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JHR 38: I-9 (2014) JOURNAL OF *0errviewed opevaccetsjoural
doi: 10.3897/JHR.38.7302 SHORT COMMUNICATION ME Hymenopter a

www.pensoft.net/journals/jhr The imernatonl Society of Hymenoptxriss, RESEARCH

New molecular evidence for fragmentation between
two distant populations of the threatened
stingless bee Melipona subnitida Ducke
(Hymenoptera, Apidae, Meliponini)

Geice R. Silva', Bruno A. Souza’, Fabia M. Pereira’,
Maria T. R. Lopes’, Sérgio E. S. Valente', Fabio M. Diniz?

| Department of Biological Sciences, Federal University of Piaui, CEP: 64049-550, Teresina, PI, Brazil
2 EMBRAPA Meio-Norte, Laboratory of Molecular Biology & Biotechnology, CP: 01, CEP: 64.006-220,
Teresina, PI, Brazil

Corresponding author: Fabio M. Diniz (fabio.diniz@embrapa.br)

Academic editor: Jack Neff | Received 18 February 2014 | Accepted 3 March 2014 | Published 12 June 2014

Citation: Silva GR, Souza BA, Pereira FM, Lopes MTR, Valente SES, Diniz FM (2014) New molecular evidence for
fragmentation between two distant populations of the threatened stingless bee Melipona subnitida Ducke (Hymenoptera,
Apidae, Meliponini). Journal of Hymenoptera Research 38: 1-9. doi: 10.3897/JHR.38.7302

Abstract

For a snapshot assessment of the genetic diversity present within Melipona subnitida, an endemic sting-
less bee distributed in the semi-arid region of northeastern Brazil, populations separated by over 1,000
km distance were analyzed by ISSR genotyping. This is a prerequisite for the establishment of efficient
management and conservation practices. From 21 ISSR primers tested, only nine revealed consistent and
polymorphic bands (loci). PCR reactions resulted in 165 loci, of which 92 were polymorphic (57.5%).
Both ®,,, (ARLEQUIN) and 0° (HICKORY) presented high values of similar magnitude (0.34, p<0.0001
and 0.33, p<0.0001, respectively), showing that these two groups were highly structured. The dendrogram
obtained by the cluster analysis and the scatter-plot of the PCoA corroborate with the data presented
by the AMOVA and 0° tests. Clear evidence of subdivision among sampling sites was also observed by
the Bayesian grouping model analysis (STRUCTURE) of the ISSR data. It is clear from this study that
conservation strategies should take into account the heterogeneity of these two separate populations, and

address actions towards their sustainability by integrating our findings with ecological tools.

Keywords

Population differentiation, Hymenoptera, Jandaira, genetic diversity, ISSR markers

Copyright Geice R. Silva et al. 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.

2 Geice R. Silva et al. / Journal of Hymenoptera Research 38: 1-9 (2014)

Introduction

Melipona subnitida Ducke (Hymenoptera, Apidae, Meliponini) is an endemic stingless
bee distributed in the Caatinga, the semi-arid region of northeastern Brazil (Michener
2007, Camargo and Pedro 2013). The species, called Jandaira by natives, has great
ecological significance as a pollinator of the local native flora and cultivated crops and
is of economic importance in honey production, which is valued for its alleged me-
dicinal properties and antibacterial activity (Cruz et al. 2004; Alves et al. 2008; Silva
and Paz 2012).

The stingless bees are currently threatened by the increasing destruction of native
semi-arid vegetation and by the intensification of agriculture in the Caatinga (Roulston
and Goodell 2011; Pereira et al. 2011). In response, small populations of stingless bees
may gradually decline, resulting in local extinction. The assessment of the genetic di-
versity present within Melipona subnitida populations is therefore a prerequisite for the
establishment of efficient management and conservation practices.

Molecular markers have proven to be decisive in elucidating diversity and differ-
ences at the DNA level in microorganisms, plants and animals (Panwar et al. 2010;
Sebastien et al. 2012; Rana et al. 2012; Bonatti et al. 2014). Among several markers,
Inter Simple Sequence Repeats (ISSR) have been used due to their low cost and high
level of polymorphism, and as an alternative to overcome reproducibility problems
commonly found in other PCR-based markers (Abbot 2001; Lima-Brito et al. 2011).
ISSRs are semiarbitrary markers based on DNA amplification by PCR in the presence
of single 15- to 20-bp long primer complementary to a target short sequence repeat
(Zietkiewicz et al. 1994). Despite the existence of few genetic studies related to sting-
less bees, it has recently been shown that ISSR markers can be useful in the analysis of
natural bee populations, contributing to the development of management strategies of
these important genetic resources (Nascimento et al. 2010; Tavares et al. 2013).

The present study uses ISSR analysis to investigate the degree of genetic differen-
tiation between two Melipona subnitida populations separated by over 1,000 km in the
Brazilian Caatinga.

Worker bees were randomly collected from natural colonies (one bee from each
of 30 colonies) distributed in 2 locations only: (1) 15 nests in Natal (NAT; coordi-
nates: 5°48'04"S, 35°11'08" W; State of Rio Grande do Norte) and (2) 15 nests in //ha
das Canarias, Parnaiba River Delta (PAR; coordinates: 2°46'39"S, 41°51'59"W; on
the border of the states of Piauf and Maranhao) in Brazil (Figure 1A). All the samples
were taken to the laboratory and stored at —20°C until further use. Total genomic
DNA was extracted from each adult worker thorax using a phenol/chloroformalcohol
isoamyl (25:24:1, v:v:v) extraction of SDS/proteinase-K digested tissue of each indi-
vidual (Sambrook et al. 1989). High molecular weight DNA was isolated by ethanol
precipitation and visualized by agarose gel electrophoresis.

The extracted DNA was then amplified by PCR using twenty-one primers devel-
oped by the University of British Columbia (primer set #9). PCRs were carried out in
20 pwL-reaction volumes containing approximately 10-50 ng of DNA, 1x PCR buffer

New molecular evidence for fragmentation between two distant populations... 3

Coordinate 2

PAR: Ilha das Canarias, Parnaiba River Delta (PI/MA) NAT: Natal (RGN)

Coordinate 1

Figure |. A Sampling sites of Melipona subnitida: NAT (coordinates: 5°48'04"S, 35°11'08"W; State
of Rio Grande do Norte) and PAR (coordinates: 2°46'39"S, 41°51'59"W; on the border of the States
of Piaui and Maranhao) B Clustering analysis using UPGMA for M. subnitida genotypes included in this
study based on DICE similarity coefficient values. Numbers indicate bootstrap values for nodes retained
by more than 50% of bootstrap replicates (1000 replications) C Scatter-plot of the principal coordinate
analysis (PCoA) using ISSR loci. m PAR genotypes; @ NAT genotypes D Bar plot from Inferred popu-
lation structure of using the Bayesian grouping admixture model-based program STRUCTURE (K = 2).

(40 mM Tris-HCl; 100 mM KC), 1.5 uM of primer, 6.25 mM MgCl, 1.5 uM of
each dNTP, 1.25 U of Invitrogen Taq DNA polymerase. All amplifications were car-
ried out on a VERITI™ Gradient Thermalcycler (APPLIED BIOSYSTEMS). The
following PCR conditions were used: an initial denaturation at 94°C for 1.5 min, fol-
lowed by 45 cycles of 94°C/40 s, 36°C/1 min. and 72°C/2 min., and a final extension
of 72°C/5 min. ISSR markers were screened by silver nitrate detection on denatured
6% polyacrylamide gels, which were scored for absence (0) and presence (1) of bands
across genotypes and entered into a binary matrix.

Sample polymorphism was estimated as the percentage of polymorphic bands
(PPB) in the total number of bands. The program HICKORY v.1.1, which imple-
ments the Bayesian method (Holsinger et al. 2002), was used for estimating 0° (F,,
analogue), heterozygosity (H,), and the inbreeding coefhcient (/), an F,, analogue for
dominant markers. Analysis of Molecular Variance (AMOVA) was conducted using

4 Geice R. Silva et al. / Journal of Hymenoptera Research 38: 1-9 (2014)

all the amplified loci to check the occurrence of population structure among sampling
localities using ARLEQUIN v.3.11 (Excofher et al. 2006). The use of different algo-
rithms for the calculation of F.,. analogues was an additional effort to check for the
reliability of the data presented by ISSR markers.

Further, a Bayesian grouping admixture model (burn-in length 100,000 iteractions;
run length 100,000; K= 1 to 8 subpopulations tested) was used to infer the number of
subpopulations (software STRUCTURE 2.3.3; Pritchard et al. 2000). These results
were analyzed using STRUCTURE HARVESTERWeb v.0.6.9 (Evanno et al. 2005).

The similarity among samples within populations was estimated using PAST v1.34
(Hammer et al. 2001) according to Dice’s coefficient. Cluster analysis using the un-
weighted pair-group method arithmetic average (UPGMA) and multidimensional
principal coordinate analysis (PCoA) were performed on the data set to reveal the
degree of genetic differentiation between sites.

From 21 primers initially screened for their ability to generate ISSR loci, only
nine revealed consistent and polymorphic bands (loci) with 30 Jandaira worker bees.
The other 12 ISSR markers were monomorphic or had unreliable amplification and
therefore are not included in the genetic diversity analysis. Polymorphic ISSR prim-
ers were also considered reproducible after repeated PCRs, under the same reaction
conditions and, therefore, selected for genotyping (Table 1). PCR reactions involving
these nine primers resulted in 165 loci (bands) or 18.3 bands/primer, of which 92
were polymorphic (10.2 polymorphic bands/primer) ranging in size from 250 to 1636
bp, corresponding to an average polymorphism of 57.5%. Genotyping showed that
most of the detected loci were polymorphic. Overall ISSR polymorphism in Melipona
subnitida was similar to that of M. quadrifasciata Lepeletier (67%) (Nascimento et al.
2010). The number of polymorphic bands per primer ranged from 5 (UBC-884 and
UBC-888) to 23 (UBC-899).

ISSR genotyping revealed differences in genetic diversity based on the percentage
of polymorphic bands (% PPB), which was also estimated separately for each popula-
tion. Result suggests that the NAT population (80.7%) is characterized by a higher
genetic diversity than the PAR population (64.9%), which in theory might give the
NAT population an increased ability to adapt to selective pressures.

The 0°, fand H, values obtained from four different models of population struc-
ture using the Bayesian analysis are shown in Table 2. Of the models applied to the
ISSR dataset, the full model, in which 0 and fare estimated simultaneously, was
preferred primarily because of its smaller deviant information criterion (DIC) value
(657.47), with a difference of more than six units required to indicate strong support
over all the other models (Holsinger et al. 2002). In the Bayesian approach 0° (ana-
logue to Wright's F...), f (analogue to F,.), and H, (average panmictic heterozygosity
across populations) were estimated to be 0.33, 0.31 and 0.29, respectively, indicating a
pronounced genetic differentiation between populations, possibly caused by restricted
gene flow and random genetic drift (Epperson 1995).

The analysis of molecular variance also provided additional support to the evidence
of population differentiation in Melipona subnitida. The AMOVA analysis indicated

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Table 2. Summary of genetic variability, partitioning of diversity and limits for credible interval by Bayes-

ian statistical procedures.

H
Models is $ DIC

Mean | 2.5% 97.5% | Mean | 2.5 % | 97.5 %
Full | 0.3096 | 0.0455 0.2925 | 0.2565 | 0.3254 | 657.465
f=0 0 - | - | 0.2737 | 0.2099 | 0.3451 | 0.3272 | 0.3122 | 0.3418 | 665.837
08 | 0.3733 | 0.1702 0.3546 | 0.3319 | 0.3761 | 1107.250
fftee | 0.4997 | 0.0224 | 0.9742 | 0.3578 | 0.2807 | 0.4389 | 0.2771 | 0.2467 | 0.3098 | 700.287

0® is analogous to Wright's F

fis analogous to F.., H, is the average panmictic heterozygosity across

sT? Is?
populations, and DIC is deviance information criterion.

Table 3. Hierarchical analysis of molecular variance (AMOVA) detected by the ISSR genotyping.

Variance Percentage of total

Source of variation df Sum of squares ‘ P-value*
components variance (%)

Among populations 1 96.333 5.697 34.35 < 0.0001

PamonpancviCaalse oat. saa 00 10.886 65.65 0.014

within populations

TOTAL 29 401.133 16.583

Fixation index F .. index = 0.3435

*p-values calculated from a random permutation test (10,000 permutations).

that most of the genetic variation found in M. subnitida samples could be attributed
to differences among individuals within populations (approximately 66% of the vari-
ance), but also a large part of the variation (34.35%) was due to differences among
localities. The inbreeding coefficient (f= 0.31) provided by HICKORY agrees with
the results obtained by the AMOVA, as moderate endogamy might be expected for
strongly structured populations.

Both 0° (HICKORY) and @,,. (ARLEQUIN) presented high values, but of similar
magnitude and significance (0.33, p<0.0001 and 0.34, p<0.0001, respectively), show-
ing that these two populations are highly structured (Tables 2-3). These two different
approaches showed a general agreement among the results.

A high degree of population differentiation has also been observed for Melipona
quadrifasciata populations based on ISSR patterns (Nascimento et al. 2010) and
rufiventris Lepeletier (Tavares et al. 2007) based on allozyme, microsatellite and random
amplified polymorphic DNA (RAPD) molecular markers. Both studies were conducted
within the State of Minas Gerais (Brazil). However, no correlation was found between
the first internal transcribed spacer (ITS1) sequence divergence of M. subnitida popula-
tions and geographical distances in northeastern Brazil, which might be explained by
the extremely high mutation rates of the ITS region in M. subnitida (Cruz et al. 2006).

Genetic differentiation within Melipona subnitida populations was probably be-
cause of low gene flow, caused by limited dispersal ability (Engels and Imperatriz-

Fonseca 1998; Tavares et al. 2007), the large distance separating the NAT and PAR

New molecular evidence for fragmentation between two distant populations... 7

populations, and extensive disturbances of population dynamics due to anthropogenic
habitat degradation and fragmentation (Quezada-Euan et al. 2007). More recently,
mtDNA data has also pointed to genetic differentiation between these M. subnitida
populations from Rio Grande do Norte (Mossor6 city) and Piaui (Parnaiba city, on
the border of Maranhd4o state) (Bonatti et al. 2014).

Furthermore, the dendrogram obtained by the cluster analysis (Figure 1B) and the
scatter-plot of the PCoA (Figure 1C) revealed a clear separation of the species in two
main clusters confirming a significant molecular genetic difference between the two
populations. This topology corroborates the data presented by the Bayesian 0° and
AMOVA. A clear evidence of subdivision among sampling sites was also observed by
the Bayesian grouping model analysis of the ISSR data (Figure 1D).

This study provides additional evidence that ISSR markers can be useful tools in
defining population genetic substructuring in Melipona species. More importantly,
the distinctiveness of populations in these two regions suggests that the VAT and PAR
populations of M. subnitida have separate evolutionary histories. It is clear from this
study that conservation strategies should take into account the heterogeneity of these
two separate populations, and that actions should be addressed towards their sustain-
ability by integrating our findings with ecological tools. Failing to do so would risk
decimating the entire bee population by uncontrolled human activities in the region.

Acknowledgements

The authors are grateful to Mr. José Maria Vieira-Neto for help in field collection.
This work was supported by grants from the Northeast Bank of Brazil (BNB/ETENE/
FUNDECT) and Embrapa (Macroprograma 2, SEG Code: 02.11.01.029.00.00).

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