NORTH-WESTERN JOURNAL OF ZOOLOGY 13 (2): 220-226 ©NwjZ, Oradea, Romania, 2017 Article No.: e161204 http://biozoojournals.ro/nwjz/index.html
Allozyme variability in three Eupelmus species (Hymenoptera: Eupelmidae) from Bulgaria
Miroslav ANTOV1, Ivan STOYANOV2, Anelia STOJANOVA1 and Teodora STAYKOVA2*
1. Plovdiv University „Paisii Hilendarski”, Department of Zoology, Plovdiv, Bulgaria. 2. Plovdiv University „Paisii Hilendarski”, Department of Developmental Biology, Section of Genetics, Plovdiv, Bulgaria. *Corresponding author, T. Staykova, Tel: +359 261 549; Fax: +359 32 261-566; E-mail: [email protected]
Received: 01. April 2016 / Accepted: 04. November 2016 / Available online: 24. November 2016 / Printed: December 2017
Abstract. Four enzyme systems i.e. malate dehydrogenase (MDH), malic enzyme (ME), acid phosphatase (ACP) and aspartate aminotransferase (AST) were studied in three parasitoid species of the genus Eupelmus (E. aseculatus (Kalina, 1981)), E. urozonus Dalman, 1820 and E. microzonus Förster, 1860) using PAGE. Only MDH and ME produced clear bands. No bands were obtained for ACP and AST. MDH enzyme was found to be useful to distinguish between Eupelmus species. Polymorphism with four alleles of the Mdh-1 locus and monomorphism of the Mdh-2 and Me loci were observed. Intraspecific and interspecific variability of Mdh-1 alleles in the studied species from Bulgaria were detected. Mdh-159 allele was unique to E. aseculatus. The degree of polymorphism and heterozygosity of each species were calculated. Cluster analysis, based on Nei’s genetic distance confirmed that E. aseculatus, which belongs to subgenus Macroneura is divergent from E. urozonus and E. microzonus, which belong to subgenus Eupelmus.
Key words: Hymenoptera, Eupelmus, allozymes, population genetics.
Introduction methods (morphometric analysis, allozyme elec- trophoresis and evaluation of host preferences) for Eupelmus Dalman, 1820 is a genus of cosmopolitan investigation of two cryptic eupelmid species. The parasitic wasps which consists over 300 species results of the allozyme analysis clearly showed the (Noyes 2016). Gibson & Fusu (2016) in their recent usefulness of the electrophoretic method. paper recognized 104 species from the Palaeartic Allozyme electrophoresis is an effective tech- region. Gibson (1995) distinguished three subgen- nique that has been applied successfully for de- era within the genus Eupelmus Dalman: Eupelmus, termining the genetic variation of many organisms Episolindelia Girault and Macroneura Walker. Spe- (Gómez 1998, Staykova et al. 2015, Stoyanov et al. cies of Eupelmus are mostly primary or secondary 2015), and can be helpful to differentiate between ectoparasitoids of the preimaginal stages of a wide closely related species in cases when it is not pos- range of insect hosts that are concealed in pro- sible to do so using morphological characters (Pin- tected habitats such as seeds, grass stems, galls or tureau 1993, Pinto et al. 2003). Allozyme electro- cocoons (Gibson 1990, 2011, Lotfalizadeh & phoresis could be used as a quick and cheap Ghadirzadeh 2016). method of finding population markers, even when The identification of the Eupelmus parasitoids dealing with small insects (Loxdale & Lushai is very difficult because of the extreme sexual di- 1998). morphism typical for subfamily Eupelminae (Gib- Enzyme markers have been extensively ap- son 1990, 1995, 2011, Lotfalizadeh et al. 2006) and plied in taxonomic, systematic and population ge- the existence of species groups that contain very netics entomological studies (Atanassova et al. close morphologically species (Gibson & Fusu 1998, Kitthawee et al. 1999, Pinto et al. 2003, Byrne 2016). Morphological observations realized by the & Toscano 2006). The use of isoenzyme markers is recent identification keys (Gibson 1995, Gibson of great importance because it has made easier 2011, Al khatib et al. 2014, Gibson & Fusu 2016) studying the diverse areas of biology covering remain valuable to carry out a first diagnosis. questions of phylogeny, evolution, ecology and Due to difficulties in identifying the species of population dynamics (Loxdale & den Hollander the genus Eupelmus based on morphological fea- 1989, Smith & Wayne 1996, Symondson & Liddell tures, other useful methods such as molecular (Al 1996). Even if they have been largely superseded khatib et al. 2014, 2016), cytogenetic (Fusu 2008) by DNA-based markers, allozymes are still a very and electrophoretic (Fusu 2010) are used. Fusu useful technique when it is not possible to develop (2010) applied a combination of three different microsatellite markers (Ridgway 2005).
Allozyme variability in Eupelmus species 221
There is insufficient information about isoen- ing the Gendist and Neighbor programs in the PHYLIP zyme and allozyme polymorphism and its use for (Felsenstein 1993) software package. population genetics studies in the Eupelmus parasi- toids. Due to this fact the purpose of this study was to investigate intraspecific and interspecific Results variability of three Eupelmus species and to deter- mine the degree of their genetic diversity using al- No staining occurred for the ACP and AST enzy- lozyme markers. matic activities in all tested samples. The MDH and ME produced clear bands which were used for a detailed study. The banding patterns on Fig- Materials and Methods ure 1 and 2 represent enzyme electromorphs. Polymorphism was observed at MDH only. Eupelmus samples A total of four different alleles at the Mdh-1 Three eupelmid species: Eupelmus urozonus, Eupelmus mi- locus (cytozolic form of MDH) were detected in crozonus and Eupelmus aseculatus were used for polyacri- lamide gel electrophoresis (PAGE). The insects were the studied species (Table 1). One species - E. reared from galls in laboratory conditions. Eupelmus uro- aseculatus showed a unique fixed allele at the zonus was reared from Rosa galls (Osogovo Mts: Granitsa Mdh-1 locus. Village N 42°15'30.0''/E 022°44'26.2'') and E. microzonus Mdh-1100 and Mdh-178 alleles were present in and E. aseculatus - from Hypecoum imberbe galls (Thracian both E. urozonus and E. microzonus, with Mdh-178 Lowland: Plovdiv, Dzhendem Tepe locality N allele occurring more frequently (Table 1). In E. 42°08'11.2''/E 024°43'55.6''). Identification of the species urozonus this allele was recorded with exception- was done using keys of Kalina (1981), Gibson (2011), Al 84 khatib et al. (2014), Gibson & Fusu (2016). The specimens ally high frequency (0.969). Mdh-1 allele was de- were aspirated with a pooter anaesthetized with ethyl tected in E. microzonus only. It was much rarer acetate and stored at - 20°C prior to electrophoretic analy- compared to the Mdh-178 allele for this species. sis. A total of 130 imagoes (males and females) from the Mdh-159 only occurred in E. aseculatus, which ap- three species were studied for four enzymatic systems us- peared to be fixed for this allele. ing polyacrylamide vertical gel electrophoresis (mean For the Mdh-2 locus (mitochondrial form of sample size per locus ranged from 38.7 to 46.0) (Table 1). MDH) all specimens tested from the three species
100 Enzyme electrophoresis were homozygous for a single allele Mdh-2 and The whole body of each specimen was squashed with presented a single monomorphic band. quartz sand in 0.8 M trisphosphate buffer at pH=6.7, and Only one allele of the malic enzyme (Me) locus laid for extraction for 18 hours at 4C. Then the samples occurred in E. urozonus, E. microzonus and E. asecu- were centrifuged for 15 minutes at 5000 rpm before latus (Table 1). All three species were fixed for the PAGE. Ten μl of each sample were applied into the gel. Me100 allele. The electrophoretic separation was achieved using 7.5% No sex related differences of MDH and ME polyacrylamide vertical gel at 4.5 mA/cm for 3 hours, to- spectra were observed by PAGE. gether with concentrating gels (3.3%) at pH=6.7, and 0.05 M tris 0.2 M glycine electrode buffer at pH=8.3 (Davis In the three Eupelmus species, mean observed 1964, Maurer 1971). The following enzymatic activities heterozygosities (Ho) ranged from 0 to 0.085 - for were tested: malate dehydrogenase (MDH - ЕС 1.1.1.37), E. aseculatus (no polymorphism was observed) and malic enzyme (ME - EC 1.1.1.40), acid phosphatase (ACP - E. microzonus, respectively (Table 2). In E. urozonus EC 3.1.3.2) and aspartate aminotransferase (AST - ЕС the expected heterozygosity (He) and the observed 2.6.1.1). Buffers and histochemical staining for each en- one (Ho) had the same value (0.020). E. microzonus zyme were as in Shaw & Prasad (1970), Staykova et al. showed a mean observed heterozygote deficiency (2010) and Schmidtke & Engel (1972). compared with the Hardy-Weinberg expectation Statistical analyses (Ho =0.085, He =0.123). After the discovery of the isoenzyme activity regions, the Significant differences (P˂0.05) in genotype phenotypes of the discovered loci were recorded. Allele frequencies were seen at the Mdh-1 locus for E. frequencies, mean number of alleles per locus, proportion microzonus. Chi-Square (DF=3) test showed that of polymorphic loci, observed (Ho) and expected (He) het- the deviations were a result of excess of homozy- erozygosity, deviation from the Hardy-Weinberg equilib- gotes and deficiency of heterozygotes (Table 3). rium and Nei’s genetic distance (D) (Nei 1972) were cal- culated using BIOSYS-1 (Swofford & Selander 1981). The The comparison of the same locus showed these to cladogram was constructed using Nei’s (1972) genetic dis- be not significantly different in E. urozonus. tance and the UPGMA (Sneath & Sokal 1973) method us- In the analysed Eupelmus species using malate
222 M. Antov et al.
Figure 1. Malate dehydrogenase spectra of Eupelmus parasitoids in 7.5% PAGE: from left to right 1-13, 16-19 – E. micro- zonus; 14-15 - E. aseculatus.
Table 1. Allele frequencies at enzyme loci (Malate dehydrogenase – Mdh-1, Mdh-2 and Malic enzyme - Me) in Eupelmus species tested.
E. urozonus (42 ♀ + 7 ♂) E. microzonus (24 ♀ + 15 ♂) E. aseculatus (42 ♀) Mean sample size per locus 46.0±3.0 38.7±0.3 40.7±0.7 Locus/allele Frequencies Mdh-1 Mdh-1100 0.031 0.103 0.000 Mdh-184 0.000 0.115 0.000 Mdh-178 0.969 0.782 0.000 Mdh-159 0.000 0.000 1.000 Mdh-2 Mdh-2100 1.000 1.000 1.000 Me Me100 1.000 1.000 1.000
Table 2. Mean number of alleles per locus, proportion of polymorphic loci, observed (Ho) and expected (He) heterozy- gosity.
Species Mean no. of alleles per locus Percent Polymorphic loci (P=0.99) Ho He E. urozonus 1.3±0.3 33.3 0. 020±0.020 0. 020±0.020 E. microzonus 1.7 ±0.7 33.3 0. 085±0.085 0. 123±0.123 E. aseculatus 1.0±0.0 0.0 0. 000±0.000 0. 000±0.000
dehydrogenase and malic enzyme loci the mean number of alleles ranged from 1.0 (for E. asecula- tus) to 1.7 (for E. microzonus) (Table 2). The degree of polymorphism (according to the criterion 0.99) was 33.3% for E. urozonus and E. microzonus, and 0% for E. aseculatus (no polymorphic loci). Accord- ing to Selander 1976 (as cited by Lázzari 1992) many populations of animal species are polymor- phic at 25-50% of their enzyme loci, whereas indi- viduals are, on average, heterozygous at 5-15% of their loci.
The values of genetic distance (Nei 1972) cal- Figure 2. Malic enzyme spectra of Eupelmus parasitoids culated using the allele frequencies were 0.008 (be- in 7.5% PAGE: from left to right 1-7 - E. microzonus. tween E. urozonus and E. microzonus), 0.341 (be-
Allozyme variability in Eupelmus species 223
Table 3. Chi-square test for deviation from Hardy-Weinberg equilibrium.
Species Locus Genotype Observed frequency Expected frequency Chi-square DF P E. urozonus Mdh-1 Mdh-1100/100 0 0.046 0.049 1 0.825 Mdh-178/100 2 2.908 Mdh-178/78 46 46.046 E. microzonus Mdh-1 Mdh-1100/100 0 0.410 13.581 3 0.004* Mdh-184/100 3 0.923 Mdh-178/100 5 6.256 Mdh-184/84 2 0.519 Mdh-178/84 2 7.038 Mdh-178/78 27 23.853 E. aseculatus No polymorphic loci
*Frequencies significantly different at P ˂ 0.05
Table 4. Nei’s (1972) genetic distance (below diagonal) variability of Eupelmus parasitoids. Fusu (2010) based on isoenzymes. observed distinct polymorphism of Me locus with
Species E. urozonus E. microzonus E. aseculatus four alleles and monomorphism of Mdh-1 and E. urozonus ***** Mdh-2 loci in Eupelmus vesicularis by means of cel- E. microzonus 0.008 ***** lulose acetate electrophoresis. Me, Mdh-1 and E. aseculatus 0.395 0.341 ***** Mdh-2 loci identified in our investigation corre-
sponds to Me, Mdh-1 and Mdh-2 loci described by tween E. aseculatus and E. microzonus) and 0.395 Fusu (2010). The results for variability of Me and (between E. urozonus and E. aseculatus) (Table 4). Mdh-1 loci in E. urozonus, E. microzonus and E. In the UPGMA dendrogram the studied spe- aseculatus, however were different from these in E. cies are grouped in two clades (Fig. 3). Eupelmus vesicularis. The Mdh-1 locus showed distinct urozonus and E. microzonus species were clustered polymorphism among the studied species with a in one clade and E. aseculatus was separate. diagnostic allele (Mdh-159) for one of them - E. aseculatus. Lack of polymorphism of the Me locus in all three species was found. The Mdh-2 locus was monomorphic in E. urozonus, E. microzonus Figure 3. UPGMA dendro- and E. aseculatus, which is in accordance with re- gram of three Eupelmus sults reported for E. vesicularis (Fusu 2010). species based on Nei’s Low levels of genetic variation in Hymenop- genetic distance. tera have been reported from many authors (Sny- der 1974, Metcalf et al. 1975, Pamilo et al. 1978, Berkelhamer 1983). This phenomenon has been Discussion explained by the haplodiploid breeding system and eusociality among studied species Isoenzymes could be used for population genetic (Suomalainen 1962, Crozier 1970, Snyder 1974, characterisation of some morphologically very Pamilo & Crozier 1981). In our study we exclude similar species and their differentiation. According the eusociality as a probable factor justifying the to Wool et al. (1989) isozyme patterns are specific low levels of genetic variability because Eupelmus and may be applied to identify adults of species, species are solitary. Thus the main focus should be which are often difficult or impossible to identify directed to haplodiploidy. Comparing the average morphologically. Pintureau (1993) defined mor- value of expected heterozygosity (He= 0.047) for phological characters used for differentiating Eupelmus species with the same one observed in
Trichogramma (Chalcidoidea: Trichogrammatidae) diplodiploid insects (He= 0.135) and other hymen- species as often variable and showed that bio- opterans (He= 0.037) (see Berkelhamer 1983) we chemical characters can be used for distinguishing concluded that the Eupelmus species studied are between morphologically close forms. significantly less variable than diplodiploid insects
The results described in this study indicate (He= 0.047 ˂ He= 0.135) and the average heterozy- that MDH isozyme markers could be successfully gosity in Hymenoptera is not greater than that of applied to examine the species-specific genetic the Eupelmus species tested (He= 0.037 ˂ He= 0.047)
224 M. Antov et al. which is in accordance with Berkelhamer (1983). tence of morphologically-similar species within These data show that haplodiploidy may be the the taxon. main reason for the low levels of genetic variabil- MDH may be useful for studying the species- ity in Hymenoptera but there are other factors specific genetic variability of Eupelmus. The spe- which in combination with haplodiploidy will cies from two subgenera Eupelmus and Macroneura lead to reinforcement of this effect: small effective are clearly distinguishable electrophoretically at population size (Pamilo et al. 1978), inbreeding the Mdh-1 locus. The Mdh-159 allele is absent from (Wagner & Briscoe 1983) and genetic bottlenecks the species of subgenus Eupelmus - E. urozonus and (Menken 1991). E. microzonus. Three main mechanisms of movement are Malic enzyme was not suitable to distinguish typical for chalcidoids: flying, walking and jump- between the tested species because the Me locus ing (Gibson 1986). Females of both E. urozonus and shared the same electromorph in all three Eu- E. microzonus are macropterous, while E. aseculatus pelmus species. females are brachypterous and are incapable of Genetic similarity between E. urozonus and E. flight, hence their individuals have reduced mobil- microzonus was much higher than that found be- ity. tween E. aseculatus and each of the other two spe- The existence of males in both E. urozonus and cies. The dendrogram constructed using the UP- E. microzonus means that they are arrhenotokous. GMA cluster analysis confirmed that E. urozonus is Males of E. aseculatus are unknown (Kalina 1981) most closely related with E. microzonus. E. asecula- and the species is probably thelytokous. The ex- tus, which belongs to subgenus Macroneura is di- planations for observed monomorphism in E. vergent from E. urozonus and E. microzonus, which aseculatus lead to two predictions. First, if it is the- belong to subgenus Eupelmus. lytokous that means it reproduces by some form of automixis, which can have many consequences for the amount and structure of genetic variation Conclusions in the population and will lead to increase of ho- mozygosity in the population (Leach et al. 2009, The results obtained in this study reveal new in- Rabeling & Kronauer 2013). Second, if males are formation about the genetic variability of E. asecu- present then inbreeding can also explain the ob- latus, E. urozonus and E. microzonus from different served monomorphism. According to Wagner & areas of Bulgaria on the base of allozyme analysis. Briscoe (1983) another factor that lead to the ob- At present there are no other studies that geneti- served phenomenon is a reduced heterozygote cally compare these three species. The discrimina- advantage.The E. microzonus population had a tion of the species from two different Eupelmus mean heterozygote deficiency compared with subgenera by the MDH allozyme pattern is in ac- Hardy-Weinberg expectation. This probably re- cordance with their taxonomic separation. These sults from reproduction by arrhenotokous parthe- markers would be used in combination with mor- nogenesis associated with the small effective phological and biological information in the fur- population size which could lead to inbreeding ther study of Eupelmus parasitoids. explaining reduced heterozygote advantage. Ac- cording to Gibson et al. (1997) all chalcidoids have a haploid-diploid mechanism of sex determination Acknowledgments. This work has been financed by the that occurs in two types of parthenogenesis (ar- “Fund for Scientific Researches” at Plovdiv University rhenotoky and thelytoky). Genetic variations is “Paisii Hilendarski” – contract MU 15-BF-013. Many expected to be low in parthenogenetic organisms thanks to Dr. Gary Gibson (CNC, Ottawa), Dr. Rudi (Steiner et al. 1985). Kitthawee et al. (1999) also de- Berkelhamer (University of California, Berkeley, tected significant deviations from the Hardy- California) and Professor David Wool (Tel-Aviv Weinberg expectations in the Nakornratchasima University) for the provided literature. We are very female population. They observed a deficiency of thankful to Ivanka Popova and Teodora Stoyanova for language notes. heterozygotes for nine of ten polymorphic loci and offered the following six possible explanations: the existence of null alleles; probability of misclassifi- cation of the individuals; thelytoky; selection against heterozygotes; inbreeding and the exis-
Allozyme variability in Eupelmus species 225
References longicaudata (Hymenoptera: Braconidae) in Thailand. Genetica 105: 125-131. Al Khatib, F., Cruaud, A., Fusu, L., Genson, G., Rasplus, J.-Y., Ris, Lázzari, S.M.N. (1992): Characterization and discrimination of three N., Delvare, G. (2016): Multilocus phylogeny and ecological Rhopalosiphum species (Homoptera: Aphididae) based on differentiation of the “Eupelmus urozonus species group” isozymes. Revista Brasileira de Zoologia 9 (1/2): 139-151. (Hymenoptera, Eupelmidae) in the West-Palaearctic. BioMed Leach, I.M., Pannebakker, B.A., Schneider, M.V., Driessen, G., van Central Evolutionary Biology 16(13): 1-20. de Zande, L., Beukeboom, L.W. (2009): Thelytoky in Al khatib, F., Fusu, L., Cruaud, A., Gibson, G., Borowiec, N., Hymenoptera with Venturia canescens and Leptopilina clavipes as Rasplus, J.Y., Ris, N., Delvare, G. (2014): An integrative case studies. pp.347-375. In: Schön, I., Martens, K., van Dijk, P. approach to species discrimination in the Eupelmus urozonus (eds.), Lost Sex: The Evolutionary Biology of Parthenogenesis. complex (Hymenoptera, Eupelmidae), with the description of 11 Springer, The Netherlands. new species from the Western Palaearctic. Systematic Lotfalizadeh, H., Ghadirzadeh, L. (2016): Review of Iranian Entomology 39: 806-862. Eupelmidae (Hymenoptera: Chalcidoidea) with five new Atanassova, P., Brookes, C.P., Loxdale, H.D., Powell, W. (1998): records. Journal of Insect Biodiversity and Systematics 2(1): 155– Electrophoretic study of five aphid parasitoid species of the 166. genus Aphidius (Hymenoptera: Braconidae), including evidence Lotfalizadeh, H., Rasplus J.Y., Delvare, G. (2006): Rose gall wasps for reproductively isolated sympatric populations and a cryptic and their associated fauna (Hymenoptera) in Iran. Redia 139: species. Bulletin of Entomological Research 88: 3-13. 73–85. Berkelhamer, R.C. (1983): Intraspecific genetic variation and Loxdale, H.D., den Hollander, J. (Eds) (1989): Electrophoretic haplodiploidy, eusociality, and polygyny in the Hymenoptera. studies on agricultural pests. Systematics Association Special Evolution 37: 540-545. Volume No. 39. Clarendon Press, Oxford, UK. Byrne, F.J., Toscano, N.C. (2006): Detection of Gonatocerus ashmeadi Loxdale, H.D., Lushai, G. (1998): Molecular markers in entomology. (Hymenoptera: Mymaridae) parasitism of Homalodisca coagulata Bulletin of Entomological Research 88: 577-600. (Homoptera: Cicadellidae) eggs by polyacrylamide gel Maurer, G. (1971): Disc electrophoresis. Mir, Moscow [in Russian]. electrophoresis of esterases. Biological Control 36: 197-202. Menken, S.B.J. (1991): Does haplodiploidy explain reduced levels of Crozier, R.H. (1970): On the potential for genetic variability in genetic variability in hymenoptera? Proceedings of the Section haplodiploidy. Genetica 41: 551-556. Experimental and Applied Entomology, Nederlandse Davis, B.J. (1964): Disc electrophoresis - II Method and application Entomologische Vereniging Amsterdam 2: 172-178. to human serum proteins. Annals of the New York Academy of Metcalf, R.A., Marlin, J.C., Whitt, G.S. (1975): Low levels of genetic Sciences 121: 404-427. heterozygosity in Hymenoptera. Nature 257: 792-794. Felsenstein, J. (1993): PHYLIP (Phylogeny Inference Package). Nei, M. (1972): Genetic distance between populations. The Version 3.5C Distributed by the author. Department of Genetics, American Naturalist 106: 283-292. University of Washington, Seattle, W. A. Noyes, J.S. (2016): Universal Chalcidoidea database. World Wide Fusu, L. (2008): The usefulness of chromosomes of parasitic wasps Web electronic publication.
226 M. Antov et al.
Staykova, T., Ivanova, E., Tzenov, P., Vasileva, Y., Arkova- Symondson, W.O.C., Liddell, J.E. (Eds) (1996): The ecology of Pantaleeva, D., Grekov, D., Avramova, K. (2015): Genetic agricultural pests: biochemical approaches. The Systematics analysis of isoenzyme polymorphism in silkworm (Bombyx mori Associatian Special Volume Series 53. Chapman & Hall, L.) (Lepidoptera: Bombycidae) strains and phylogenetic London. relationships. Acta Zoologica Bulgarica 67(1): 117-125. Wagner, A.E., Briscoe, D.A. (1983): An absence of enzyme Steiner, W.W.M., Voegtlin, D.J., Irwin, M.E., Kampmeier, G. (1985): variability within two species of Trigona (Hymenoptera). Electrophoretic comparison of aphid species: detecting Heredity 50(2): 97-103. differences based on taxonomic status and host plant. Wool, D., Gerling, D., Nolt, B.L., Constantino, L.M., Bellotti, A.C., Comparative Biochemistry and Physiology 81(2): 295-299. Morales, F.J. (1989): The use of electrophoresis for identification Stoyanov, I., Staykova, T., Stojanova, A., Vasileva, P., Ivanova, E. of adult whiteflies (Homoptera, Aleyrodidae) in Israel and (2015): Isoenzymic genetic variability in populations of Messor Colombia. Journal of Applied Entomology 107(1-5): 344-350. structor (Hymenoptera: Formicidae) from Bulgaria. Acta Zoologica Bulgarica 67(3): 337-344. Suomalainen, E. (1962): Significance of parthenogenesis in the evolution of insects. Annual Review of Entomology 7: 349-366. Swofford, D.L., Selander, R.B. (1981): BIOSYS-1: A computer program for the analysis of allelic variation in genetics Rel. 1.0 Department of Genetics and Development University of Illinois at Urbana-Champaign. Urbana, Illinois 60801, USA.