The Journal of Heredity 2001:92(6)
© 2001 The American Genetic Association 92:519-521
Brief Communication |
Partial Cytochrome b Sequences for Six Hymenoptera of the Eastern United States
From the Bee Research Laboratory, USDA, ARS, Bldg. 476, BARC-East, Beltsville, MD 20705. Portions of this research were completed as part of an Oakland Mills High School, Columbia, MD, mentor program (Gardner).
Address correspondence to Anita M. Collins at the address above or e-mail: collinsa{at}ba.ars.usda.gov.
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Abstract |
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Mitochondrial DNA (mtDNA) haplotypes have been commonly used to determine honeybee subspecies relationships. To see if these markers would also be useful for comparisons of other Hymenoptera, we collected workers of six local species: Vespa crabro, the European hornet; Bombus impatiens, a bumblebee; Vespula germanica, the German yellow jacket; Polistes fuscatus, a paper wasp; Halictus ligatus, an alkali bee; and an unspecified Megachile, a leafcutting bee. MtDNA was isolated and digested with six endonucleases (AvaI, BglII, EcoRI, HindIII, HinfI, XbaI). The digested DNA was electrophoresed and visualized on agarose gels with comparison to a standard fragment marker and similarly treated honeybee mtDNA. The fragments obtained were also purified and sequenced. Phylogenetic relationships between six wasp and bee species, Apis mellifera, and several other similar aculeate Hymenoptera were determined. Newly defined DNA sequences were posted to GenBank (AF281169–AF281174).
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Introduction |
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Mitochondrial DNA (mtDNA) is a short, circular molecule containing a double strand of nonnuclear DNA that has been widely used as a tool for identification and comparison in honeybees (Apis mellifera L.) (Schmitz and Moritz 1990; Smith and Brown 1988). Known markers have been used for distinguishing between Africanized and European honeybees (Hall and Muralidharan 1989; Hall and Smith 1991; Sheppard et al. 1991, 1994; Smith et al. 1989) and to study population genetics, biogeography, and phylogeny of the genus Apis (Meixner et al. 1993; Smith 1991; Smith and Hagen 1996; Smith et al. 1991), specifically A. mellifera (Franck et al. 1998; Garnery et al. 1995; Smith and Brown 1990). The honeybee mtDNA has been completely sequenced (16,343 bp) (Crozier and Crozier 1993).
A limited number of other Hymenopteran species have been studied in similar ways. Restriction fragment length polymorphism (RFLP) methods were used (Sappal et al. 1995) to differentiate several species of Trichogrammatidae. DNA sequences of the cytochrome b locus, as well as 16S and 28S ribosomal RNA, were used to determine the phylogeny of some Braconidae species (Dowton and Austin 1994; Dowton et al. 1998; Gimeno et al. 1997). Dowton et al. (1998) also included two members of the same Aculeate Hymenoptera genera we sampled (Apis and Polistes). Danforth et al. (1998) distinguished two cryptic species of Halictus using sequence differentiation of another mtDNA locus, cytochrome oxidase I. Koulianos (1999) and Koulianos and Schmid-Hempel (2000) inferred phylogenetic relationships within the bumblebees, Bombus, based on cytochrome b and cytochrome oxidase I sequences.
The purpose of this experiment was to see if a group of restriction endonucleases (AvaI, BglII, EcoRI, HindIII, HinfI, XbaI) that have been used with A. mellifera (Crozier and Crozier 1993; Sheppard et al. 1996; Smith and Brown 1988) would be useful for distinguishing other Hymenopteran species of the mideastern United States.
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Materials and Methods |
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Insect Collection
Hymenoptera were collected from several locations near Columbia, MD, during the months of August and September 1997. The samples were killed by freezing at 5°C and stored in an ultracold freezer (-80°C) at the Bee Research Laboratory, USDA, ARS, Beltsville, MD. Species collected were identified by local experts as Vespa crabro, the European hornet; Bombus impatiens, a bumblebee; Vespula germanica, the German yellow jacket; Polistes fuscatus, a paper wasp; Halictus ligatus, an alkali bee; and an unspecified Megachile, a leafcutting bee. Voucher specimens are being held at the Bee Research Laboratory.
DNA Extraction and PCR Procedures
MtDNA was extracted using a modified phenolic extraction method (Sheppard and McPheron 1991). For each extraction, one thorax (or one-half thorax for the large species) was ground in a 1.5 ml microcentrifuge tube with 250 µl of lysis solution. The extracted samples were amplified using the polymerase chain reaction (PCR) on a Perkin-Elmer Cetus DNA Thermal Cycler for 35 cycles at 94°C for 1 min, 46°C for 1 min 15 s, 64°C for 2 min, and then held at 0°C. The reaction was performed using 7 µl of sterile water, 5 µl of reaction buffer, 8 µl of dNTPs mixture (200 µM final concentration of each), 2.5 µl of each primer (1 µM final), 0.5 µl of Taq polymerase, and 1 µl of template DNA. We used the BglII primer pair (Crozier and Crozier 1993), which amplifies a section of cytochrome b that includes polymorphic restriction sites for A. mellifera. The primer sequences are as follows (Sheppard et al. 1994):
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The PCR products (15 µl) were digested with each of the restriction enzymes separately, then electrophoresed on a 1.5% agarose gel, stained with ethidium bromide, and visualized under ultraviolet (UV) light.
Purification and Sequencing
A representative sample from each species was chosen, PCR was performed as before, and the product was electrophoresed and visualized to assess quality and quantity. Thirty microliters of the PCR product were purified for sequencing using a Nucleo-Trap PCR purification kit (CLONTECH Laboratories Inc., Palo Alto, CA). The purified sample was sent to the Center for Agricultural Biotechnology, University of Maryland, College Park, MD, and sequenced in both directions using dye-primer sequencing reactions and an AB1377 sequencing machine (PE Biosystems).
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Analysis |
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Bands visualized in electrophoretic gels were compared with an internal size standard, phiX174 digested with HaeIII restriction endonuclease (Boehringer Mannheim Biochemica, Indianapolis, IN) and with a European honeybee pattern. The forward and reverse sequences obtained for each species were edited and aligned using Omiga 2.0 (Oxford Molecular Products 1998) and the best overall sequence determined. The experimental sequences and representative sequences from similar aculeate Hymenoptera in GenBank (Table 1) were aligned and cropped to include overlapping base pairs only. A single nonaculeate Hymenopteran, Bracon sp., was used as an outgroup. Heuristic search algorithms (PAUP 4.02b) were used to generate a most parsimonious phylogenetic tree from the data (Swofford 1998).
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Results |
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The amplified segment of cytochrome b mtDNA in each species was 530 bp in length. When aligned with the A. mellifera ligustica sequence (Crozier and Crozier 1993) the matching region fell between their base pairs 11395 and 11893. BglII and HinfI were the only two restriction enzymes of the six tested that cut any of the amplified mtDNA. BglII cut fragments from the species Halictus ligatus and A. mellifera to produce lengths of 320 bp and 210 bp. The restriction enzyme HinfI cut both Vespula germanica and Polistes fuscatus mtDNA to produce fragment lengths of 340 bp and 190 bp. A HinfI site was found within the Halictus sequence, but probably produced a fragment too small to be visualized on the gel. Vespa crabro DNA appears to have been cut by both endonucleases: with BglII there is a band at 530 bp and a faint one at less than 100 bp; with HinfI there is band at 530 bp, 410 bp, and a very faint one about 120 bp. This suggests that only some of the amplified copies are being cut successfully. Only one individual of the 11 B. impatiens samples was cut by HinfI. Megachile sp. fragments were not cut by any of the restriction enzymes. AvaI, EcoRI, HindIII, and XbaI did not cut these sections of cytochrome b in any of the taxa screened.
The phylogenetic analysis for the seven specimen species, eight other aculeate Hymenoptera, and the Bracon outgroup sample generated a single most-parsimonious tree with a length of 712 steps and a consistency index of 0.5281 (Figure 1). For the bootstrap method with heuristic search, subsampling across taxa was done, and 10,000 bootstrap replicates were run. A total of 211 informative sites were found. All bases were equally weighted. Bootstrap statistics are included in the diagram.
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Discussion |
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These six endonucleases would not be particularly useful for determining the phylogenetic relationships of this group of organisms. Only two of the six endonucleases used in this analysis had cut sites in the mtDNA PCR products produced using the BglII primer. In two of the species we found within-species variation. One B. impatiens sample was cut by HinfI, while the other 10 were not. One V. germanica sample produced band sizes of 410 bp and 120 bp when cut by HinfI instead of 340 bp and 190 bp, as found in the remaining 13 samples.
Hymenoptera generally express low levels of genetic variability because of their haplodiploid biology (Pamilo et al. 1997), but the lack of variability among our specimens probably has more to do with our limited sampling than with existing population variation. Accordingly, the species where we found variation were also the ones with the largest sample sizes. Further, collections were done in only a few locations, with most species found in only one, and it is possible that we were collecting nest mates for these social species. More extensive collection will be necessary to determine within-species population variation for these genetic markers.
The sequences found were all portions of the cytochrome b locus. The cumulative differences in the sequences of the partial gene from our samples and similar species found in GenBank gave a phylogenetic relationship that generally follows the accepted grouping for Hymenoptera. The outgroup (Bracon sp.), the group of wasps (V. crabro, V. germanica, Vespula vulgaris, and P. fuscatus), and the grouping of bees (10 remaining species) each fell on a separate branch. However, the placement of Megachile sp., Anthophora acervorum, and Euglossa imperialis as more closely related to the Apis species than the other bees was unexpected. Jermiin and Crozier (1994), working with the cytochrome b locus in ants, suggest that in Hymenoptera this locus might diverge evolutionarily at a different rate than others that have been studied. Sequence comparisons using this locus might lead to different phylogenetic relationships than with some other commonly used loci.
GenBank accession numbers for the newly sequenced species are AF281169–AF281174. The sequences reported for Megachile sp., H. ligatus, and P. fuscatus represent the first in their respective genera for the mtDNA genome.
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Acknowledgments |
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We wish to thank Suzanne Batra and Jeff Aldrich, ARS, USDA, for identification of specimens, and Jay Evans, ARS, USDA, for assistance with GenBank and PAUP software. Thank you to Deborah R. Smith and Jay Evans for review of the manuscript.
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Footnotes |
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Corresponding Editor: Martin Tracey
Received December 12, 2000
Accepted September 5, 2001
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References |
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