Zoodiversity_01_2021.indb UDC 595.768.2(477.4) GENETIC DIFFERENTIATION OF UKRAINIAN POPULATIONS OF EUSOMUS OVULUM (COLEOPTERA, CURCULIONIDAE): EVIDENCE OF MULTIPLE HYBRID SPECIATION S. Yu. Morozov-Leonov1*, V. Yu. Nazarenko2 Schmalhausen Institute of Zoology NAS of Ukraine vul. B. Khmelnytskogo, 15, Kyiv, 01030, Ukraine 1E-mail: morleone2000@yahoo.com 2E-mail: nazarenko@izan.kiev.ua *Corresponding author S. Yu. Morozov-Leonov (https://orcid.org/0000-0003-1784-7753) V. Yu. Nazarenko (https://orcid.org/0000-0003-4245-5049) Genetic Diff erentiation of Ukrainian Populations of Eusomus ovulum (Coleoptera, Curculionidae): Evidence of Multiple Hybrid Speciation.  Morozov-Leonov, S. Yu., Nazarenko, V. Yu. — Th e clonal structure of populations of the weevil Eusomus ovulum Germar, 1824 (Coleoptera, Curculionidae) from several regions of Ukraine was analyzed. Th e signifi cant diff erentiation between populations from dif- ferent region was demonstrated. Th e hypothesis of multiple origins of the hybrid form E. ovulum from several parental species is proposed. K e y w o r d s : weevil, Curculionidae, Eusomus, allozyme, polyclonal structure, multiple hybrid speciation. Introduction Natural hybridization is widely distributed in nature (Arnold, 2003; Avise, 2008). Th e consequences of hybridization are various. Hybrids are oft en sterile (Feng et al., 2020). Numerous fertile hybrids can be an important component of the populations of parental species (Konopiński, Amirowicz, 2018.). Finally, hybridization generates sometimes a new genetic form that diff ers from both parental species and is capable to reproduce autonomically (hybrid speciation) (Mallet, 2007; Vallejo-Marín, Hiscock, 2016). In particular, many hybrid forms with a species status are known among weevils (Coleoptera, Curculionidae) (Kajtoch, Lachowska- Cierlik, 2009; Stenberg, Lokki, Saura, 2000). Th e hybrid forms of weevils are known to reproduce by parthenogenesis. Th ere is the opinion that parthenogenesis is a very eff ective mode to provide fertility within such hybrid forms (Dedukh et al., 2020). One of the consequences of parthenogenetic reproduction is the clonal structure of the off spring. Parthenogenetic hybrid forms of weevils with a species status can be monoclonal or polyclonal (Nazarenko, Morozov-Leonov, 2018). Th e monoclonality of the hybrid form has a simple explanation. A single clone may come from a single Zoodiversity, 55(1): 9–16, 2021 DOI 10.15407/zoo2021.01.009 10 S. Yu. Morozov-Leonov, V. Yu. Nazarenko hybrid female. Th e origin of polyclonality is not so simple. First, the emergence of new clones can be a result of mutations (Vorburger, 2008). However, the study of the genetic variability of many clonal forms shows that their polyclonality is the result of multiple hybridization of the parental species (Collares-Pereira and Coelho, 2010; Mezhzherin et al., 2019; Bogart, 2019). It is obvious that the hybrid form, which occurred as a result of repeated acts of hybridization of the parental species, has a high level of genetic variability and a signifi cant evolutionary potential. Th erefore, for a correct assessment of the evolutionary prospects of any polyclonal genetic form, it is necessary to reconstruct its origin. Among the model species suitable for evolutionary genetic studies, weevils of the family Curculionidae are very promising (Stenberg et al., 2003; Stenberg, Lundmark, 2004). First, this family contains both Mendelian species and parthenogenetic ones. Second, these parthenogenetic forms are known to be polyploid hybrids. Th us, the study of the genetic structure of populations of parthenogenetic forms of weevils will make it possible to assess the evolutionary prospects of both hybrid forms and clonal reproduction. Previous studies have revealed the complex polyclonal structure of many parthenogenetic forms of weevils. Th us, the studies of the well-known weevil O. scaber showed the existence of noticeable genetic diff erentiation between geographically distant populations (Stenberg et al., 1997, 2000). More recent studies have demonstrated the interpopulation diff erentiation of other parthenogenetic forms of weevils, such as Polydrusus inustus (Kajtoch, Lachowska-Cierlik, Mazur, 2009; Kajtoch, Korotyaev, Lachowska-Cierlik, 2011), Eusomus ovulum (Mazur et al., 2016), and Strophosoma melanogrammum (Kotásková, Kolasa, Kajtoch, 2018). Th e value of interpopulation diff erentiation could be small (in case of E. ovulum) or relatively high (between P. inustus populations). Earlier, we detected genetic diff erentiation of geographically distant Otiorhynchus ligustici populations (Morozov-Leonov, Nazarenko, 2016). However, the populations we studied were within the same geographic region and diff ered in a single gene. Th is diff erence may be the result of mutation and successful reproduction of a mutant clone. Th erefore, the aim of our study was to analyze the genetic variability of populations of the polyclonal parthenogenetic weevil species that are located within several geographically distant regions. Material and methods For a detailed analysis, we used a widespread polyclonal species Eusomus ovulum Germar, 1824 (Coleop- tera, Curculionidae, Sciaphilini) (Nazarenko, Morozov-Leonov, 2018; Mazur et al., 2016). Th e material for this work was collected in 2017–2019. Th e distance between the most distant samples is approx. 700 km, from north to south 400 km. Samples were taken from 13 populations located on the territory of western, central, southern and eastern Ukraine (table 1, fi g. 1) (decimal latitude and longitude of each data collection point are indicated in parentheses). No. 1 — vicinity of Lutsk (50.734, 25.324), 24 specimens; No. 2 — near Buscha, 13 specimens (50.303,26.237); No. 3 — near Buderash, 20 specimens (50.303, 26.237); No. 4 — near Novomalyn, 18 spec- imens (50.300, 26.351); No. 5 — Lisnyky, 11 specimens (50.294, 30.529); No. 6 — Pidhirtsi, 61 specimens (50.245, 30.550); No. 7 — Baryshivka, 11 specimens (50.363, 31.279); No. 8 — near Rankovyy, 61 specimens (50.236, 31.724); No. 9 — Tashyne, 20 specimens (46.902, 31.120); No. 10 — Tyagynka, 46 specimens (46.784, 33.036); No. 11 — Askaniya Nova, 27 specimens (46.454, 33.870); No. 12 — Gaydary, 24 specimens (49.626, 36.331); No. 13 — Siversky Donets fl oodplain, 10 specimens (49.631, 36.299). Fig. 1. Geographic localization of Eusomus ovulum samples. 11Genetic Diff erentiation of Ukrainian Populations of Eusomus ovulum… We have studied the electrophoretic variability of esterases (Es-1, 2, 3, 4, 5) that are known to be highly polymorphic within weevil species (Morozov-Leonov, Nazarenko, 2016; Nazarenko, Morozov-Leonov, 2018). Th e thoracic segments of every weevil were frozen during 12 hours, then used in the acrylamide electrophoresis. Sample preparation, electrophoretic analysis of esterases, the data interpretation were performed by standard methods (Harris, Hopkinson, 1976). All heterozygous phenotypes are indicated without clarifi cation of the gene dosage. Such designation is used because phenotypes like 100/100/113 and 100/113/113 sometimes cannot be reliably distinguished (fi g. 2). Th erefore, we designated all heterozygous phenotypes like as 100/113. Results A l l e l i c v a r i a b i l i t y o f t h e s t u d i e d g e n e s . Of the fi ve studied genes encoding nonspecifi c esterases, 4 are represented by several alleles (table 1, fi g. 2). Th e gene encoding Es-1 is represented by two alleles in the studied samples - Es-10 and Es-191 (fi g. 1). All identifi ed genotypes were homozygous Es-10/0 (clone Eo4) and Es-191/91 (all other clones). Th e Es-10 allele was detected in samples 9–12 only (southern and eastern Ukraine). Th e gene encoding Es-2 does not show variability detected electrophoretically. In all samples, it is represented by a single allele Es-282. Th e Es-3 gene is represented by two alleles (Es-3113 and Es-3118). Th e homozygous genotype Es-3118/118 is characteristic for Eo5 clone. All other clones have a heterozygous genotype Es-3113/118. Th e Es-4 gene is represented by three alleles (Es-489, Es-4100, Es-4113). Th e Es-489 allele was found in the Es-489/100 genotype only that is characteristic for Eo5 clone. Th e homozygous genotype Es-4100/100 is characteristic for Eo2 clone. All other clones (Eo1, Eo3, Eo4) have a heterozygous genotype Es-4100/113. Th e Es-5 gene is represented by three alleles Es-50, Es-581 and Es-5100. Allele Es-581 in the heterozygous genotype Es-581/100 is characteristic only for clone Eo5. Allele Es-50 is found in T a b l e 1 . Th e electrophoretic phenotypes of clones in Eusomus ovulum samples from Ukrainian populations Clone Sample T ot al No. Gene Es-1 Es-2 Es-3 Es-4 Es-5 1 2 3 4 5 6 7 8 9 10 11 12 13 Eo1 91 82 113/118 100/113 100 23 13 20 18 11 58 11 61 18 22 2 257 Eo2 91 82 113/118 100 100 3 3 Eo3 91 82 113/118 100/113 0 1 19 20 Eo4 0 82 113/118 100/113 0 2 24 27 3 56 Eo5 91 82 118 89/100 81/100 10 10 n 24 13 20 18 11 61 11 61 20 46 27 24 10 346 Fig. 2. Th e electrophoretic spectra of the esterases in the Eusomus ovulum specimens. 12 S. Yu. Morozov-Leonov, V. Yu. Nazarenko the homozygous genotype Es-50/0 and is characteristic for clones Eo3, Eo4. Th e allele Es-5100 (genotype Es-5100/100) is typical for clones Eo1, Eo2. Po l y c l o n a l s t r u c t u r e o f E u s o m u s o v u l u m p o p u l a t i o n s i n U k r a i n e . In the Ukrainian populations of Eusomus ovulum, 5 clones were identifi ed (table 1, fi g. 3). More- over, the Eo2 clone was rare — its frequency is 0.009. Th e frequencies of other clones vary from 0.029 (Eo5) to 0.743 (Eo1). Th e number of clones within a single sample varies from 3 (sample 12) or 2 (samples 1, 6, 9, 10) to 1 (all other samples). Two of the identifi ed clones were found in one sample only. Th ese are clones Eo2 (sample 6) and Eo5 (sample 13). Clone Eo4 was identifi ed in samples 9-12. Th e most numerous clone Eo1 was found in almost all samples, except for samples 11 and 13. G e n e t i c d i f f e r e n t i a t i o n o f E u s o m u s o v u l u m c l o n e s i n U k r a i n i a n p o p u l a t i o n s . Analysis of the studied genes variation allows to divide the identifi ed clones into three groups. Th e fi rst group includes clone Eo1 and clones Eo2 and Eo3 that are genetically close (table 1). Th ey have a similar set of alleles and diff er from clone Eo1 in one gene (Es-4 for clone Eo2 and Es-5 for clone Eo3). Clone Eo4 should be referred to the second group. It also has a set of alleles that is similar to Eo1 clone, but diff ers from it in the genotypic composition of two genes (Es-1 and Es-5). Finally, the third group consists of a single clone Eo5. Its allelic set diff ers from all others in the presence of alleles detected only in this clone (Es-489 and Es-581). F e a t u r e s o f t h e g e o g r a p h i c a l d i s t r i b u t i o n o f t h e i d e n t i f i e d c l o n e s . None of the identifi ed clones were found in all studied samples. Th e most numerous clone Eo1 was found in all regions, except for the most eastern one (the Siversky Donets fl oodplain). A local clone Eo2 was identifi ed in one of the samples from central Ukraine. Clone Eo3 is located on the right shore of the Siversky Donets River. Th e discovery of a single specimen Fig. 3. Polyclonal structure of studied Eusomus ovlulum samples inUkraine. 13Genetic Diff erentiation of Ukrainian Populations of Eusomus ovulum… carrying this clone on the territory of western Ukraine requires a more detailed analysis of populations from there. Clone Eo4 is widespread in samples from southern and eastern Ukraine, except for sample 13, which is located farther than all others in the eastern direction. Clone Eo5 was recorded in the sample from the Siversky Donets fl oodplain. Discussion A l l e l i c v a r i a b i l i t y o f t h e s t u d i e d g e n e s . Clonal forms of weevils are characterized by constant heterozygosity for some of the studied genes (Nazarenko, Morozov-Leonov, 2018). Our data, resulting from this study, confi rm this pattern, which clearly marks the hybrid nature of parthenogenetic forms of weevils (Kajtoch, Lachowska-Cierlik, 2009). P o l y c l o n a l s t r u c t u r e o f E u s o m u s o v u l u m p o p u l a t i o n s i n U k r a i n e . Th e Eusomus ovulum species as a whole can be characterized as having a polyclonal structure, in contrast to some previously studied related monoclonal forms of hybrid origin (Nazarenko, Morozov-Leonov, 2018). Our results, obtained using electrophoretic analysis of enzymes, are consistent with those of other researchers who have studied the variability of DNA sequences (Mazur et al., 2016). In both cases, variability of the studied characters and interpopulation diff erentiation were found. G e n e t i c d i f f e r e n t i a t i o n o f U k r a i n i a n p o p u l a t i o n s o f t h e p a r t h e n o g e n e t - i c c l o n a l f o r m E u s o m u s o v u l u m a n d t h e i r o r i g i n . Analysis of the allelic variation within the studied E. ovulum populations requires an explanation. First of all, it is necessary to off er a reasonable explanation for the existence of the Eo5 clone, which diff ers markedly from all other clones detected in the Ukrainian populations of E. ovulum. Th ree hypotheses can be proposed to explain the observed interpopulation diff erentiation of this species. First, clone Eo5 may be of mutant origin. Th e successful expansion of a clone derived from a single mutant specimen is theoretically possible. In nature, the cases are known of a single clone successful dispersal in many populations within a large geographic region (Hotz et al., 2008). Moreover, there are cases when in some populations the frequency of an initially rare (hemi) clone increases signifi cantly (Morozov-Leonov, 2017). However, in that case of E. ovulum, the hypothesis of the mutant origin of Eo5 suggests a two-step process of the appearance within one clone of two mutations in two diff erent genes (Es-4 and Es-5). Th e probaility of such a coordinated mutagenesis of two diff erent genes seems to be low. A more reliable test of this hypothesis requires the study of other E. ovulum populations located in eastern Ukraine. Second, clones of E. ovulum could arise from hybridization of two polymorphic Men- delian species. Th is hypothesis provides the simplest explanation for the observed poly- clonal structure of the studied weevil species. Is it known that many polymorphic hybrid forms are derived from polymorphic ancestral species (Arioli, Jakob, Reyer, 2010). Howev- er, in this case, the problem of explaining the signifi cant interpopulation diff erentiation of parthenogenetic E. ovulum remains in the form of a problem of signifi cant diff erentiation of its ancestral species. Th e weak point of this hypothesis is the lack of data on the clonal forms of E. ovulum that are genetically intermediate between both groups of clones (Eo1-4 and Eo5). To confi rm or deny this hypothesis, a detailed genetic analysis of the popula- tions of the Mendelian species that is ancestral for the parthenogenetic form E. ovulum, is required. Unfortunately, at the moment, no populations of the species ancestral for E. ovu- lum have been found, unlike O. scaber, for example, where populations of the ancestral Mendelian species are known (Stenberg et al., 2003; Stenberg, Lundmark, 2004). Finally, third, clones of E. ovulum could have come from hybridization of more than two parental Mendelian species. It can be assumed that one Mendelian ancestor species (spe- 14 S. Yu. Morozov-Leonov, V. Yu. Nazarenko cies A) was common to all existing clones (table 2). Th e polymorphism of this species for two genes (Es-1, Es-5) allowed it to generate more than one clone of hybrid origin. Th is species, as we suppose, has repeatedly hybridized with at least two other species. Th e second hypo- thetical ancestor species (species B), as shown by the obtained data, was polymorphic in three genes (Es-1, Es-4, Es-5). Th e hybridization “species A x species B” has occurred several times. In some populations, the alleles Es-1100 and Es-5100 prevailed. Th ere, the hybridization “species A x species B” gave rise to clones Eo1, Eo2, spreading in western and central Ukraine. In other populations of this species, the Es-50 allele prevailed. Accordingly, the hybridization “species A x species B” gave rise to clones Eo3 and Eo4, living in the territory of eastern Ukraine. Fi- nally, in some populations, species B had the Es-10, Es-50 alleles only. As a result, the hybrid form in this region is monoclonal and is represented by the clone Eo4 (southern Ukraine). Th e third Mendelian species, hypothetically involved in hybridization (species C), was genetically distant from the two previous species. Th e hybridization “species A x species C gave rise to the clone Eo5, found in the fl oodplain of the Siversky Donets only and being genetically distant from the parapatric clones Eo3 and Eo4. In fact, the third hypothesis is the development of the second one, provided that the dif- ferentiation between the populations of one of the ancestral species has reached the species level. An objection to the hypothesis of E. ovulum origin from more than 2 ancestral spe- cies may be the fact that there is no reliable morphological diff erentiation between diff erent clones of this form. It should be noted that there are many known examples of extreme morphological similarities between genetically separate species. For example, such pairs of species are detected among snails (Rama Rao et al., 2018), fi sh (Winston, 1995; Ivankov, Kaplunenko, Borisovets, 2016) and even mammals (Stoffb erg, Jacobs, Miller-Butterworth, 2004). However, a detailed analysis of the morphological variation across E. ovulum species area seems to be very promising, looking at its genetic diff erentiation. E c o l o g i c a l p r e f e r e n c e s o f E u s o m u s o v u l u m d i f f e r e n t c l o n e s . Our data show that the hybrid form Eusomus ovulum does not have a strictly limited ecological niche. Clones Eo1, Eo2 were found within the forest and forest-steppe zones. Clones Eo3, Eo4 are localized within the steppe zone. Finally, the Eo5 clone was found only in the wetland. A T a b l e 2 .Hypothetical reconstruction of E. ovulum clones origin on the territory of Ukraine Species A Hybrid clone Other parental species Alleles Gametes Number Genotype Gametes Alleles Es-10, Es-191 Es-3118 Es-4100 Es-50, Es-5100 Es-191 Es-3118 Es-4100 Es-5100 Eo5 Es-191/91 Es-3118/118 Es-489/100 Es-581/100 Es-191 Es-3118 Es-489 Es-581 Es-191 Es-3118 Es-489 Es-581 Sp ec ie s C Eo1 Es-191/91 Es-3113/118 Es-4100/113 Es-5100/100 Es-191 Es-3113 Es-4113 Es-5100 Es-10, Es-191 Es-3113 Es-4100, Es-4113 Es-50, Es-5100 Sp ec ie s B Eo2 Es-191/91 Es-3113/118 Es-4100/100 Es-5100/100 Es-191 Es-3113 Es-4100 Es-5100 Es-191 Es-3118 Es-4100 Es-50 Eo3 Es-191/91 Es-3113/118 Es-4100/113 Es-50/0 Es-191 Es-3113 Es-4113 Es-50 Es-10 Es-3118 Es-4100 Es-50 Eo4 Es-10/0 Es-3113/118 Es-4100/113 Es-50/0 Es-10 Es-3113 Es-4113 Es-50 15Genetic Diff erentiation of Ukrainian Populations of Eusomus ovulum… clear correlation between the clone genotype and the habitat is an additional argument in support for the hypothesis of the parthenogenetic polyclonal form Eusomus ovulum multiple hybrid origin. 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