Research Article |
Corresponding author: Michael Mikát ( michael.mikat@gmail.com ) Academic editor: Christopher K. Starr
© 2023 Michael Mikát, Jakub Straka.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Mikát M, Straka J (2023) Genetic evidence for parthenogenesis in the small carpenter bee Ceratina dallatoreana (Apidae, Ceratinini) in its native distribution range. Journal of Hymenoptera Research 95: 199-213. https://doi.org/10.3897/jhr.95.87165
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Arrhenotoky is the typical mode of reproduction in Hymenoptera. Diploid females develop from fertilized eggs, whereas haploid males originate from unfertilized eggs. However, some taxa of Hymenoptera have evolved thelytoky, in which diploid females originate parthenogenetically from unfertilized diploid eggs. In contrast to some other hymenopteran lineages, like ants and parasitic wasps, thelytoky is generally very rare in bees.
Here, we evaluated the frequency of thelytoky in the small carpenter bee Ceratina dallatoreana, which was previously assumed to be thelytokous. By comparing genotypes of microsatellite loci between mothers and their offspring, we found that all female offspring were genetically identical to their mothers. We conclude that parthenogenesis is the prevailing and perhaps obligate mode of reproduction in C. dallatoreana. We also classify the cytological mode of this parthenogenesis as apomixis, or automictic parthenogenesis with central fusion and extremely reduced or non-existing recombination, because offspring showed no decrease of heterozygosity.
Because sociality is influenced by relatedness and Ceratina are ancestrally facultatively social, the high relatedness afforded by parthenogenesis should associate with social living in the nest. In accordance with previous work, however, we found no social nests of C. dallatoreana.
Apidae, heterozygosity, relatedness, sociality, thelytoky, Xylocopinae
Sexual reproduction predominates in animals (
Different types of parthenogenesis most likely evolved by different mechanisms, with each type showing characteristic modes of inheritance (
Cytological mechanisms of parthenogenesis influence the genetic diversity and heterozygosity in the population (
A haplodiploid sex determination system is widespread among Hymenoptera (
Thelytokous reproduction has evolved repeatedly in Hymenoptera (
C. dallatoreana females nest in broken dead stems with pith, constructing a linear series of cells (
We collected nests of C. dallatoreana in several locations across its native area of distribution, in Cyprus (2018, 2019), Italy (Puglia and Lazio regions, 2013 and 2017), Greece (Crete 2018, 2020), Albania (2018) and Tajikistan (2019) (Fig.
Nests were collected from natural nesting opportunities, as well as in stems broken or cut by human management. The most common nesting substrates were Rubus spp. and Foeniculum vulgare. In Cyprus and Crete we cut stems of these plants to increase nest density for ease of sampling several months later. To ensure that all inhabitants were inside the nest, nests were collected in the evening after 18:00 local time. Nests were opened lengthwise with garden clippers, and the number of adults, number of juveniles, and the stages of juveniles were noted. All individuals were preserved in 96% ethanol for further analysis.
We isolated DNA using the Chelex protocol (
We selected 12 female C. dallatoreana (9 from Cyprus and 3 from Tajikistan) for testing of microsatellite loci. We used microsatellite primers developed for C. nigrolabiata (
We evaluated results of amplification and obtained four possibilities for each locus: a) a locus was successfully amplified in all cases and was polymorphic (eight loci). b) a locus was successfully amplified in some cases and was polymorphic (two loci), c) a locus was amplified in all cases but was not polymorphic (three loci), or d) amplification of locus failed (one locus, Suppl. material
Polymorphic and reliable loci were loci numbers 30, 23, 8, 67, 17, 36, 9, and 12 (Suppl. material
We used Type-it Multiplex PCR Master Mix (Quiagen) according to the manufacturer’s protocol. Primers of six microsatellite loci were use in a concentration of 0.05 μmol/l. We used these PCR conditions: 95 °C for 15 min; 30 cycles of 94 °C for 30 sec, 60 °C for 90 sec, 72 °C for 60 sec; and finally 60 °C for 30 min. After PCR, we mixed 0.8 μl of PCR product with 8.8 μl of formamide and 0.4 μl of marker Liz 500 Size scanner (Applied Biosystems). We heated the mixture to 95 °C for 5 min and then cooled it to 12 °C. Fragmentation analysis was performed on a 16-capillary sequencer at the Laboratory of DNA Sequencing at the Biological section of Faculty of Science, Charles University, Prague. Identification of alleles was performed in Gene Marker (Soft Genetics) software.
We included mothers from nests and additional individuals in this analysis. We did not include offspring, as they had the same genotypes as their mothers. For each locus, we checked if an individual had one allele (homozygote) or two alleles (heterozygote). Individuals were considered diploid when heterozygous at least one locus. Individuals with only one allele at each locus were considered haploid. We analyzed 132 females (30 from Crete, 64 from Cyprus, 11 from Georgia, 12 from Italy, 9 from Tajikistan, 3 from Albania, and 3 from North Macedonia). We also analyzed one gynandromorph (individual with female head morphology and male abdomen morphology) from Tajikistan.
We counted multilocus genotypes for different localities. As one locality we defined an area where collected samples are at most ten kilometers from each other. In this analysis, we included adult females. We present only data from localities where at least three adult females were genotyped.
We used adult females for this analysis. We samples from two populations: Lefkara village, Cyprus (n = 50), and Georgioupoli village, Crete (n = 26). All individuals were collected at most 10 kilometers from each other in the same population. We calculated observed and expected heterozygosity using software Genepop, version 4.7.5. (
We compared the genotype of each mother with offspring from the same nest. We analyzed 188 offspring from 59 nests in total. For this analysis, we selected nests in which the mother and immature brood were present – nests in stages active brood nests or full brood nest. Nests in the active brood stage are nests where the mother currently perform provisioning of brood cells. These nests contained currently provisioned brood cells and in outermost brood cell was egg or this brood cell was only partially provisioned (
We collected C. dallatoreana samples across its native range. In total, we found 476 adult females, one gynandromorph and no adult males. Of the adult females, we collected 253 in Cyprus, 137 in Crete, three in Albania, three in North Macedonia, 36 in Italy, 30 in Georgia, and 14 in Tajikistan. We found the gynandromorph in Tajikistan.
All analyzed adult females from the maternal generation (n = 132) were heterozygotes in at least one locus. One female was a heterozygote in only one locus, while all others (n = 131) were heterozygotes in at least two loci. Thus, we determined that C. dallatoreana females are diploid. The gynandromorph was homozygous in all loci, therefore we considered this individual to be haploid.
We generally detected high heterozygosity in our studied loci. Average heterozygosity across all locations and loci was 56.25%. However, heterozygosity differs between loci, with the highest proportion of heterozygotes at locus 36 (97.06%), and lowest proportion at locus 12 (4.41%). The proportion of heterozygotes in each locus across different geographical areas is shown in Table
Proportion of heterozygotes at each studied locus by geographical area. The category other includes samples from Albania (n = 3) and North Macedonia (n = 3).
Proportion of heterozygotes in locus | ||||||||
---|---|---|---|---|---|---|---|---|
Country | N | 17 | 36 | 23 | 9 | 12 | 67 | mean |
Crete | 30 | 0.33 | 1.00 | 0.73 | 1.00 | 0.03 | 0.97 | 0.68 |
Cyprus | 64 | 0.16 | 0.98 | 0.16 | 0.58 | 0.00 | 0.97 | 0.47 |
Georgia | 11 | 0.73 | 0.91 | 0.64 | 0.72 | 0.18 | 0.82 | 0.67 |
Italy | 12 | 0.58 | 0.83 | 0.25 | 0.75 | 0.00 | 0.50 | 0.49 |
Tajikistan | 9 | 0.33 | 1.00 | 1.00 | 0.78 | 0.00 | 1.00 | 0.69 |
Other | 6 | 0.50 | 1.00 | 0.33 | 1.00 | 0.00 | 0.67 | 0.58 |
Total | 132 | 0.32 | 0.97 | 0.41 | 0.73 | 0.04 | 0.90 | 0.56 |
Allele frequency deviated from Hardy-Weinberg equilibrium for all loci in Georgioupoli (Crete) and in three of five variable loci in Lefkara (Cyprus). Heterozygosity was increased in some loci but decreased in others. Observed heterozygosity was significantly higher than expected for loci 36 and 9 in Georgioupoli (Crete) and 36 and 67 in Lefkara (Cyprus), but significantly lower for loci 17, 23 and 12 in Georgioupoli (Crete) and 17 in Lefkara (Cyprus) (Table
Comparison of expected (HetEXP) and observed (HetOBS) proportions of heterozygotes. P-values of statistical tests from expected frequencies are shown: p(excess) = p-value of heterozygote excess test, p(deficit) = p-value of heterozygote deficit test, p(HW) = p-value test of difference from Hardy-Weinberg equilibrium in allele frequency. All calculation performed in software Genepop. Bold indicates significant values. Locus 12 in Cyprus population had only one allele, therefore excess or deficit of heterozygotes could not be calculated.
Lefkara (Cyprus), N=50 | ||||||
---|---|---|---|---|---|---|
Locus | p(deficit) | p(excess) | p(HW) | HetEXP | HetOBS | n alelles |
17 | 0.0000 | 1.0000 | 0.0000 | 0.59 | 0.18 | 5 |
36 | 1.0000 | 0.0000 | 0.0000 | 0.54 | 0.98 | 4 |
23 | 1.0000 | 0.7339 | 1.0000 | 0.15 | 0.16 | 3 |
9 | 0.4326 | 0.5688 | 0.0900 | 0.63 | 0.58 | 3 |
12 | NA | NA | NA | 0.00 | 0.00 | 1 |
67 | 1.0000 | 0.0000 | 0.0000 | 0.64 | 0.96 | 3 |
Georgioupoli (Crete), N=26 | ||||||
Locus | p(deficit) | p(excess) | p(HW) | HetEXP | HetOBS | n alelles |
17 | 0.0027 | 0.9974 | 0.0000 | 0.61 | 0.27 | 6 |
36 | 1.0000 | 0.0048 | 0.0000 | 0.82 | 1.00 | 9 |
23 | 0.0176 | 0.9824 | 0.0000 | 0.70 | 0.69 | 4 |
9 | 1.0000 | 0.0000 | 0.0000 | 0.63 | 1.00 | 4 |
12 | 0.0007 | 1.0000 | 0.0003 | 0.15 | 0.00 | 3 |
67 | 0.3422 | 0.6578 | 0.0000 | 0.72 | 0.96 | 6 |
We found a high diversity of multilocus genotypes. In all localities, we collected at least two multilocus genotypes. In Cyprus, we collected the largest sample in Lefkara (n = 50). In this location, we collected females with 26 multilocus genotypes. Eighteen of these genotypes were collected only once. Two of the most common genotypes had a frequency of 14.0% (7/50). We sampled another three locations in Cyprus: Agios Theodoros (n = 4, three multilocus genotypes); Mathiatis (n = 3, three multilocus genotypes); and Pyrgos (n = 5, four genotypes).
In Crete, we collected the largest sample in Georgioupoli (n = 26). In this location, we found 15 multilocus genotypes. The most common genotype had frequency 36.4% (9/26). Another sampled location was Chania airport, where we found three multilocus genotypes in three sampled individuals.
In Italy, we sampled at Pescariello (n = 5, five genotypes), Cassino (n = 3, two genotypes) and Santa Marinella (n = 3, three genotypes). In Georgia, we analyzed females from Vashlovani (n = 4, three genotypes) and Kvareli (n = 3, three genotypes). In Tajikistan, we analyzed samples from Shariston (n = 4, two genotypes).
Almost all offspring genotypes were identical to those of their mother (97.87%, 184/188 out of 59 nests). The same genotype in mother and offspring was found in all nests in Albania (two nests, 10 offspring), Crete (six nests, 23 offspring), and Italy (10 nests, 38 offspring). In Cyprus, we found one out of 89 offspring (32 nests) with a different genotype than its mother and in Tajikistan we found 3 such offspring out of 28 offspring (9 nests). However, all offspring for which we detected different genotypes from those of the mother had much lower detection peak for multiple microsatellite loci than most of the analyzed individuals. All four individuals contained at least one allele which was not shared with their mother. Two individuals from Tajikistan had both alleles different from the mother at least one locus. One individual from Cyprus and two from Tajikistan had a unique allele not found in any other individual. We can also exclude the effect of null alleles, because all four individuals were heterozygotes at least one locus with alleles that would disagree with the maternal genotype. Therefore, the apparent differences between offspring and maternal genotypes was the result of genotyping errors.
Previously, Ceratina dallatoreana was believed to reproduce parthenogenetically (
Thelytokous parthenogenesis is rare in bees. Outside of Apis mellifera capensis (
Although we found several offspring with genotypes that were not identical to genotypes of mothers, we suspect that these cases were the result of genotyping errors such as allelic dropout or false alleles. Situations in which offspring showed different genotypes from the mother were usually not compatible with scenarios of sexual reproduction. These results were also incompatible with any mode of parthenogenesis, because we detected alleles in offspring that were not detected in the mother. In two of four cases, offspring bear in at least one locus both alleles different from the mother. In case of parthenogenesis we can suppose allele loss, but not the rise of novel alleles.
Offspring resulting from parthenogenesis should bear only alleles shared by their mother. However, the cytology of parthenogenesis determines the rate of loss of heterozygosity from mother to offspring (
We have shown that thelytokous parthenogenesis is the prevailing mode of reproduction in C. dallatoreana. However, there remains the question of whether sexual reproduction is only extremely rare or not occur at all. The existence of males is rarely reported for this species, but most of the reports of males could have been confused with closely related species (
The best documented examples of thelytoky in aculeate Hymenoptera are found among advanced eusocial species, and features of thelytoky are influenced by their social organization (
We are grateful to Daniel Benda, Karolína Dobešová, Klára Daňková, Slavomír Dobrotka, Zuzana Dobrotková, Karolína Fazekašová, Tereza Fraňková, Jiří Houska, Jiří Janoušek, Lukáš Janošík, Celie Korittová, Tereza Maxerová, Miroslav Mikát, Blanka Mikátová, Jindra Mrozek, Daniela Reiterová, Tadeáš Ryšan, Vít Procházka, Vojtěch Waldhauser and Jitka Waldhauserová for assistance in the field. We are also grateful to Vít Bureš and Celie Korritová for collecting additional bees. We are grateful to Jesse Huisken and Ben Pyenson for feedback on the manuscript. The Grant Agency of Charles University (Grant GAUK 764119/2019) and the Specific University Research Project Integrative Animal Biology (Grant SVV 260571/2021) supported this research.
Dataset
Data type: occurences, genetic
Explanation note: Primary data used for paper Genetic evidence for parthenogenesis in small carpenter bee, Ceratina dallatoreana in its native distribution area. Dataset contains information about lotaions and dates of collected samples, lenght of microsatellite loci and infromation to which analyses was sample included.
Faunistic notes and microsatellite primers
Data type: genetic, faunistic
Explanation note: 1) notes about distribution of species C. dallatoreana 2) Features of microsatellite loci of C. dallatoreana 3) Features of successfully amplified microsatellites for C. dallatoreana 4) frequencies of alleles of microsatellite loci of C. dallatoreana 5) Comparison between expected and observed proportion of heterozygotes on reduced dataset from Lefkara (Cyprus) and Georgiopoli (Crete).