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Genetic evidence for parthenogenesis in small carpenter bee, Ceratina dallatoreana in its native
distribution area
Running title: Parthenogenesis in a solitary bee
Michael Mikát 1)2)3)* and Jakub Straka 1)
1) Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic
2) Department of Biology, Faculty of Science, York University, Toronto, Canada
3) Department of Zoology, Martin Luther University, Halle, Germany
* corresponding author: Michael.mikat@gmail.com, Department of biology, York University,
Toronto, 4700 Keele Street, 203D Lumbers Building, Toronto Ontario, Canada, M3J 1P3, + 420
721589730
Abstract
Arrhenotoky is typical mode of reproduction for Hymenoptera – females originate from fertilized
eggs, males from unfertilized eggs. However, some lineages of Hymenoptera have switched to
thelytoky, where diploid females originate instead from unfertilized diploid eggs. In the contras with
some other hymenopteran lineages, thelytoky is generally very rare in bees.
Here, we examined reproduction in the small carpenter bee Ceratina dallatoreana, which is assumed
to be thelytokous. We compared genotype of microsatellite loci between mothers and their offspring.
Offspring were genetically identical with mother in all cases. We did not detect any male offspring.
Therefore, we conclude that parthenogeny is the prevailing, and perhaps obligate, mode of
reproduction in C. dallatoreana. Offspring were clones of their mother with no observed decrease of
heterozygosity. Thus the cytological mode of parthenogenesis is apomixis, or automimic with central
fusion and extremely reduced or non-existing recombination.
Ceratina bees are originally facultatively eusocial, therefore thelytoky may influence social evolution
by causing extremely high within-colony relatedness. However, to date no multifemale nests have
been recorded in C. dallatoreana.
Keywords: Apidae, thelytoky, social behavior, heterozygosity, relatedness, Xylocopinae
Introduction
Sexual reproduction predominates in animals, however, parthenogenesis has evolved repeatedly in
many lineages (Engelstädter, 2008; Gokhman & Kuznetsova, 2018; Neiman & Schwander, 2011;
Normark, 2003; Thierry, 2013). Strictly parthenogenetic lineages are usually young (Fujita et al.,
2020; Neiman et al., 2009), and though obligate parthenogenesis can be successful in short-term, in
the long-term sexual reproduction is more successful (Neiman & Schwander, 2011; Thierry, 2013).
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Parthenogenesis probably most often evolved not as an adaptive trait, but as by a product of
hybridization or manipulation by a symbiont (Tvedte et al., 2019; Vavre et al., 2004). In insects the
occurrence of parthenogenesis differs between orders, and is especially common in stick insects and
mayflies (Liegeois et al., 2021; Tvedte et al., 2019).
Parthenogenesis is very diverse and different types of parthenogenesis most likely evolved by different
evolutionary mechanisms, each having different consequences for the genetics of the taxa in which it
evolved (Engelstädter, 2008). In obligate parthenogenetic population only females exist. However, in
many species parthenogenesis is conditionally dependent and present together with sexual
reproduction in the same population (Liegeois et al., 2021; Normark, 2003). Such facultative or
cyclical parthenogenesis is more common than obligate parthenogenesis (Hörandl et al., 2020).
There exists several cytological mechanisms of parthenogenesis, which strongly differ in influence on
genetic diversity and heterozygosity of offspring (Engelstädter, 2017; Hörandl et al., 2020; Pearcy et
al., 2006). Parthenogenesis can be caused by absence of meiosis (mitotic parthenogeny, apomixis) or
with meiosis, but diploidy restored by various mechanisms (meiotic parthenogenesis) (Stenberg &
Saura, 2009). Mitotic parthenogenesis causes increased heterozygosity, since recombination leading to
loss of alleles does not occur (Schwander & Crespi, 2009; Tsutsui et al., 2014; Tvedte et al., 2019). On
the other hand, meiotic parthenogeny should lead to decreased heterozygosity, because diploidy is
restored by endomitriosis (leading to zero heterozygosity) or fusion of products of meiosis, having a
similar outcome to self-fertilization – heterozygosity of offspring may be the same or smaller than that
of a parent in each locus, and it is always lower on average. Typically, automictic parthenogenesis
with terminal fusion (fusion of sister pronuclei) leads to a rapid decrease of heterozygosity (Alavi et
al., 2018; Engelstädter, 2017). However, heterozygosity can be retained in the case of meiotic
parthenogeny with central fusion, if crossing over is not present in meiosis (Engelstädter, 2017;
Stenberg & Saura, 2009). In these cases, there always merge different products of meiosis I, which
contain complementary half of genetic information of mother. Result of meiotic parthenogeny with
central fusion without recombination is clone of mother similarly as in mitotic parthenogeny
(Engelstädter, 2017).
For Hymenoptera, a haplodiploid sex determination system (Kooi et al., 2017; Normark, 2003). Male
offspring originate from unfertilized eggs, and are therefore haploid, females from fertilized eggs, and
are therefore is diploid (Kooi et al., 2017; Stubblefield & Seger, 1994). Unmated females thus produce
only male offspring (Shukla et al., 2013) and the sex of offspring depends the decision to fertilize an
egg or not in mated females (Gerber & Klostermeyer, 1970; Stubblefield & Seger, 1994). Arrhenotoky
can be a predisposition for evolution of thelytoky (Vorburger, 2014). Thelytokous reproduction has
evolved repeatedly in many hymenopteran lineages (Kooi et al., 2017; Vorburger, 2014). It is well-
documented in many sapflies and parasitic Hymenoptera (especially Chalcidoidea, Cynipoidea and
Ichnumoidea), but is found less frequently within aculeate Hymenoptera (Kooi et al., 2017). Within
aculeate Hymenoptera, the best evidence tor thelytoky is from social species (Goudie & Oldroyd,
2018; Wenseleers & Van Oystaeyen, 2011). Thelytoky evolved repeatedly in ants and has been
documented in at least 50 species to date (Goudie & Oldroyd, 2018; Heinze, 2008; Rabeling &
Kronauer, 2013). In bees, facultative thelytoky is known in Apis mellifera capensis, where workers lay
thelytokous eggs, usually not in the nest that they originated from (Goudie & Oldroyd, 2014, 2018).
However, thelytoky is very infrequent in solitary nesting and weakly social Hymenoptera, with few
exceptions (Kooi et al., 2017). Based on sex ratio, thelytoky is assumed in several Ceratina species,
including C. dallatoreana (Daly, 1966; Snelling, 2003). Parthenogeny was considered also for several
Australian Halicitd bees, however probably these species are normally sexually reproducing and
evidence for parthenogeny is insufficient (Michener, 1960).
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Ceratina dallatoreana (Apidae: Xylocopinae) nests in broken dead stems with pith (Daly, 1966).
Within these stems females build a series of linear cells (Daly, 1966). Although facultative sociality is
common in this genus (Groom & Rehan, 2018; Rehan et al., 2009; Sakagami & Maeta, 1977), social
nests have not been detected in this species to date (Daly, 1966; Mikát et al., 2022). This species is
distributed in the most of Mediterranean and also in central Asia (Terzo, 1998; Terzo and Rasmont,
2004, fig 1). It also exists as an introduced species in California (Daly, 1966). Males are extremely
rare in this species (Daly, 1966, 1983), therefore it is assumed that C. dallatoreana reproduce by
thelytokous parthenogeny.
In this study, we use microsatellite genetic markers for examination of presence and density of
parthenogenesis in different populations across the known range of C. dallatoreana and assess if
heterozygosity is in Hardy-Weinberg equilibrium, increased or decreased in this species.
Methods
We collected nests of C. dallatoreana in several locations across its area of distribution. Nests were
collected in Cyprus (2018 and 2019), Italy (regions Puglia and Lazio, 2013 and 2017), Greece (Crete
Island, 2018 and 2020), Albania (2018) and Tajikistan (2019). Coordinates of locations, where nests
were sampled are shown in supplementary dataset. Additionally, we analyzed individuals collected in
Georgia (2013-2014) and Northern Macedonia (2014)
Nests were collected from natural nesting opportunities, in stems which were broken or cut by human
management. In Cyprus and Crete we cut some stems to increase nest density for ease of sampling.
The most common nesting substrate were Rubus spp. and Foeniculum vulgare, however nests in dead
stems of other plants nests were also collected. Nests were collected in evening (after 18:00 of local
time) to ensure that all inhabitants were inside the nest. Nests were open by clippers, and nest contents
(number of adults and number and stage of juveniles) were noted. Individuals were preserved in 96%
ethanol for further analysis.
For analysis of relatedness we used only nests in active brood nest and full brood nest stages. Active
brood nests contain currently provisioned brood cells with a pollen ball, with or without an egg, in the
outermost brood cell (Mikát et al., 2021; Rehan & Richards, 2010). Full brood nests contain larva or
pupa in innermost and outermost brood cell, as females have already completed provisioning, and are
now guarding their offspring until adulthood (Mikát et al., 2021; Rehan & Richards, 2010). We used
these two stages, because for these stages it is unambigious that exchange of individuals between nests
did not occur.
In total, 59 nests with 188 offspring were analyzed, with 2 nests and 10 offspring from Albania, 6
nests and 23 offspring from Crete, 32 nests and 89 offspring from Cyprus, 10 nests and 38 offspring
from Italy and 9 nests and 28 offspring from Tajikistan.
Isolation of DNA: We isolated DNA using Chelex protocol. Isolation was done usually from part of
an individual (one or two legs from adults or pupae, part of the body of most larvae), but whole eggs
and whole bodies of small larvae were also used. Samples were transferred to microcentrifuge tubes
and dried for at least three hours. Later, we added 8
μ
l of proteinase K and 50
μ
l of 10% Chelex
solution. This mixture was vortexed and inserted into a thermo cycler. The mixture was heated to 55°C
per 50 mins and 97°C per 8 mins then cooled. The whole mixture was then vortexed and inserted into
a centrifuge. After this 30
μ
l of supernatant transferred to a well in the PCR plate.
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Optimalization of multiplex: 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 (Mikát et al., 2019). Fourteen microsatellite loci were arranged in two multiplexes. The
first multiplex was previously applied to C. nigrolabiata, C. chalybea and C. cyanea (Mikát et al.,
2019). The second multiplex contained six loci. Four were different than loci in first multiplex (17 and
36 marked by 6FAM, 9 marked by VIC and 7 marked by PET), and two loci (12 and 51) were shared
with the first multiplex but marked by a different color.
We evaluated results of amplification and obtained four possibilities for each locus: a) a locus was
successfully amplified in all cases and was polymorphic b) a locus was successfully amplified in
almost all cases and was polymorphic c) a locus was amplified in all cases, but was not polymorphic,
d) amplification of locus failed (this occurred in a high proportion of cases, table S1).
Polymorphic and reliable loci were 30, 23, 8, 67,17,36,9,12 (table S1, number system of loci is same
as for C. nigrolabiata, (Mikát et al., 2019)). However, we excluded locus 30 for overlap with same
color marked loci and locus 8 for interaction of primers with primers for another locus. Six
microsatellite loci were thus retained for final analysis (Table S1, S2).
PCR and Fragmentation analysis: We used Type-it Multiplex PCR Master Mix (Quiagen) according
the manufacture’s protocol. Primers of six microsatellite loci were use in concentration of 0.05
μ
mol/l.
We used these PCR conditions: 95°C for 15 minutes; 30 cycles of 94°C for 30 s, 60°C for 90 s, 72°C
for 60 s; and finally, 60°C for 30 min. After PCR, we mixed 0.8
μ
l of PCR product with 8.8
μ
l of
formamide and 0.4
μ
l of marker Liz 500 Size scanner (Applied Biosystems). We heated mixture to
95°C for 5 min and then cooled 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.
Analysis of ploidy and heterozygosity: We included mothers from nests and addition individuals in
this analysis. We did not include offspring (as they had same genotypes as mothers) to this analysis.
For each locus, we checked if an individual had one allele (homozygote) or two alleles (heterozygote).
Individuals were considered to be diploid when heterozygous in at least one locus. Individuals which
had only one allele in each locus were considered to be 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
Northern Macedonia). We also analyzed one gynandromorph (individual with female morphology of
head and male morphology of abdomen) from Tajikistan.
Analysis of deficit or surplus of heterozygotes: We used mothers and additionally sampled females
for this analysis. We tested females from two populations, Lefkara village, Cyprus (N=40), and
Georgiopoli village, Crete (N=26). All individuals were collected maximally ten kilometers from each
other in the same population We calculated observed and expected heterozygosity using software
Genepop, version 4.7.5. (Rousset, 2020). Finally, we tested the possible excess or deficiency of
heterozygotes also using Genopop.
Analysis of parthenogeny: We compared the genotype of each mother with the genotypes of their
offspring from the same nest, checking if they shared the same genotype. We analyzed 188 offspring
from 58 nests in total. Ten offspring from two nests were from Albania, 23 offspring from six nests
were from Crete, 89 offspring from 32 nests from Cyprus, and 28 offspring from 9 nests form
Tajikistan.
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Results
Ploidy: All analyzed females from maternal generation (n = 136) were heterozygotes in at least one
loci. One female was a heterozygote in only one locus, other females were heterozygotes in at least
two loci. Thus we determined that C. dallatoreana females are diploid.
In Tajikistan, we collected one gynandromorph individual. This gynandromorph was homozygote in
all loci, therefore we considered this individual as haploid.
Heterozygosity: We detected generally high heterozygosity in our studied loci. Average
heterozygosity across all locations and loci was 56.25%. However, heterozygosity differs between
loci, with the highest in locus 36 (97.06%), and lowest in locus 12 (4.41%, Table 1). Proportion of
each locus across different geographical areas is shown in Table 1.
Heterozygosity was increased in some loci, but decreased in others. Observed heterozygosity was
significantly higher than expected for loci 36 and 9 in Crete and 36 and 67 in Cyprus, but significantly
lower for loci 17, 23 and 12 in Crete and 17 in Cyprus (Table 2). For other loci, there was no
significant difference between observed and expected heterozygosity.
Diversity of genotypes: In Tajikistan, the most common genotype had a frequency of 44.44% (4/9),
The second most common genotype had frequency of 33.33% (3/9). Other two genotypes had a
frequency of 11.11% (1/9). In Italy, the most common genotype had frequency of 16.66%. The other
10 genotypes were found each in one individual. In Georgia, the most common genotype had a
frequency of 27% (3/11). The other 8 genotypes were detected only once. In Cyprus we found two
genotypes with a frequency of 15.625% (10/64). The third most common genotype was found with a
frequency of 12.5% (8/64). The other seven genotypes had a frequency of between 3.13-4.69%. The
other 18 genotypes were detected only once (frequency 1.57%). In Crete, the most common genotype
had a frequency of 30% (9/30). The second most common genotypes had a frequency of 10% (3/10).
There were also found genotypes with a frequency of 6.66% (2/30). We also found 14 genotypes
which were found only once.
Table 1: Proportion of heterozygotes in each studied loci by geographical area. Other includes
samples from Albania (N=3) and Northern Macedonia (N=3).
Proportion of heterozygotes in locus
Country N 17 36 23 9 12 67 average
Crete 30 0.3333 1.0000 0.7333 1.0000 0.0333 0.9667 0.6778
Cyprus 64 0.1563 0.9844 0.1563 0.5781 0.0000 0.9688 0.4740
Georgia 11 0.7372 0.9090 0.6363 0.7272 0.1818 0.8181 0.6682
Italy 12 0.5833 0.8333 0.2500 0.7500 0.0000 0.5000 0.4861
Tajikistan
9 0.3333 1.0000 1.0000 0.7778 0.0000 1.0000 0.6852
Other 6 0.5000 1.0000 0.3333 1.0000 0.0000 0.6667 0.5833
All 136 0.3235 0.9706 0.4118 0.7279 0.0441 0.8971 0.5625
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Table: Comparison between expected and observed proportion of heterozygotes. HetEXP = expected
proportion of heterozygotes, HetOBS = observed proportion of heterozygotes, p(excess) – p-value of
heterozygote excess test, p(deficit) – p-value of heterozygote deficit test. All calculation performed in
software Genepop. Bold distinguishes significant values. Locus 12 in Cyprus population had only one
allele, therefore excess or deficit of heterozygotes was not possible to calculate.
Crete(N=26)
Cyprus (N=40)
locus HetEXP
HetOBS p(excess)
p(deficit)
HetEXP
HetOBS
p(excess)
p(deficit)
17 0.6054 0.2692 0.9974
0.0027 0.5888 0.1750 1.0000 0.0000
36 0.8150 1.0000
0.0048 1.0000 0.5513 0.9750
0.0000 1.0000
23 0.6958 0.6923 0.9824
0.0176 0.1420 0.1500 0.8142 1.0000
9 0.6266 1.0000
0.0000 1.0000 0.6363 0.6250 0.4081 0.5997
12 0.1477 0.0000 1.0000
0.0007 0.0000 0.0000 NA NA
67 0.7177 0.9615 0.6578 0.3422 0.6363 0.9500 0.0000 1.0000
Relatedness:
We found that almost all offspring genotypes were identical to mother’s (97.87%, 184/188, out of 59
nests). The same genotype in mother and offspring was found in all nests in Albania (2 nests, 10
offspring), Crete (6 nests, 23 offspring), and Italy (10 nests, 38 offspring). In Cyprus, we found one
offspring with a different genotype than its mother (out of 89 offspring and 32 nests) and in Tajikistan
we found 3 such offspring (out of 9 nests and 28 offspring). However, all offspring for which we
detected different genotype than of 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 is
not shared with their mother. Two individuals from Tajikistan both had alleles different than mother in
at least one locus. In one individual from Cyprus and two from Tajikistan had a unique allele, which
was not found in any other genotyped individual. This suggests that apparent differences between
offspring and maternal genotypes were the result of genotyping errors.
Discussion
Ceratina dallatoreana was considered to reproduce parthenogenetically (Daly, 1966, 1983), however,
genetic evidence was lacking. We observed parthenogeny in several locations in Mediterranean
(Albania, Italy, Crete, Cyprus) and central Asia (Tajikistan), providing evidence for parthenogenesis
from a large part of the original geographic range of the species. As males were extremely rare in
North Africa (Daly, 1983) and also California, where the species is introduced (Daly, 1966) we can
suppose that parthenogeny is prevailing or only mode of reproduction in the whole distributional area.
Thelytokous parthenogeny is generally rare in bees and well-documented only in Apis mellifera
capensis (Goudie & Oldroyd, 2014; Rabeling & Kronauer, 2013). Outside Apis, parthenogeny has
been documented only in the Ceratina genus of small carpenter bees. This includes evidence for
parthenogeny in C. acantha ((Slobodchikoff & Daly, 1971), C. dentipes (Shell & Rehan, 2019;
Snelling, 2003, Mikát unpublished data), C. parvula (Terzo et al., 2007, Mikát, unpublished data) and
C. dallatoreana ((Daly, 1966), this study). These species are not closely related, belonging to different
subgenera (Ascher & Pickering, 2020; Rehan & Schwarz, 2015). Species which are considered to be
the most closely related to C. dallatoreana, such as C. dentiventris and C. sakagamii do not have
skewed sex ratio (Daly, 1983; Terzo, 1998), therefore parthenogeny is probably not the prevailing
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mode of reproduction in these two species. However for the species C. rasmonti, known from only
few individuals and closely related to C. dallatoreana, males are unknown (Terzo, 1998) therefore this
species can be also parthenogenetic, but larger sample size is necessary for proper evaluation. The
distribution of parthenogeny in different Ceratina lineages suggests that there is a trend for
parthenogenesis to arise in the Ceratina genus, but future research including sampling of more species
and high-resolution phylogeny is necessary for evaluation of frequency and evolution of
parthenogenesis in Ceratina.
Although we found only few offspring with genotypes that were not identical to genotypes of mothers,
we suspect that these cases were the result of genotyping errors. Situations in which offspring had
different genotypes than mother were usually not compatible with scenario of sexual reproduction.
These results were also incompatible with any mode of parthenogenesis, because we detected alleles in
offspring which are not detected in mother. In case of parthenogeny we can suppose allele loss, but not
an allele arise.
Offspring resulting from parthenogenesis should bear only alleles which also bear their mother.
However, cytology of parthenogeny determines the rate of loss of heterozygosity from mother to
offspring (Pearcy et al., 2006). We did not observe any heterozygosity loss – all offspring were
genetically identical to their mother when four improperly genotyped individuals were excluded. This
strongly supports that offspring are identical clones of their mother. This situation is compatible with
two cytological types of parthenogeny: apomixis or automixis with central fusion. Our results are fully
compatible with possibility of apomixis. Automixis with central fusion is less probable, as under this
scenario, there should be at least some heterozygosity loss due to recombination (Engelstädter, 2017;
Goudie & Oldroyd, 2014). Therefore, automixis with central fusion is possible in C. dallatoreana only
if recombination is missing, its rate is extremely low or if all six our microsatellites are very close to
the centromere. Empirical studies on organisms with central fusion automixis using microsatellites
showed at least some heterozygosity loss (Fougeyrollas et al., 2015; Rey et al., 2011). Studies of Apis
mellifera capensis show that homozygotes arise due to recombination, but they often die during early
developmental stages – therefore, high heterozygosity is preserved by selection (Goudie & Oldroyd,
2014). As we did not find any case of a homozygote offspring with a heterozygote mother (even in
offspring egg stage), apomixis is the more probable mechanism.
We showed that thelytokous parthenogenesis is the prevailing mode of reproduction in C.
dallatoreana. However, there remains a question as to whether sexual reproduction is only extremely
rare, or if it does not occur at all. Existence of males is rarely reported for this species, however, most
of reports of males could have been confused with closely related species (Daly, 1983; Terzo, 1998).
But males are undoubtedly reported from California, where C. dallatoreana is invasive and no similar
species are present (Daly, 1966). However, the existence of males alone does not prove their
involvement in reproduction. Strictly apomictic species have usually only one or a few genotypes in
one location or region (Lorenzo-Carballa & Cordero-Rivera, 2009; Ryskov et al., 2017). However,
although we detected some genotypes repeatedly, there was generally high genotype diversity in each
location, suggesting that sexual reproduction sometimes occurs in C. dallatoreana, although it is likely
very rare.
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 (Goudie &
Oldroyd, 2018). On the other hand, Ceratina bees are mostly facultatively social (Groom & Rehan,
2018; Rehan, 2020). Although most studied species are able to establish social colonies, the larger
proportion of the population is solitary and social colonies contain only two or a few females (Groom
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& Rehan, 2018; Mikát et al., 2022; Rehan et al., 2009; Sakagami & Maeta, 1977). Reversion to strict
solitarity also exist in some species (Groom & Rehan, 2018; Mikát et al., 2020). Social nests have not
been documented in C. dallatoreana to date (Daly, 1966; Mikát et al., 2022), thought the number of
nests so far analyzed does not preclude the possibility of very rare sociality in this species. Regardless,
it is clear that social nesting is at least very uncommon in this species. This is quite surprising, because
parthenogeny should promote social behavior, due to high relatedness between mother and offspring
(Hamilton, 1964). Moreover, two other species of Ceratina where parthenogeny probably occurs (C.
dentipes and C. parvula) are facultatively social (Mikát et al., 2022; Rehan et al., 2009; Terzo et al.,
2007). In last parthenogenetic species, Ceratina acantha (Slobodchikoff & Daly, 1971), social status
was not examined to this date.
Ceratina bees are generally an excellent group for study of social evolution, due to their extensive
within and between species variability in social behavior (Groom & Rehan, 2018; Rehan, 2020).
Existence of parthenogeny in many Ceratina species which are not closely related provides us with
further examples of between-species variability in relatedness, a highly important and probably key
parameter influencing social evolution (Foster et al., 2006; Hamilton, 1964; Nonacs, 2017). However,
better understating of within-colony relatedness and natural history of more species is necessary for
understanding co-evolution of thelytoky and sociality.
Acknowledgments:
We are grateful to Daniel Benda, Karolína Dobešová, Klára Da
ň
ková, Slavomír Dobrotka, Zuzana
Dobrotková, Karolína Fazekašová, Tereza Fra
ň
ková, Ji
ř
í Houska, Ji
ř
í Janoušek, Lukáš Janošík, Celie
Korittová, Tereza Maxerová, Miroslav Mikát, Blanka Mikátová, Jindra Mrozek, Daniela Reiterová,
Tadeáš Ryšan, Vít Procházka, Vojt
ě
ch Waldhauser and Jitka Waldhauserová to their assistance in
field. We are also grateful to Vít Bureš and Celie Korritová for collecting additional bees. We are
grateful to Jesse Huisken for feedback to 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.
Disclosure:
Authors do not have any conflict of interests.
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Fig 1: Native range of
Ceratina dallatoreana
. Map is based on (Terzo & Rasmont, 2004, 2011) and
new localities from this study. Red – range of
C. dallatoreana
, yellow – other lands, blue – sea and
large lakes, Black triangles – sit es where
C. dallatoreana
samples for this study were collected.
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