Genetic implications of a biological invasion: Chromosomal and DNA barcode monomorphism in Old World populations of Colorado potato beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Once introduced into new area, invasive species can be expected to have low genetic diversity due to the founder effect. Here we tested this prediction using cytogenetic and molecular analysis of Armenian and Belarusian populations of Colorado potato beetle Leptinotarsa decemlineata (Say, 1824) and by comparing the results with those of native (North America) and those introduced into Europe. This revealed that the karyotype of males from Armenia and Belarus is remarkably conserved with 2n = 35 (34 + X0), n = 17AA + X0; and includes a pair of large acrocentric chromosomes. Thus, these populations belong to the so-called acrocentric chromosome race of the Colorado potato beetle. At diakinesis there are clearly visible argentophilic signals, probably NORs (the nucleolus organizer regions) present on some autosomal bivalents, while the X chromosome was homogenously argentophilic during different stages of meiosis. C-banding revealed a small amount of constitutive heterochromatin weakly visible in the pericentromeric regions of some chromosomes. Analysis of the DNA-barcode fragment of the gene cytochrome c oxidase subunit I (COI) revealed a single haplotype (we call it “the European haplotype”) and lack of inter-population variability in all the samples collected from different locations in Armenia and Belarus. The comparison of our karyological and molecular data with that available in the literature and GenBank shows that all the populations studied from the Old World are monomorphic with respect to karyotype and the mitochondrial DNA-barcode. We assume that (1) the presence of acrocentric chromosomes in the karyotype and (2) the European haplotype of mitochondrial genome are the ancestral states for all populations in the Old World and inherited from the New World invaders who colonized Europe 100 years ago. New World populations are polymorphic with respect to karyotype and mitochondrial genes; however, the European haplotype has not yet been found in America. We believe that in the future it will be found in North America, which will shed light on the origin of populations of this dangerous pest in Eurasia.


INTRODUCTION
Colorado potato beetle Leptinotarsa decemlineata (Say, 1824) is one of the best known invasive insect species. Despite signifi cant progress in plant protection, Colorado potato beetle (CPB) is still the main pest of potato (Solanum tuberosum) worldwide. However, the beetles may damage tomatoes (Solanum lycopersicum), eggplant (Solanum melongena) and tobacco (Nicotiana tabacum), as well as feed and survive on the other plants of the family Solanaceae (Alyokhin, 2009;Liu et al., 2012).
CPB spread across the United States and Canada during the second half of the 19th century and then invaded Europe at the beginning of the 20th century (France, Bordeaux) and is currently present almost throughout the Eur-the standardized gene regions (658 bp long, 5' segment of mitochondrial cytochrome oxidase subunit I, COI) in GenBank (Table 2). This is strange, since over the past 15 years, DNA barcodes have become a universal tool not only for identifying species (Hebert et al., 2003), but also for understanding the evolution and ecology of biodiversity (Kress et al., 2015).
The aim of this study was to reveal possible similarities or differences between Armenian and Belarusian populations of Colorado potato beetle based on karyological and DNA barcode data. In addition, we were interested in testing the prediction that due to the founder effect (Barton & Charlesworth, 1984), invasive populations have a lower genetic diversity than native populations of the same species (Dlugosch & Parker, 2008;Holm et al., 2018; but see also : Roman & Darling, 2007). To test this prediction, a chromosomal and DNA barcode comparison of native (North America) and introduced (Europe) populations of CPB was carried out.

Material and sampling
Adults of Leptinotarsa decemlineata were collected in Armenia and Belarus in 2019 (Table 1) on Solanum tuberosum. For the karyological study male and female abdomens were dissected, immersed in 0.9% sodium citrate solution at room temperature for 40 min. Then the gonads were fi xed in 3 : 1 fi xative (96% ethanol : glacial acetic acid). The remaining bodies of the same specimens were fi xed in 96% ethanol for DNA study. The fi xed samples were frozen and stored at -20°C until processed.

Preparation of karyological slides
The dissected gonads were placed on slides in a drop of 70% acetic acid. Squashed chromosomal preparations were obtained using the dry ice quick-freezing technique (Conger & Fairchild, 1953).
Ag-banding was done according to the method proposed by Howell & Black (1980), with minor modifi cations. The slides were exposed to hydrolysis in 2N formic acid for 10 min, rinsed in running water and dried. Then 4-5 drops of 50% aqueous silver nitrate (AgNO 3 ) solution and 2 drops of colloidal developer solution (0.2 g gelatin, 10 ml distilled water and 0.1 ml concen- Udalov & Benkovskaya, 2011;Brechko et al., 2016). However, there are not many cytogenetic and molecular studies on L. decemlineata.
More recent studies report the fi rst data obtained using C-banding staining and in situ restriction enzyme digestion combined with the analysis of repetitive DNA for studying the organization of heterochromatin in chromosomes of L. decemlineata (Baus Lončar et al., 2005). Repetitive DNA in nuclear DNA of CPB and pericentromerically located small heterochromatic blocks on all chromosomes in its karyotype. The chromosome location of two different satellite-DNA families on mitotic and meiotic chromosomes of L. decemlineata was studied by fl uorescence in situ hybridization using LEDE-I and LEDE-II satellite DNAs as probes (Lorite et al., 2013). Positive hybridization signals in the pericentromeric region on some chromosomes, including X chromosome, is recorded.
There are several studies on CPB populations that used molecular markers (Jacobson & Hsiao, 1983;Azeredo-Espin et al., 1991, 1996Zehnder et al., 1992;Sidorenko et al., 2000;Hawthorne, 2001;Sidorenko & Berezovska, 2002;Grapputo et al., 2005;Grapputo, 2006;Lorite et al., 2013;Zhang et al., 2013;Przybylska et al., 2014;Izzo et al., 2018;Yang et al., 2020), including analyses based on sequencing the whole genome (Crossley et al., 2017;Cohen et al., 2021). These methods are promising for the investigation of intra-and interpopulation polymorphism, as well as migration routes and microevolutionary processes accompanying the formation of the current CPB range (Udalov & Benkovskaya, 2011). Nonetheless, currently there is little data on mitochondrial DNA barcodes, trated formic acid -HCOOH) were placed on each slide. The slides were covered with a coverslip and incubated on a hotplate for 3-4 min at 60°C in a moist chamber (warmed beforehand). The slides were dried after rinsing in distilled water. C-banding was revealed using the protocol of Rożek (2000). The slides were treated for 1-3 min in 0.2 N HCL at room temperature then rinsed in distilled water. Thereafter, the slides were placed in 5% Ba(OH) 2 solution at 20°C for approximately 4 min, then rinsed with distilled water. Then the slides were incubated in 2 × SSC solution (0.3 M sodium chloride containing 0.03 M trisodium citrate) at 60°C for 1 h. After rinsing in distilled water, the slides were dried and stained using 4% Giemsa solution in phosphate buffer (pH 6.8) for 8 min.

DNA extraction, PCR amplifi cation and sequencing
Total DNA was extracted from the wing muscle, using the Qiagen DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) following the protocol for Animal Tissue.
PCR conditions for COI amplifi cation were as follows: initial denaturation period of 2 min at 94°C was followed by 30 cycles of 1 min at 94°C, annealing for 30 s at 45°C and extension for 1 min 30 s at 72°C, with a fi nal extension step of 10 min at 72°C.
PCR products were purifi ed with ExoStar (GE Healthcare, Little Chalfont, UK) in accordance with the manufacturer's manual and sequenced in both directions externally by StarSEQ GmbH (Mainz, Germany).
DNA extraction, PCR amplifi cation, gel electrophoresis, PCR products purifi cation were carried out in the DNA laboratory of the Natural History Museum, University of Oslo. All sequences obtained in this study were submitted to GenBank (accession numbers MW346681-MW346686 and MW348764-MW348766). Their accession numbers and specimen vouchers are presented in Table 2. The voucher specimens and all DNA extracts were deposited in the Scientifi c Center of Zoology and Hydroecology, NAS RA.

Samples and sequence alignment
Nucleotide sequences obtained in this study were edited and aligned using BioEdit software (Hall, 1999). 27 additional DNA barcodes of L. decemlineata (3 from USA, 1 from Canada, 2 from Austria, 6 from Germany, 2 from Poland and 13 from Bashkortostan, Russia) were obtained from GenBank and added to the alignment. We edited the GenBank sequence JF889843 (Germany) by extracting its terminal part that was not properly aligned. DNA barcodes of L. haldemani, L. juncta and L. texana (11 samples) were also obtained from GenBank and added to the alignment as outgroups to root the trees (Table 2).
An additional alignment was created to study mitochondrial polymorphism in American populations. For this purpose, 82 haplotypes of the mitochondrial genome fragment that included the terminal part of the COI gene and the initial part of the COII gene were downloaded from GenBank (Crossley et al., 2017). Since this fragment does not overlap the standard DNA barcode, phylogenetic trees for it were built separately. The species Diabrotica undecimpunctata was used as an outgroup to root the trees.

Phylogenetic tree construction
The evolution model test for DNA substitutions was performed in MEGA X (Kumar et al., 2018). For the DNA barcode dataset, Tamura-Nei TN93+G was the optimal model. For the fragment that included the terminal part of the COI gene and the initial part of the COII gene, Tamura three-parameter (T92+G) was the optimal model. Maximum Likelihood phylogenetic trees were constructed for the two datasets using the substitution models found. The standard nonparametric bootstrap (Felsenstein, 1985) (100 replicates) was used to evaluate the statistical nodal support of the trees.
The Bayesian phylogenetic analysis was performed using the program MrBayes v.3.2.7 (Ronquist et al., 2012). Two runs of 10,000,000 generations with four chains (one cold and three heated) were performed for both datasets. The consensus of the obtained trees was visualized using FigTree v.1.4.4 (http://tree. bio.ed.ac.uk/software/fi gtree/).

Chromosome analysis
Nuclear divisions were found in CPB males of both Armenian and Belarusian populations. In females the karyotype could not be determined with certainty as divisions were rare and because the morphology of the chromosomes was unclear. Mitotic divisions were recorded only in beetles from Armenian populations. The male mitotic metaphase displayed 35 chromosomes including 17 autosomal pairs that constitute a decreasing size series and large meta-submetacentric X chromosome (Fig. 1a, b). All large and middle-sized chromosomal pairs were meta-and submetacentric, except for one acrocentric autosomal pair (AA2). The morphology of the small chromosomes was poorly visible, however, most likely, one autosomal pair (AA13) was acrocentric and the others biarmed.
Meiotic spermatocyte divisions were recorded in males from both Armenian and Belarusian populations. At diakinesis/metaphase I (MI) 17 autosomal bivalents and an un-paired meta-submetacentric X chromosome were observed (Figs 1c,d,e;2a,b,c). The autosomal bivalents gradually decreased in size. At diakinesis and prometaphase there were two or three ring-shaped autosomal bivalents with two chiasmata, two cross-shaped bivalents with an interstitial chiasma and the remaining bivalents were rod-shaped and most likely had one terminal chiasma.
At metaphase II there were 17 and 18 chromosomes of which the majority were biarmed meta-and submetacentric. In each daughter cell, among the large meta-and submetacentric chromosomes one acrocentric chromosome was clearly visible, the morphology of the small chromosomes was unclear (Figs 1f, g; 2d, e).
At meiosis, Ag-banding revealed that the X chromosome was brightly homogenously argentophilic (Figs 1c,d,f;2a,b,d). In addition, at diakinesis Ag-positive signals, probably NORs (the nucleolus organizer regions) were clearly visible on two ring-shaped and one rod-shaped bivalents; moreover, small and weak argentophilic signals were observed on two rod-shaped bivalents (Figs 1с; 2a, b). On the autosomal chromosomes at prometaphase-metaphase I and metaphase II there were no distinct Ag-positive signals.
The C-banding revealed a small amount of constitutive heterochromatin weakly visible in the pericentromeric regions of some chromosomes that did not form distinct blocks (Figs 1e, g; 2c, e). At prometaphase-metaphase I (Figs 1e, 2c), small pericentromeric block of C-heterochromatin was visible on the unpaired X chromosome, while at metaphase II (Figs 1g, 2e) it was unclear which of the large two-armed chromosomes was the X chromosome.
Thus, the karyotype of the males of Colorado potato beetle from Armenia and Belarus is 2n = 35 (34 + X0), n = 17AA + X0. In beetles from all the populations studied, a pair of large acrocentric chromosomes was present.

Comparison of the sequences and phylogenetic analyses
Comparative analysis of the sequencing results revealed no nucleotide substitutions between samples from four Armenian and fi ve Belarusian populations of L. decemlineata. Moreover, analysis of all available DNA barcodes revealed that this variant of the DNA barcode (herein called "European haplotype") was present in all the samples from Austria, Germany and Poland and in most (7 out of 13) samples from Bashkortostan (Russia). In 6 out of 13 Bashkortostan samples, few nucleotide substitutions are reported (Udalov & Benkovskaya, 2010); however, in our opinion, it remains unclear whether these substitutions were real or sequencing errors. Examination of the DNA barcode alignment also revealed four transitions differentiating the European samples from the four American sequences (Table 3).
Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic analyses of the Leptinotarsa DNA barcodes revealed the topology shown in Fig. 3.
In both BI and ML trees, the sequence HQ605769 from North Dakota, USA appeared as a sister to the clade that included all the European sequences (Fig. 3). Together, the North Dakota sequence and the European sequences Table 3. Nucleotides in four positions on the COI gene fragment differentiating the European DNA-barcodes from the four available Nearctic samples.  formed a sister clade to the clade consisting of the other three North American CPB sequences. The samples from three other species of Leptinotarsa (L. haldemani, L. juncta and L. texana) formed three separate clades, of which L. juncta and L. texana were sister clades, while L. haldemani was another separate clade. In addition, the sequence HQ984330 of the specimen mentioned in GenBank as Leptinotarsa sp. belonged to the last clade and, therefore, to the same species.
Additional phylogenetic analyses were conducted to study mitochondrial polymorphism in American populations. For this purpose, 82 haplotypes of the mitochondrial genome fragment that included the terminal part of the COI gene and the initial part of the COII gene were used. BI and ML analyses of these haplotypes revealed the topology shown in Fig. 4. In contrast to European samples (Fig. 3), these analyses showed a high level of mitochondrial polymorphism in North American populations and revealed several major haplogroups (Fig. 4), with a divergence level between them of up to 4%.
According to T.H. Hsiao (1985), the Colorado potato beetle is a chromosomally polymorphic species in North America. There are three chromosomal "races": (1) the metacentric "race", in which all autosomes are metacentric (Mexico, USA), (2) the acrocentric "race", derived from the metacentric "race" by the pericentric inversion in the second pair of autosomes (USA, Europe) and (3) the heterozygous meta-acrocentric "race" (USA, Canada). More recent studies revealed a large acrocentric chromosome pair in the chromosomes of a population from Canena in Spain, thus, confi rming the presence of the acrocentric "race" of CPB in Europe (Lorite et al., 2013).
Our studies did not reveal chromosomal polymorphism in Armenian and Belarusian populations of CPB. The large autosomal acrocentric pair was found in the karyotypes of all the populations of L. decemlineata studied. Therefore, we assume that both Armenian and Belarusian populations of CPB belong to the acrocentric "race", which according to literature data, is peculiar to European populations.
Up till now, karyological studies on CPB were carried out using mainly conventional staining techniques. In the present paper, Ag-banding was used to study the karyotype of the Colorado potato beetle for the fi rst time. This revealed that the X chromosome was homogenously argentophilic during the different stages of meiosis in all the populations studied, which is most likely due to the presence of an argentophilic substance (proteins). At diakinesis there were clearly visible argentophilic signals (probably NORs) located on some autosomal bivalents: the two ringshaped and one rod-shaped bivalents. Weak argentophilic signals were also detected on a few bivalents.
Baus Lončar et al. (2005) report for the fi rst time the Cbanding staining of the chromosomes of L. decemlineata, which is confi rmed by our study in which the chromosomes had a small amount of constitutive heterochromatin located pericentromerically. This observation is consistent with the data for other beetles. It is known that in most species of the order Coleoptera large C-blocks on chromosomes are uncommon and only recorded in a few species (for references see Rożek et al., 2004;Karagyan et al., 2012).
In the current study, analysis of the DNA barcode fragment of the COI gene revealed lack of interpopulation variability in all samples of CPB collected from different locations in Armenia and Belarus. Thus, there is only a single DNA barcode haplotype. Moreover, analysis of sequences from Austria, Germany and Poland available in the literature (Hendrich et al., 2015;Rulik et al., 2017) indicate that this DNA barcode haplotype ("European haplotype") is the only variant known from Western Europe. A more complicated situation is found in Bashkortostan (Russia) where the European haplotype is present in 7 of 13 studied samples (Udalov & Benkovskaya, 2010). In 6 out of 13 Bashkortostan samples, there are few nucleotide substitutions (Udalov & Benkovskaya, 2010); however, in our opinion, it is unclear whether these substitutions are real or sequencing errors. Anyway, the European haplotype is the only or the predominant one in all populations of the Old World studied. Therefore, we hypothesize that the European haplotype is the ancestral state for all populations of the Old World and inherited from the New World invaders who colonized Europe 100 years ago.
In contrast to Europe, the DNA analysis of American samples, both carried out earlier (Izzo et al., 2018) and in the current study, reveal an extremely high level of polymorphism. It should be noted, however, that the analysis of American samples is based on the other fragment of the mitochondrial genome, which makes it diffi cult to directly compare the American and European data and prevents the phylogeographic analysis of the entire dataset (America + Europe). For this reason, we cannot identify the North American population that was the ancestor of the European lineage of the Colorado potato beetle. Nevertheless, we believe that the population-ancestor will be found in North America in the near future and will shed light on the origin of the populations of this dangerous pest in Eurasia.