Department of Entomology, Cornell University, Ithaca, New York
Correspondence: E-mail: brady.sean{at}nmnh.si.edu.
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Abstract |
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Key Words: elongation factor-1 (EF-1
) intron gain bees Apiformes Colletidae phylogeny
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Introduction |
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One trend already evident is that some spliceosomal introns have originated relatively recently in the evolutionary history of eukaryotes (Hankeln et al. 1997; Logsdon 1998; Tarrío, Rodríguez-Trelles, and Ayala 1998). Such cases of recent intron gain have been used to bolster the "introns-late" theory, which maintains that spliceosomal introns are introduced into previously continuous protein-coding regions (Rogers 1990; Cavalier-Smith 1991; Palmer and Logsdon 1991). Although proponents of the alternative "introns-early" theory (Doolittle 1978; Gilbert 1979) contend that deletion of introns ancestral to all eukaryotes is mainly responsible for contemporary intron distributions among taxa, they also recognize that intron insertion occurs to some degree (de Souza et al. 1998; Fedorov, Merican, and Gilbert 2002). Well-documented specific cases of recent intron gain, however, are few in the literature.
In this paper we report the discovery of a novel intron in the F1 copy of elongation factor 1 (EF-1
). This nuclear gene encodes a protein involved in the GTP-dependent binding of charged tRNAs to the acceptor site of the ribosome during translation (Maroni 1993). EF-1
is known to possess two paralogous copies in bees (Danforth and Ji 1998), other Hymenoptera (present study), beetles (Jordal 2002), and flies (Hovemann et al. 1988), whereas only a single copy has been reported from other arthropod taxa.
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Materials and Methods |
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Our sampling included all seven currently recognized families of bees (all supraspecific bee taxonomy in this paper follows Michener (2000), and each family contained nearly all constituent subfamilies (18 of 21 subfamilies [table S1 in Supplementary Material online lists all species sampled]). This coverage included bees from virtually all continents, including Africa, Australia, Eurasia, North America, and South America. Additionally, within each individual subfamily, our sampling often was quite dense at lower taxonomic levels. We also included members from the close outgroups to bees, the wasp families Crabronidae and Sphecidae (Alexander and Michener 1995), as well as the more distantly related Formicidae (ants).
All sequence and phylogenetic analyses were performed using PAUP* version 4.0b10 (Swofford 2002). To verify that all sequences obtained in this study belong to the F1 homolog of EF-1, we constructed distance and parsimony trees of our sequences (exons only) combined with the known F1 copy in Apis mellifera (GenBank accession number X52884) and F2 copies of this gene from a representative of each bee family (GenBank accession numbers AF015267, AF435383, AY230129, and AY230131 and unpublished data). The distance tree was calculated using Kimura two-parameter distances (Kimura 1980) analyzed under the neighbor-joining clustering algorithm (Saitou and Nei 1987). The parsimony tree was inferred using a heuristic search strategy of 100 random addition replicates of TBR branch swapping. We ran an additional parsimony analysis using only bee F1 sequences. In all cases, we assessed robustness of clades using the nonparametric bootstrap (Felsenstein 1985).
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Results |
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Alignment of these sequences and comparison with Apis mellifera indicate the presence of an intron in all 12 species of Colletidae surveyed (fig. 2). This intron is absent from all other species of bees (37 spp.), wasps (four spp.), and ants (three spp.) sampled. The colletid intron occurs at position 313/314 relative to the Apis mellifera coding sequence (fig. 1). The alignment reveals strict conservation of the intron splice site (fig. 2); we find no evidence of intron sliding. The initial and final 2 bp of all intron sequences match the canonical GT/AG template for spliceosomal introns (Lewin 2000). The intron is inserted in symmetrical phase 1 (i.e., between codon positions 1 and 2). The AT content is much higher in intron sequences (64%) compared with the surrounding exon sequences (44%), a condition commonly reported (Csank, Taylor, and Martindale 1990; Kwiatowski, Skarecky, and Ayala 1992). However, there is no substantial difference in exon AT content between taxa with introns (43%) compared with those without introns (44%).
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Discussion |
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We found that this intron is absent from all other bees in our study. Our sampling broadly covered the taxonomic diversity of bees, suggesting the complete absence of this intron from all noncolletid bees. This conclusion is consistent with two recent phylogenetic studies (Leys, Cooper, and Schwarz 2002; Schwarz, Bull, and Cooper 2003) that report sequences from 59 species in 12 genera from the bee subfamily Xylocopinae (see table S2 in Supplementary Material online), all of which lack this intron. This intron also is absent from all wasp and ants sampled in the current study, and does not occur in Drosophila (Hovemann et al. 1988) or beetles (Jordal 2002). Furthermore, an intron is absent from the equivalent position in species from other arthropod orders with only a single reported EF-1 copy, including Collembola (Carapelli et al. 2000), Diplura (Carapelli et al. 2000), Hemiptera (aphids [Normark 1999; von Dohlen, Kurosu, and Aoki 2002]), Lepidoptera (Cho et al. 1995; Mitchell et al. 1997), and Odonata (damselflies [Jordan, Simon, and Polhemus 2003]). The fact that this intron is known to be absent from so many arthropod lineages provides strong evidence that its presence only in colletid bees is the result of a comparatively recent insertion event.
Another intron has been documented in a position 11 bp downstream from that in Colletidae (based on sequence alignment [results not shown]) in the following taxa: Artemia (brine shrimp) (Lenstra et al. 1986), Scutigera (myriapod), Acerentomon (proturan), and several genera of Collembola (final three groups from Carapelli et al. [2000]). Although intron sliding has been invoked by some to explain slightly discordant intron positions between taxa, this phenomenon has been very difficult to demonstrate for putative sliding events greater than 1 bp to several bp (Stoltzfus et al. 1997; Sato et al. 1999; Rogozin, Lyons-Weiler, and Koonin 2000). Because of the larger positional discrepancy of the apterygotan intron and its complete absence from all pterygote insects sampled, we interpret it as a nonhomologous intron whose position is coincidentally close to the intron found in Colletidae.
Colletidae is a diverse family containing over 2,000 species that, along with the family Stenotritidae (21 species endemic to Australia), traditionally have been considered the most "primitive" bees (Michener 1944, 1979, 2000). In the most recent morphological analysis of bee family relationships (Alexander and Michener 1995), their phylogenetic affinities were not well established. The comparative distribution of the intron reported in this study provides a new character supporting monophyly of the colletid bees. Furthermore, some authors argue that stenotritids are derived from within the Colletidae (McGinley 1981; Alexander and Michener 1995; Engel 2001), whereas others contend that Stenotritidae are a distinct group based on glossal morphology (McGinley 1980), nesting biology (Houston 1975), and embryology (Torchio 1984). Our results support the latter hypothesis because stenotritids lack this unique intron (fig. 3).
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The possibility of parallel insertions or multiple losses warrants caution when using comparative intron distributions either to argue for recent, unique insertion events or to use those distributions for phylogenetic information. Parallel insertions evidently are rare, with the first unambiguous case recently being uncovered between Drosophila and plants (Tarrío, Rodríguez-Trelles, and Ayala 2003). Intron deletion can threaten the utility of using intron insertions for phylogenetic information in cases where such deletion is not recognized as a secondary event (e.g., Krzywinski and Besansky 2002; Wada et al. 2002). Some proponents of the introns-early hypothesis maintain that intron loss in general may be more common than intron gain. For example, genomic comparisons among human, mouse, and rat intron distributions show six clear-cut cases of intron loss but none of intron gain (Roy, Fedorov, and Gilbert 2003). Asymmetrical intron positions in unicellular organisms have also been interpreted as indicative of predominant intron loss (Mourier and Jeffares 2003). On the other hand, phylogenetic analyses of 205 intron positions in a variety of organisms reveal that 77% show a pattern consistent with a single intron gain with no subsequent loss (Stoltzfus et al. 1997). On a more detailed level, comparisons of three genes involved in recombination in plants, animals, and fungi suggest that insertions occur three times more often than deletions (Hartung, Blattner, and Puchta 2002). The present uncertainty over the relative rates of intron gain versus loss mandates thorough taxonomic coverage of an intron distribution before it is used as evidence of a unique insertion event, a requirement we believe we have fulfilled in this case.
We cannot as of yet precisely determine the origination date of this intron insertion. The fossil representation of Colletidae is limited to two species from early Miocene Dominican amber, which may be relatively derived taxa (Michener and Poinar 1996; Engel 1999). Date estimates for the origin of bees place an upper bound at approximately 140 to 120 MYA (Grimaldi 1999; Engel 2001). Given this poor fossil representation and the currently poor phylogenetic resolution of bee families and subfamilies, a more precise age estimate for the origin of this intron will have to await future application of fossil-calibrated molecular dating techniques (e.g., Sanderson 2002; Thorne and Kishino 2002) on a comprehensive, robust bee phylogeny.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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