains up to 2400 m (Fibiger, 1993) which is in
accordance to the mountain zone of Mt. Biokovo. In Bulgaria recently it was found at elevation of 739 m in
‘Dragoman’ Natura 2000 protected area (Beshkov & Nahirnić-Beshkova, 2021).
Ch. fimbriola in Croatia exists so far in the monograph dealing with the Noctuidae fauna of Mt.
Durmitor in Montenegro (Carnelutti et al., 1991). In it, Ch. fimbriola is mentioned as present in Croatia, as
was the case with other citations for neighboring countries in this work, but the exact source of this citation
is not clear, as for most records of Noctuidae from the surrounding countries no exact records or used
literature were provided. Up to our knowledge, this species has not been recorded in the country so far. Also,
it was not possible to check the correct identification of this species in the collection of Carnelutti, which is
still not available to the public. Accordingly, for the species to be included in the moth fauna of Croatia it
should be confirmed in the future.
Chersotis fimbriola on the Balkan Peninsula is represented by two ssp.: Chersotis fimbriola thurneri
Varga, Gyulai, Ronkay, L. & Ronkay, G., 2013 (=fimbriola forsteri Thurner, 1967, nom. praeocc.) presented
in Southern and Central Balkans (Greece, North Macedonia, Albania) (Fig. 4, a-e) and Chersotis fimbriola
niculescui Rákosy, 1996, endemic for N. Dobrogea in Romania. Specimens from Bulgaria: Balchik, reported
in the literature as Ch. fimbriola belong to another species: Ch. laeta macini (see in Beshkov, 2000 and in
Varga et al., 2013) as well as here in the Introduction. Both species Ch. fimbriola and Ch. laeta sometimes is
difficult to identified correctly only on appearance, although they differ in shape of the wings, size and color.
For correct identification examination of genitalia, or even DNA barcoding is necessary. Male genitalia
differ in shape of the valvae and size of the saccular process and the clasper, but most reliable difference is
the size of the cornuti of the vesica. In the female genitalia the most diagnostic feature is the shape of the
ostium bursae.
In Europe, four distinct subspecies of Ch. laeta have been recognized so far (Fibiger, 1993);
Chersotis laeta leonhardi (Rebel, 1904) described from Maklen pass in Bosnia & Herzegovina (Rebel, 1904)
Chersotis laeta achaiana Thurner, 1967 described from Mt. Chelmos in Greece (Thurner, 1967), Chersotis
laeta cretica Hacker & Varga, 1990 described from Crete in Greece and Chersotis laeta macini Rákosy,
Stangelmaier & Wieser, 1996 described from Macin Mountain range in N. Dobrogea (Rákosy, 1996).
Here Chersotis laeta achaiana Thurner, 1967, previously known only from Southern Greece is
reported as a new for Albania from Tomorr Mts (Fig. 5: a, b, e).
According to Hacker & Varga (1990) Chersotis laeta leonhardi is distributed in Bosnia &
Herzegovina, North Macedonia, Albania and Greek Macedonia. Recently it was reported from W Serbia
(Beshkov & Nahirnić, 2020). While Ch. laeta leonhardi (Rebel, 1904) has been described from Bosnia &
Herzegovina, it is lacking on the distribution map for this species in Europe (Fibiger, 1993). In the checklist
of Lepidoptera of Bosnia & Herzegovina it is erroneously attributed to Ch. fimbriola leonhardi (see in Lelo,
2004). Our records from Mt. Biokovo represent the first observations of this species in Croatia and further
expand its known range toward west. On Mt. Biokovo we recorded it only in the peak zone, from about 1500
m a.s.l. to the peak of Sv. Jure located at 1762 m a.s.l. Light trapping at the lower altitudes did not reveal the
presence of this species. Due to that, it can be regarded as high mountain species also in Croatia. As Mt.
Biokovo is an isolated mountain with strong Mediterranean influence, and is itself the third highest mountain
in Croatia, it is not likely that Ch. laeta leonhardi will be recorded on other mountains in the country.
GENUS CHERSOTIS IN CROATIA
98
Figure 3. a) Ch. anatolica, male. Albania, Tomorr Mts, Abaz Ali Top, 2379m, N40.6361, E020.1615, 14.VII.2018, S.
Beshkov & A. Nahirnić leg.; b) Ch. elegans, male. AL, Korca County, west from Pepellash Village, 1318m,
N40°28'55"; E020°41'27", 22.VIII. 2017, S. Beshkov & A. Nahirnić leg.; c) Ch. anatolica, male genitalia with everted
vesica. Gen. prep. 1./04.III.2019, S. Beshkov. AL, Tomorr Mts, Abaz Ali Top, 2379m, N40.6361, E020.1615,
11.VIII.2018, S. Beshkov, A. Nahirnić & C. Plant leg.; d) Ch. elegans, male genitalia with everted vesica. Gen. prep.
1./03.III.2019, S. Beshkov. AL, Mt. Tomorr, Abaz Aliu Top below to south, 2026 m, N40°37'19", E020°10'42",
10.VIII.2018, S. Beshkov, A. Nahirnić & C. Plant leg., e) Ch. elegans, female genitalia. Gen. prep. 9./14.II.2018, S.
Beshkov. AL, Mali i Thatë (=Galičica Mts), above Bregas Village, 1485m, N40°48'34"; E020°50'05", 20.VIII.2017, S.
Beshkov & A. Nahirnić leg., f) & g) Ch. elegans, everted vesica, Gen. prep. 2./22.III.2017, S. Beshkov. Serbia,
Slivovicki Vis, N43°08'29", E022°23'12”, 03.IX.2016, S. Beshkov, A. Nahirnić leg.
KOREN ET AL.
Ecologica Montenegrina, 48, 2021, 86-108 99
Figure 4. a) Ch. fimbriola thurneri, female. Albania, Korca County, above Boboshticë Village, 1225m, N40°32'26";
E020°47'31", 08.VII.2016, S. Beshkov & A. Nahirnić leg., b) Ch. fimbriola thurneri, male genitalia with everted vesica.
Gen. prep. 4./22.XII.2020, S. Beshkov. AL, Korca County, above Boboshticë, 1225m, N40°32'26"; E020°47'31",
08.VII.2016, S. Beshkov & A. Nahirnić leg. c.) Ch. fimbriola ssp., male genitalia with everted vesica. Gen. prep.
6./22.XII.2020, S. Beshkov. North Macedonia, above Demir Kapija Town, 244m, N41°22'58"; E022°11'45",
10.VI.2018, S. Beshkov & A. Nahirnić leg. d) Ch. fimbriola thurneri, male genitalia with everted vesica. Gen. prep.
5./22.XII.2020, S. Beshkov. AL, Korca County, above Drenovë Village, 1050m, N40°35'18"; E020°48'23", 7.VII.2016,
S. Beshkov & A. Nahirnić leg. e) Ch. fimbriola ssp.female genitalia. Gen. prep. 1./22.XI.1999, S. Beshkov. SW
Bulgaria, Pirin Mts, Yane Sandansky Chaler, 1200 m alt., 29.VII.1969, det. and in coll. of Al. Slivov in IBER (Sofia).
GENUS CHERSOTIS IN CROATIA
100
However, despite its limited distribution, it is usually very common and sometimes even dominant
noctuid species with several dozen specimens attracted each night.
Figure 5. a) & b) Ch. laeta achaiana, males. Albania, Tomorr Mts, below, Abaz Ali Top, 1730m, N40.6111,
E020.1885, 13.VII.2018, S. Beshkov & A. Nahirnić leg., c) & d) Ch. laeta macini, males. Romania, Dobrogea, Jud.
Tulcea, Enisala, 02.6.2016, L. Szekely leg. e) Ch. laeta achaiana, male genitalia with everted vesica. Gen. prep.
2./21.XII.2020, S. Beshkov. AL, Tomorr Mts, below, Abaz Ali Top, 1730m, N40.6111, E020.1885, 13.VII.2018, S.
Beshkov & A. Nahirnić leg. f) Ch. laeta macini, male genitalia with everted vesica. Gen. prep. 1./21.XII.2020, S.
Beshkov. Romania, Dobrogea, Jud. Tulcea, Enisala, 02.6.2016, L. Szekely leg.
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Ecologica Montenegrina, 48, 2021, 86-108 101
Figure 6. a) Ch. laeta leonhardi. Croatia, Mt. Biokovo, below Sv. Jure, 43°20'26.2"N 17°03'08.0"E, 1620 m
a.s.l.,30.VIII.2018, T. Koren leg. b) Ch. laeta leonhardi. Albania, Tirana County, Dajt Mt, Shkallë Village, 893m,
N41°19'49"; E019°57'55", 29.VI.2017, S. Beshkov & A. Nahirnić leg. c) Ch. laeta leonhardi. AL, Tomorr Mts, below
Abaz Ali Top, 1730m, N40.6111, E020.1885, 13.VII.2018, S. Beshkov & A. Nahirnić leg., d) Ch. laeta leonhardi. AL,
Bjeshkët e Nemuna Mts (=Prokletije Mts), Radohima Mt., between Qafa e Thorës pass and Theth village, south-east of
Shtegu peak, 1657m, N42.3854, E019.7502, 19.VII.2018, S. Beshkov & A. Nahirnić leg., e) Ch. laeta leonhardi, male
genitalia with everted vesica. Gen. prep. 3./21.XII.2020, S. Beshkov. AL, Bjeshkët e Nemuna Mts (=Prokletije Mts),
Radohima Mt., between Qafa e Thorës pass and Theth village, south-east of Shtegu peak, 1657m, N42.3854,
E019.7502, 19.VII.2018, S. Beshkov & A. Nahirnić leg., f) Ch. laeta leonhardi, male genitalia with everted vesica.
Gen. prep. 2./22.XII.2020, S. Beshkov. AL, Korca County, above Drenovë Village, 1050m, N40°35'18"; E020°48'23",
7.VII.2016, S. Beshkov & A. Nahirnić leg.
GENUS CHERSOTIS IN CROATIA
102
Species Ch. margaritacea was recorded for Croatia over 150 years ago in Dalmatia (Mann, 1869).
During later research presence of the species in Croatia was confirmed several times: Zadar (Galvagni,
1909), Vis island (Galvagni, 1909), Mt. Učka (Rebel, 1912), Senj (Stauder, 1925), Đurđevački pjesci
(Kranjčev, 1985), Mt. Biokovo (Mladinov & Kučinić, 1993; Kučinić & Lorković, 1998), Island Krk
(Habeler, 2003).
This is a West Palaearctic-Central Asian species inhabiting parts of central and southern Europe, NW
Africa (Morocco), Georgia, Azerbaijan, Armenia, Turkmenistan, Central Asia and Russia (Fibiger, 1993;
Varga et al., 2013). Adults fly from July to October (Fibiger, 1993). It is one of the widest distributed species
of the genus Chersotis in Croatia. It is present in all three biogeographical regions and the only species of the
genus that can be found also on the Adriatic islands (e.g. Vis - Galvagni, 1909). All the new records fall
inside the known distribution in the country. We recorded it in the mountain habitats meadows and forest
edges, which is in accordance to the known habitat preference (Fibiger, 1993).
After being recorded for the first time in XIX. Century (Josipdol, Mann, 1867), Ch. multangula was
recorded several times in fauna of Croatia: Mt. Biokovo (Kučinić & Lorković, 1998), Mt. Strahinščica
(Koren & Gomboc, 2015), Mt. Ivanjščica (Koren & Gomboc, 2015).
This species inhabits alpine and subalpine, open steppe-like zones of mountains, usually between
1000 and 2000m (Fibiger, 1993), but in Bulgaria it is found on the Black Sea coast near Balchik (Beshkov,
2000). Adults fly from June to September. The distribution of this species in Croatia is also very wide and
include all three biogeographical regions, but with very scarce records (see Koren & Gomboc, 2015). During
our survey we recorded this species on several new areas including Mt. Dinara, Mt. Lička Plješivica, Mt.
Velebit and Mt. Papuk. We also confirmed its occurrence on Mt. Biokovo where it is a common species in
the zone above 1500 m a.s.l.
Species Ch. rectangula was determined for fauna of Croatia during recent research in north-east part
of Croatia on Mt Ivanščica (Koren & Gomboc, 2015). This is a Western Palearctic species with fragmented
distribution across Europe. Outside Europe it is known from Russia, Turkmenistan, Kirghizia, the Caucasus
area, Armenia, Turkey and north-west Iran (Fibiger, 1993). The Siberian and Central Asian data are
uncertain (Varga et al., 2013). Ch. rectangula is a xero-montane species that inhabits bushy habitats, mainly
in the subalpine zone, but also can be found in rocky grassland at lower altitudes between 600 and 2400
meters above sea level (Dufay, 1971) or even on the sea coast of Bulgaria (Beshkov, 2000). Adults fly from
late June to mid-September, and are attracted to light and bait traps (Fibiger, 1990). Previous to this survey it
was known only from the upper zone of Mt. Ivanjščica (Koren & Gomboc, 2015). The record of a specimen
from Mt. Lička Plješevica expands the known distribution in Croatia further to the south. Still, it seems that
it is a rare and local species in the country, possibly requiring the inclusion in the future Red Data Books or
lists of Noctuoidea of the country.
The knowledge about the distribution of the genus Chersotis in Croatia has been significantly
expanded due to this survey. Chersotis elegans has been recorded for the second time in the country, almost
a century after the first record from Mt. Učka (Rebel, 1912). For Ch. rectangula, a newly recorded species in
the country’s fauna, a second locality on Mt. Plješevica has been found. Chersotis cuprea has been
confirmed on Mt. Velebit and for the first time recorded on four additional mountain chains. For the two
commonest species, Ch. multangula and Ch. margaritacea, additional records significantly widened their
distribution in the country. Chersotis laeta leonhardi has been for the first time recorded in Croatia,
widening its known distribution in the Balkan peninsula toward west. Chersotis laeta achaiana Thurner,
1967, previously known as an endemic for the Southern Greece is reported as a new for Albania from
Tomorr Mts, above Ujanik, Abaz Ali Top, 1730m, N40.6111, E020.1885, 13.VII.2018, S. Beshkov & A.
Nahirnić leg.
The use of the barcoding method and the BOLD library confirmed the correct identification of all six
confirmed Chersotis species. In addition, Ch. elegans as well as Ch. laeta leonhardi and Ch. laeta achaiana
were barcoded for the first time and added to the BOLD library (Ratnasingham & Hebert, 2007).
Our research can be regarded as one of the first published results of the DNA barcoding of
Lepidoptera of Croatia and Albania, which is aimed at solving potential taxonomical problems, especially in
the most diverse family, Noctuidae. As is the case with Trichoptera in Croatia, for which the barcoding
already proved as a useful tool to reveal species sets (Cerjanec et al., 2020; Ćukušić et al., 2017; Kučinić et
al., 2013, 2017, 2019a, 2019b, 2020) of certain areas or habitat types like springs (Kučinić et al., 2020) as
well the confirmation of rare and potentially endangered species (Cerjanec et al., 2020; Kučinić et al., 2017),
the DNA barcoding of Lepidoptera of Croatia will also continue in the future.
KOREN ET AL.
Ecologica Montenegrina, 48, 2021, 86-108 103
Figure 7. a) Ch. margaritacea, female. Albania, Korca County, above Zvezdë, 1088m, N40°43'59"; E020°52'49",
23.IX.2016, S. Beshkov, A. Vaso & A. Nahirnić leg., b) Ch. margaritacea female. AL, Mali i Thatë (=Galičica Mts),
above Bregas Village, 1485m, N40°48'34"; E020°50'05", 20.VIII.2017, S. Beshkov & A. Nahirnić leg., c) Ch.
multangula, male. AL, Korca County, Krusheve, 11.VII.2018., d) Ch.multangula, male. Al, Lure Mts, 1687m,
9.VII.2019, e) Ch. multangula, male genitalia with everted vesica. AL, Mali i Thatë (=Galičica Mts), above Bregas
Village, 1485m, N40°48'34"; E020°50'05", 20.VIII.2017, S. Beshkov & A. Nahirnić leg. Gen. prep. 8./14.II.2018, S.
Beshkov, f) Ch. multangula, female genitalia. SW Bulgaria, Maleshevska Planina Mts, below Alaborun top, 980m,
10.VIII.2000, S. Beshkov leg.
GENUS CHERSOTIS IN CROATIA
104
Conclusions
Barcoding is one of the most useful methods for the correct identification in Lepidoptera due to the fact that
the used gene region is generally characterized by low intraspecific variation and much higher divergence
between species (Zahiri et al., 2014; Zahiri et al., 2017). Our study supports the conclusion that, when used
with care and in conjunction with other techniques (integrative taxonomy), DNA barcodes provide powerful
addition to the tools available for taxonomic work on animals (Mutanen et al., 2016; Vitecek et al., 2017).
Still, some open questions remain, especially the subspecific status of Ch. laeta macini and Ch. laeta
achaiana, as well as the relationship between Ch. fimbriola from central Europe to Ch. fimbriola thurneri
from the Balkans. These cases should be investigated in the future, with additional molecular markers and/or
morphological analysis on a greater number of specimens from different localities. Specifically, regarding
unresolved or poorly supported relationships, the multilocus molecular approach would be necessary to
surpass the limitation of the DNA barcode methodology regarding the resolution of deep phylogenies.
Acknowledgments
This research is a part of the scientific project “DNA barcoding of Croatian faunal biodiversity” (IP-2016-
06-9988) funded by the Croatian Science Foundation and financial support of the University of Zagreb. We
would like to thank Mladen Vajdić and Natural History Museum Zagreb for allowing us access to Vajdić
Collection.
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