J Insect Conserv DOI 10.1007/s10841-014-9639-3
ORIGINAL PAPER
Conservation study of an endangered stingless bee (Melipona capixaba—Hymenoptera: Apidae) with restricted distribution in Brazil Juliano Nogueira • Josemar de Carvalho Ramos • Juliana Benevenuto Taˆnia Maria Fernandes-Saloma˜o • Helder Canto Resende • Lucio Antonio de Oliveira Campos • Mara Garcia Tavares
•
Received: 19 August 2013 / Accepted: 16 June 2014 Ó Springer International Publishing Switzerland 2014
Abstract Melipona capixaba, popularly known as ‘‘uruc¸u preta’’, is a stingless bee restricted to the mountainous Atlantic Rainforest areas of Espı´rito Santo State, Brazil. Due to the endemism and small population size, this species discovered in 1994 is now considered ‘‘vulnerable to extinction’’. Using ISSR, PCR–RFLP and microsatellites markers, we studied the genetic variability and structure of M. capixaba from 88 colonies collected throughout the distribution area of the species within Espı´rito Santo State. The microsatellite, ISSR and mitochondrial haplotype analyses showed that M. capixaba has low genetic variability compared to other insect species. The molecular analyses also indicated a high genetic similarity among the M. capixaba samples, with no clear pattern of structuring. The analyses of molecular variance results indicated that most of the total genetic variation in M. capixaba was explained by the genetic diversity within local populations. Results suggest that the analyzed samples could be treated as a single population for preservation purposes. Thus, given its endemism, local adaptation and low number of natural colonies, efforts for the conservation of M. capixaba should focus on preservation and increasing the number of colonies in the wild, so that M. capixaba can
Electronic supplementary material The online version of this article (doi:10.1007/s10841-014-9639-3) contains supplementary material, which is available to authorized users. J. Nogueira J. C. Ramos J. Benevenuto T. M. Fernandes-Saloma˜o H. C. Resende L. A. O. Campos M. G. Tavares (&) Departamento de Biologia Geral, Universidade Federal de Vic¸osa, Av. P H Rolfs, s/n, CEP: 36.570-000 Vic¸osa, Minas Gerais, Brazil e-mail:
[email protected]
support constant captures and the effects of habitat deforestation in Espı´rito Santo State. Keywords Conservation genetics Habitat fragmentation ISSR Meliponini Microsatellites
Introduction Melipona capixaba (Moure and Camargo 1994), popularly known as ‘‘uruc¸u preta’’, is a stingless bee restricted to the mountainous Atlantic Rainforest areas of Espı´rito Santo State, Brazil (Melo 1996), being found in the municipalities of Afonso Cla´udio, Alfredo Chaves, Brejetuba, Castelo, Conceic¸a˜o do Castelo, Domingos Martins, Marechal Floriano, Vargem Alta, Venda Nova do Imigrante e Santa Maria de Jetiba´ (Resende 2012). This region is characterized by vegetation of the humid tropical rainforest type (Atlantic Rainforest), altitudes between 800 and 1,200 m and average annual temperatures around 18–23 °C (Melo 1996; Resende 2012). The originally geographic distribution of M. capixaba is unknown, but Serra et al. (2012), using different models to describe the potential distribution of this species, verified that at present, M. capixaba occupies a small fraction of its potential distribution range. According to these authors, several factors (human influence, geographic barriers, absence of essential food source and hollow trees for nest and heat on the lowland regions) may prevent its dispersion to its potential distribution area. The authors also argument that maybe, M. capixaba be a recent species that were not capable, yet, to reach all suitable areas available. Whatever the reasons responsible for this discrepancy, presently this species can be found in an area of only 3,450 Km2 (Resende 2012). This region does seems to be the current area
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of occurrence of this species because after 4 years of collection efforts in different areas of Espı´rito Santo State, by two independent studies, we have not identified new places of its occurrence (Resende 2012; Serra et al. 2012). Melipona capixaba workers generally are robust (approximately 10.8 mm of total length), with a predominantly dark brown head and mesosoma, very smooth and shiny mesonotum and black shiny terga (Moure and Camargo 1994). Morphologically, M. capixaba is very similar to M. scutellaris (Latreille), a species found in the Atlantic Rainforest in northeastern Brazil (Moure and Camargo 1994). These two species are also very similar genetically (Fernandes-Saloma˜o et al. 2005) and there is evidence of hybridization when their colonies are kept together (Nascimento et al. 2000). Nests of M. capixaba are usually found in hollows of old trees and show entrance ranging from very simple to very elaborate, located close to the ground or over 2 m tall (Melo 1996). Although this species easily adapts to rational boxes, rational creation is not well accepted by local meliponicultors that prefer keeping colonies in rustic wooden boxes or in the original pieces of trees. In these conditions, colonies can be keep with relative success at altitudes between 700 and 800 m, in regions near native forests and mild temperatures. Bellow 700 m high, management requires special care as supplementary feeding with honey of other bee species and, bellow 600 m, this stingless bee do not survive, especially in warmer regions, even with proper management (Resende 2012). Due to the endemism and small population size, M. capixaba was only discovery in 1994 and is now considered ‘‘vulnerable to extinction’’, especially considering the deforestation of the Atlantic Rainforest (Silveira et al. 2008). The Atlantic Rainforest deforestation started with the colonization process, mainly due to coffee crops and wood extraction (Thomaz 2010; Juvanhol et al. 2011). The original extension of this bioma was 1,315,460 km2 (IBGE 2008) but at present, less than 8 % of the Atlantic Rainforest remains (Fundac¸a˜o SOS Mata Atlaˆntica and INPE 2013). For the Espı´rito Santo State, in particular, from the 4,614,814 ha (100 % of the State area) originally occupied by the Atlantic Rainforest, currently there is only 510,752 ha (*11 %) (Fundac¸a˜o SOS Mata Atlaˆntica and INPE 2013). Recently, however, some governmental politicians have been implemented in order to minimize the impacts of this process, such as the indication of some reminiscents as priority areas for conservation (Thomaz 2010). Most reminiscent fragments (83 %) present in the occurrence area of M. capixaba, however, are very small (0–5 ha) (Thomaz 2010; Resende 2012), which could difficult the specie’s dispersal and survival. These data highlight the potential extinction risk and the need to preserve the remaining colonies of M. capixaba.
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Management and conservation programs should consider the demographic history, genetic variability and structure of species (Crandall et al. 2000), which could also assist in making decisions about the translocation of M. capixaba colonies in an effort to avoid potential deleterious consequences of inbreeding, as well as deciding which colonies should remain genetically isolated. Molecular markers have been employed to characterize population genetic variability/differentiation and/or the genetic status of some stingless bee species. In many cases, the data were used to determine the conservation strategies used for the particular bee population, which include M. beecheii (Quezada-Eua´n et al. 2007; May-Itza´ et al. 2012), M. rufiventris (Tavares et al. 2007), Plebeia remota (Francisco et al. 2008); M. yucatanica (May-Itza´ et al. 2010) and Scaptotrigona hellwegeri (Quezada-Eua´n et al. 2012). Thus, in the present study, we used microsatellites, ISSR and mitochondrial markers to characterize the genetic diversity of M. capixaba colonies present in the central region of Espı´rito Santo State, Brazil. These analyses can help to verify if the remaining colonies of M. capixaba can be treated as a single population or does each locality have distinct stocks that should be managed separately in conservation programs. This issue must be considered in order to develop effective conservation strategies for this threatened endemic stingless bee species.
Materials and methods Field sampling Adult workers of Melipona capixaba were collected throughout the distribution area of the species within Espı´rito Santo State. Samples from eighty-eight colonies from 21 localities in the municipalities of Domingos Martins (DM), Conceic¸a˜o do Castelo (CC), Venda Nova do Imigrante (VN), Alfredo Chaves (AC), and Vargem Alta (VA) were analyzed (Table 1; Fig. 1). Most samples were provided by meliponicultors that kept colonies (in wood boxes or in the original trunks) on their own property or in the neighbourhood; only four samples were obtained from colonies located in native forest. However, according to the meliponicultors that provided samples, such nests were directly removed from the nature and do not represent artificial split of original colonies. During sampling, bees were collected at the entrance to their colonies and stored in absolute ethanol at -80 °C until DNA extraction. Voucher specimens were deposited in the insect collection at the Central Apiary of the Universidade Federal de Vic¸osa (Vic¸osa, Minas Gerais, Brazil).
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DNA extraction and amplification The total DNA from the head and mesothorax of a single individual from each colony of M. capixaba was extracted as described by Fernandes-Saloma˜o et al. (2005). Genomic DNA was amplified using four microsatellites primers specifically designed for M. capixaba (Table 2). These microsatellites are described here for the first time and Table 1 Municipalities, codes, geographic coordinates, number of Melipona capixaba colonies sampled (N) and the mitochondrial haplotypes identified in each municipality Municipality
Lat. (S)
Long. (W)
N
Haplotypes
Domingos Martins (DM) Conceic¸a˜o do Castelo (CC)
-20°240
-41°050
33
I, II, IV, V
-20°270
-41°270
10
II, IV, V, VI, VII
Venda Nova do Imigrante (VN)
-20°450
-41°060
15
I, III, IV, V
Alfredo Chaves (AC)
-20°480
-40°930
16
I, II, III, IV, V
Vargem Alta (VA)
-20°540
-40°980
14
I, III
were designed through amplifications using eight different ISSR primers (807, 815, 866, 827, 834, 841, 890 and C –Wmed Representac¸o˜es Ltda) and an individual of M. capixaba. The sequence of these microsatellite primers were designed using the online program OligoExplorer 1.2 and their optimal annealing temperature was determined through gradient test using a Mastercycler Gradient Thermocycler (Eppendorf Ò). Of the 11 loci analyzed, the four polymorphic (P = 36.4 %) were used in the present study. The microsatellites amplification mix (12.5 ll) contained approximately 10 ng of genomic DNA, 0.6 ll of primer F (modified at the 5 ‘end with the addition of the sequence of the M13 universal primer: 50 -TTTTCCCAGT CACGA-30 according to Schuelke 2000) at 0.1 mM, 0.6 ll of primer R at 1.0 mM, 1.0 ll of M13 universal primer labeled with fluorescence (FAM-6-carboxy-fluorescein or HEX hexacloro-6-carboxy-fluorescein) at 1.0 mM, 0.8 ll of dNTP at 200 lM, 2.5 ll of 1X PCR buffer, 1.0 ll of MgCl2, at 1.5 mM and 0.5 U GoTaqÒ Flexi DNA polymerase (Promega). Amplifications were performed at an initial denaturation step of 94 °C/3 min, followed by 10 cycles of 94 °C/15 s, specific annealing temperature of each primer/20 s, 72 °C/30 s. Subsequently, 25 cycles of
Fig. 1 Map of Brazil and Espı´rito Santo State showing the high regions of the Atlantic Rainforest (dark gray) where the five populations of Melipona capixaba were sampled. The black circles represent the 21 localities where samples were obtained from the municipalities of Domingos Martins, Conceic¸a˜o do Castelo, Venda Nova do Imigrante, Vargem Alta and Alfredo Chaves
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J Insect Conserv Table 2 Characteristics of the four microsatellite loci and ten ISSR primers utilized for the analysis of Melipona capixaba
Locus
Primers sequence (50 -30 )
Ta (°C)
F: 50 -CGTCAATCCTCCCTGAGCTA-30
68.5
4
64.0
10
72.5
k
Repet motif
Allele size range (bp)
CG2TG(CG)5
205–255
(GAAG)3
164–224
3
(T)10
222–224
64.0
2
(GTTC)2GT
104–128
SSR Mcap1
R: 50 -GGGAGGGTGTGTACTTTTGC-30 Mcap2
F: 50 -GCACAGTGGGGTGGAAAGAG -30 R: 50 -CGCCCCCCGTTTGATTATCG -30
Mcap5
F: 50 -CGTTCGGAGGAAAACGCTCG -30 R: 50 -CCATCCAATAGCTTCGGCCA -30
Mcap13
F: 50 - CCAGCGTCTTTCCTCGTCGA-30 0
R: 5 - CGCTTCATCCGTTTCTTGGG-3
0
ISSR
F and R, forward and reverse primers, respectively; Ta: annealing temperature; k: number of alleles
807 808
AGAGAGAGAGAGAGAGT AGAGAGAGAGAGAGAGC
54.8 49.6
14 12
815
CRCTCTCTCTCTCTCTG
52.0
16
827
ACACACACACACACACG
54.8
11
836
AGAGAGAGAGAGAGAGYA
55.4
13
841
GAGAGAGAGAGAGAGAYC
50.7
9
866
CTCCTCCTCCTCCTCCTC
58.4
7
890
VHVGTGTGTGTGTGTGT
49.6
20
C
GTGGTGGTGGTGGTGRG
53.4
8
Terry
GTGGTGGTGGTGRC
54.8
8
89 °C/15 s, 53 °C/20 s, 72 °C/30 s and a final extension of 72 °C/30 min. PCR products were measured using an MegaBACE automated capillary sequencer. Allelic sizes were scored against the size standard ET 550R (GE Healthcare Life Sciences) and the peaks analyzed using the MegaBACE Fragment Profiler program (GE Healthcare Life Sciences). The ISSR PCR reactions were performed in a final volume of 25 ll containing approximately 10 ng template DNA, 1 ll of each primer at 5 lM, 2 ll of dNTPs at 100 mM, 5 ll reaction buffer (Promega), 1.5 lL 25 mM MgCl2, 0.25 lL formamide and 1 U GoTaqÒ Flexi DNA polymerase (Promega). Ten primers were used in the amplifications: 807, 808, 815, 827, 836, 841, 866, 890, C and Terry (Wmed Representac¸o˜es Ltda). The PCR reaction consisted of an initial denaturation of 3 min at 95 °C and 40 cycles of 1 min at 92 °C, 2 min at the specific annealing temperature of each primer, and 2 min at 72 °C, with a final extension step at 72 °C for 7 min. The ISSR amplification products were resolved in 1.5 % horizontal agarose gels in 1X TBE buffer (40 mM Tris–acetate, 1 mM EDTA pH 8.0) with 100 V for 4 h and stained with ethidium bromide. A negative control and a 1 Kb marker (Invitrogen) were used as a molecular standard in all the reactions. The ISSR profiles were visualized under UV light. Primers COX-1 (1,389–1,411 pb of the tRNATrp gene from Melipona bicolor) and COX-4 (3,202–3,228 pb of the
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COII gene from M. bicolor) (Silvestre et al. 2008) were used to amplify the complete sequence from the tRNATyr/ COI/tRNALeu2 genes. The reactions (25 lL) contained approximately 50 ng DNA, 0.8 lL of each primer at 10 lM, 2.5 lL of 25 mM MgCl2, 2 lL of dNTPs at 100 mM, 5 lL of reaction buffer 10X and 1.5 U GoTaqÒ Flexi DNA polymerase (Promega). The PCR reaction consisted of an initial denaturation of 5 min at 94 °C and 34 cycles of 1 min at 94 °C, 1 min and 20 s at 50 °C, and 2 min at 64 °C, with a final extension step at 64 °C for 10 min. The amplicon was digested with the restriction enzymes DraI, HinfI, KspAI, MspI and SspI in a mixture with final volume of 25 ll containing 10–15 lL of the PCR product, 2.5 ll of the specific buffer 10 9 for each restriction enzyme and 6 U of the enzyme. The mixture was incubated for 6 h in a Mastercycler Gradient (EppendorfÒ) thermocycler at the ideal temperature for each enzyme, followed by 15–20 min at the heat inactivation temperature of the enzyme. The digestion products were resolved in 2 % agarose gel or 12 % polyacrylamide gel and stained with ethidium bromide solution. The RFLP patterns were visualized under UV light. A 1 kb marker (Invitrogen) was used as a molecular standard in all the reactions. The RFLP profiles were visualized under UV light. The polymorphic RFLP patterns obtained from each enzyme were named (A, B, C and D) according to the order of observation of each one.
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Data analysis For analysis of the molecular data, a multiallelic matrix was constructed for microsatellites, while binary matrices were constructed based on the presence (1) and absence (0) of amplified DNA fragments (ISSR) or haplotypes (mtDNA). All microsatellites loci were assessed using MICROCHECKER 2.2.3 (Van Oosterhout et al. 2004) to check for null alleles, scoring errors and large allele dropout, carried out by 1,000 randomizations. The Popgene version 1.32 program (Yeh et al. 1999) was used to calculate the following estimators of genetic diversity for the microsatellite loci: mean number of alleles/locus (A), allele frequency, observed (Ho) and expected (He) heterozygosities. For ISSR loci, the parameters estimated were: mean expected heterozygosity (He) (Nei 1973), Shannon diversity index (I) (Shannon and Weaver 1949) and the percentage of polymorphic loci (P). Nucleotide diversity (h) and haplotype diversity (I) were estimated for mtDNA using the REAP software (McElroy et al. 1991). The genetic distance between samples was estimated for the microsatellite loci according to Nei (1978). For the ISSR markers, it was initially calculated considering the Jaccard similarity index (Sij) within and among pairs of samples. The genetic similarity (Sij) was then converted to genetic distances (Dij) using the arithmetic complement of similarity given by the expression: Dij = 1 - Sij. The genetic distance among mitochondrial haplotypes (d), in turn, was determined according to Nei and Tajima (1981) and Nei and Miller (1990) using the REAP software (McElroy et al. 1991). A 2D graphic dispersion analysis and a cluster analysis using the unweighted pair group mean algorithm (UPGMA) were used to investigate the relationships between samples. The projection efficiencies of the distances in the plane were estimated according to the correlation between the original distances and those showed in the graph. The genetic structure of samples was determined using the hierarchical analyses of molecular variance procedure (AMOVA) (Excoffier et al. 1992). We also calculate the fixation index (FIS) for each locus. These analyses were done using the GENES program (Cruz 2013). In order to obtain sample sizes large enough for population genetic analyses, colonies from the same locality were pooled and considered to be a population for most of the statistical analyses. However, the 2D graphic dispersion and one of the UPGMA analyses carried out were conducted without pooling samples.
Results The number of alleles in the microsatellite loci ranged from 2 to 10, with an average of 4.75 alleles/locus (Table 2).
Table 3 Estimates of the microsatellites, ISSR and mtDNA genetic parameters in five Melipona capixaba populations Microsatellites
ISSR
mtDNA
Ho
He
He1
I
P (%)
h
I2
DM
0.06
0.41
0.17
0.26
50.00
0.21
0.30
CC
0.05
0.33
0.14
0.20
36.44
0.22
0.32
VN AC
0.00 0.00
0.37 0.28
0.15 0.14
0.22 0.20
42.37 39.83
0.22 0.03
0.32 0.03
VA
0.00
0.32
0.14
0.20
39.83
0.25
0.38
Mean
0.02
0.34
0.15
0.22
41.69
0.18
0.27
Codes are the same as in Table 1. Ho and He: observed and expected heterozygosity computed using Levene (1949). He1: Nei’s expected heterozygosity. I: Shannon’s indice. P: percentage of polymorphic loci. h: genic diversity and I2: haplotype diversity
The allelic frequencies may be seen in Online Resource 1. It is noteworthy that exclusive alleles were detected in the samples from Domingos Martins (alleles A, B and G/Mcap2), Conceic¸a˜o do Castelo (allele I/Mcap2) and Vargem Alta (allele B/Mcap1). The average observed and expected heterozygosity values for M. capixaba were 0.02 (range 0.00–0.06) and 0.34 (range 0.28–0.41), respectively (Table 3). The v2 test (P \ 0.05) rejected the hypothesis that samples were in Hardy–Weinberg equilibrium, showing a marked excess of homozygotes, a result that can be attributed to the small population size of the M. capixaba samples presently extant in the Espı´rito Santo State. The inbreeding coefficient FIS (overall FIS = 0.93) confirmed this excess of homozygotes. MICROCHECKER analysis suggested the presence of null alleles at Mcap1 and Mcap2 locus, which can result in excess of homozygotes for these loci. For all loci, however, there is no evidence for scoring error due to stuttering or for large allele dropout. The use of the ten selected ISSR primers resulted in a total of 118 bands, ranging from seven (primer 866) to 20 bands (primer 890) per primer, with a mean of 12 bands/ primer (Table 2). The highest percentage of polymorphism was observed in samples from Domingos Martins (50.0 %) and Venda Nova do Imigrante (42.37 %), while Conceic¸a˜o do Castelo presented the smallest percentage (36.44 %). The values of the Nei genetic diversity and the Shannon index ranged from 0.14 to 0.17 and from 0.20 to 0.26, respectively (Table 3). The RFLP analysis detected seven mitochondrial haplotypes, with a variation of 2–5 haplotypes per locality (Table 1). Despite their ample distribution, haplotypes I, IV and V were most frequent, being present in approximately 75 % of the analyzed samples. Exclusive haplotypes (VI and VII) were detected only in samples from Conceic¸a˜o do Castelo. The values of the nucleotide diversity and the haplotype diversity ranged from 0.03 to 0.25 and from 0.03 to 0.38, respectively (Table 3).
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The genetic distances between samples, calculated considering the microsatellite and the ISSR markers, was low, ranging from 0.003 to 0.118 and from 0.168 to 0.199, respectively. The distances between the mitochondrial haplotypes were also low, ranging from 0.018 to 0.235 (Online Resource 2). The 2D graphic dispersion (Fig. 2) and the cluster analysis (data not shown), constructed from the ISSR, microsatellites and mtDNA genetic distance matrices, did not indicate the presence of defined groups. The correlation values of the 2D dispersion analysis were 96.1, 89.3 and 94.8 % for microsatellites, ISSR and mtDNA markers, respectively. These values confirm the good adjustment between the genetic and the distances graphically represented. Consistent with this, the hierarchical analysis of molecular variance indicated that 98.3, 93.0 and 55.5 % of the total genetic variation in M. capixaba determined by microsatellites, ISSR and mtDNA respectively, was explained by the genetic diversity within local populations.
Discussion The microsatellite, ISSR and mitochondrial haplotype analyses showed that M. capixaba has low genetic variability compared to other Hymenopterans. For instance, in the present study the proportion of ISSR polymorphic loci was 50 %, while Paplauskiene et al. (2006) verified that from the 60 DNA fragments amplified from 10 to 12 individuals of Apis mellifera carnica (Pollman) and A. m. caucasica (Gorbachev), 66.7 % were polymorphic. Similarly, Nascimento et al. (2010), who investigated 61 colonies of M. quadrisfasciata from eight locations in the state of Minas Gerais/Brazil, found that from the 119 ISSR bands obtained, 80 (68 %) were polymorphic. Additionally, although the average number of microsatellite alleles detected in the present study (4.75) was higher than that detected in M. bicolor (Lepeletier) (3.88— Peters et al. 1998), Trigona carbonaria (Smith) (3.6— Green et al. 2001) and M. seminigra merrillae (Cockerell) (3.7—Francini et al. 2009), the mean observed heterozygosity in M. capixaba (Ho: 0.02) was much lower than that detected in other species of stingless bees analyzed with species-specific primers. The values found for M. bicolor, M. s. merrillae and Nannotrigona testaceicornis (Lepeletier), for instance, were 0.40, 0.34 and 0.59, respectively (Peters et al. 1998; Francini et al. 2009; Oliveira et al. 2009). However, it has long been known that Hymenoptera (and especially social species) have low levels of heterozygosity compared to most others insects (Graur 1985).
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Fig. 2 Dispersion analysis plot related to the five populations of Melipona capixaba evaluated by microsatellites (a; r (correlation): 0.96), ISSR (b, r: 0.89) and mtPCR-RFLP (c, r: 0.95) markers. Individuals from different populations are represented by: cross (Domingos Martins), dark circles (Conceic¸a˜o do Castelo), dark squares (Venda Nova do Imigrante), dark triangles (Vargem Alta), and dark stars (Alfredo Chaves). The open rectangles represent the centroids for each population while the X and Y axes represent the measures relative to the genetic distances expressed by the arithmetic complement of the Jaccard index
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Additionally, comparisons between the genetic variability found in the present study and other Hymenopteran species must consider the fact that, like most stingless bees, colonies of M. capixaba possess a single queen mated with a single male (Kerr et al. 1962; Da Silva et al. 1972; Peters et al. 1999; Strassmann 2001; Palmer et al. 2002). The excess of homozygote observed in microsatellite loci may be result from the presence of null alleles at two of the four loci used, as suggested by the MICROCHECKER test. However, Dabrowski et al. (2014) demonstrated that different methods of assessment and detection of null alleles, including MICROCHECKER, resulted in false positives. According to these authors, detection of null alleles using indirect methods, such as those based on observed deviations from HWE, is susceptible to errors, given that these methods are based on assumptions that are commonly violated in natural populations. Thus, the marked excess of homozygotes should be the result of the small population size of the M. capixaba, and not an artefact of putative null alleles. The 2D dispersion analyses also indicated a high genetic similarity among the M. capixaba samples tested, showing a mixing of samples from different localities, with no clear pattern of structuring. Microsatellite and ISSR dendrograms, when considering each colony individually further showed that, usually, not even the samples from the same locality grouped in a single cluster. Colonies from Domingos Martins, Alfredo Chaves and Venda Nova do Imigrante, for example, were distributed on different clusters of the dendrograms. Similarly, dendrograms considering the samples grouped in five populations did not indicate different groups (data not shown). Accordingly, low genetic distance values were found, which were consistent with the restricted distribution of M. capixaba and the fact that many alleles were shared by most samples. The AMOVA results suggested that the genetic differences detected among samples were not enough to consider them as distinct stocks. When considering the microsatellites/ ISSR and the PCR–RFLP markers, it is worth mentioning that the differences observed among the AMOVA values, are due to their specific characteristics, i.e., nuclear X mitochondrial markers. Due to the differential inheritance of the mtDNA (haploid and uniparental), a four times higher level of genetic structuring is expected for mitochondrial genes (Palumbi et al. 2001). Thus, our results suggest that the analyzed samples have low genetic diversity and could be treated as a single population for preservation purposes. However, M. capixaba, is well adapted to the biotic uniqueness of its occurrence area (altitude, climate, vegetation and temperature) which may be relevant adaptive factors to explain its endemism. Thus, these factors must be considered in any preservation program. To our knowledge
M. capixaba colonies do not develop in regions with warm temperatures and altitudes lower than 600 m, even when appropriately managed. Indeed, in our sampling expeditions, we have not registered the presence of M. capixaba colonies in the regions around Domingos Martins, where lower altitudes and warmer and wetter climate predominate. Additionally, some beekeepers reported failed attempts to maintain colonies in the municipalities of Guarapari, Marataı´zes and Vito´ria in the Espı´rito Santo coast (below 600 m altitude) and also in warmer areas of the municipality of Santa Teresa. Finally, experimental colonies introduced in the meliponaries of the Federal University of Uberlandia and Federal University of Vic¸osa did not resist for long time, probably due to the low altitude of these sites, the warmer climate and different vegetation compared with the natural distribution range of the species (Resende 2012). Despite the considerations above about altitude and temperature, and the occurrence of M. capixaba, we should keep in mind that climatic conditions and vegetation change regionally over time. Thus, the limited geographic distribution of M. capixaba found today could be a remnant of a wider distribution in the past, which was reduced due to large deforestation of the Atlantic Forest, due to agroindustrial expansion and urbanization. Deforestation has been considered the main threat to M. capixaba, since stingless bee species depend on suitable trees to obtain nest sites and floral resources (pollen and nectar) to supply their nutritional needs (Silveira et al. 2008). Furthermore, deforestation can isolate populations in the forest fragments, increasing endogamy and, consequently, diminishing even more the species genetic diversity (as observed in the present study). Thus, these pressures are likely to increase the vulnerability of this species. The fact that some beekeepers are used to open colonies (in order to collect honey) and leave the brood combs open to the environment also contributed to accelerate population reduction. Meliponicultors also use to collect colonies from the forest and keep them on their properties for hobby. But, although this species can easily adapt to handling in rational boxes, which could be an opportunity to their rescue and preservation, local meliponicultors prefer maintain colonies in pieces of the original trunks or in rustic wooden boxes. This inadequate manipulation associated with the maintenance of colonies in inappropriate locations and without supplementary alimentation may contribute to the death of colonies (Resende 2012). In this sense, it is not uncommon to hear reports from local meliponicultors that their colonies ‘‘left’’, instead of acknowledging that they died. Thus, it is necessary to provide information to the local population about the importance of using appropriate breeding techniques for the conservation and management of this species. This
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awareness is crucial when one considers that many of the remaining colonies are on private properties. Our sampling experience also demonstrated that it is not easy to localize and sample wild M. capixaba colonies in the wilderness. Several fragments of Atlantic rainforest in the region were not sampled due to difficulties in accessing them. Therefore, despite the habitat fragmentation and the lack of large continuous ecological corridors among the localities sampled, some fragments, even from small size, could potentially allow genetic flow via males, which could explain the homogeneous and low genetic differentiation observed across samples. This could represent a key mechanism by which panmixis is achieved and so, the survival of species would be warranted. Studies related to drones flight distances, however, are scarces. Kerr et al. (1962) verified that Scaptotrigona postica males could fly about 600 m, while Carvalho-Zilse and Kerr (2004) reported that for M. scutellaris males, the maximum flight distance is approximately 800–1,000 m. In contrast, Kraus et al. (2008) verified that two drone congregation areas of Scaptorigona mexicana, separated by more than 10 km away, showed a low population sub-differentiation, suggesting a gene flow over larger distances mediated by drones. Thus, if males of stingless bee can travel the distances separating two or more fragments, fragmentation alone and in this context may not represent a risk for the species survival. However, we should not forget others risks associated with the fragmentation process, such as lack of nests sites and food resources, as mention above. In stingless bees, however, the dispersion of genes depends not only on the distance of flight of the males, but also on the swarming distance to form a new nest. It is well known that new stingless bee colonies depend on the mother colony for some time to provide food and material for the construction of the new colony (Inoue et al. 1984). In this regard, the maximum distance flight of workers is also important and Arau´jo et al. (2004) verified that the maximum flight distance for some stingless bees is a function of body size. In this study, the estimated flight distances for large-sized species such as M. bicolor and M. scutellaris were greater than 2 km. Similarly, a flight distances of 2,000 and 2,470 m were estimated for M. quadrifasciata and M. compressipes, respectively (Kerr 1987). Thus, since M. capixaba workers are as large and robust as the bees species mentioned above, they probably can fly over a similar distance. Taking this into account, these species would be effectively isolated (in a fragmented habitat) if forest fragments were greater than 2 km apart each other (Arau´jo et al. 2004). Alves et al. (2011), however, showed that colonies of M. scutellaris subjected to a severe bottleneck, which resulted in a drastic decrease in the genetic variability and in an increase in the production of diploid males, were capable
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of survive for over 10 years, when adequately managed. Thus, according to the authors, breeding from a small stock of colonies may have less severe consequences for the stingless bees, than previously suspected. This could explain how stingless bee populations with low genetic variability have survived though the continuous and sharp increased deforestation of different habitats. Another factor that could be favouring the mixture of alleles from different regions and influencing the distribution of the genetic variability among them, are the multiplication of M. capixaba swarms in rational boxes (started in the 1990s) and the practice of transport and trade of colonies, both that are considered common in the sampled region. Thus, although the sampled colonies may not necessarily exactly mirror the genetic variability of wild ones, they may represent the majority of the remaining colonies of M. capixaba and gene flow may occur between captive and wild populations. Thus, given its endemism, local adaptation and low number of natural colonies, efforts for the conservation of M. capixaba should focus on maintaining the highest possible number of colonies, particularly those with exclusive alleles, so that M. capixaba population can support constant captures and the effects of habitat deforestation in the Espı´rito Santo State. Thus, one factor to be considered during the elaboration of preservation strategies for M. capixaba is conservation/ recuperation of local forests. In this sense, Resende et al. (2008) suggest that M. capixaba can be an effective pollinator of orchids in their range, as they found some workers carrying pollinia of the genus Maxillaria (Ruiz & Pavo´n) and Xylobium (Lindley) attached to their scuttella. This finding reinforces the need to implement species conservation efforts, as several species of Orchidaceae endemic to the Atlantic Forest of the Espı´rito Santo are also threatened with extinction (Espı´rito Santo, Decree No. 1499, June 14, 2005). In addition, Luz et al. (2011) identified 33 types of pollen grains used as food source by M. capixaba, with Myrtaceae and Melastomataceae being their main pollen sources. These factors may help in the directing efforts to recover the vegetation in these areas. On the other hand, considering that many colonies of M. capixaba belong to meliponicultors, implementation of conservation meliponars in environmental preservation areas, located within the natural range of this species (such as the Pedra Azul State Park and the Forno Grande State Park) may represent an important preservation strategy. Furthermore, they should be created in partnership with the local communities in order to emphasize the importance of protecting and maintaining colonies in their natural habitat and, concomitantly increasing the number of colonies from local populations. To conclude, considering that the loss of genetic variability can decrease the potential of different species to
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survive in the face of environmental changes, conservation strategies should concentrate on preservation and increasing the remaining M. capixaba populations in the wild. Acknowledgments The authors are grateful to the Brazilian agencies FAPEMIG (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais), CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico), CAPES (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior) and UFV (Universidade Federal de Vic¸osa) for financial support. We are also grateful to the beekeepers that provided the bee samples used in this study, to Iris Raimundo Stanciola for the technical assistance during sampling expeditions and to Dr. Cosme Damia˜o Cruz from Universidade Federal de Vic¸osa for statistical assistance with the GENES program.
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