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MBE Advance Access originally published online on December 5, 2006
Molecular Biology and Evolution 2007 24(3):632-639; doi:10.1093/molbev/msl192
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© The Author 2006. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Research Articles

Bats with hATs: Evidence for Recent DNA Transposon Activity in Genus Myotis

David A. Ray*, Heidi J. T. Pagan*, Michelle L. Thompson* and Richard D. Stevens{dagger}

* Department of Biology, West Virginia University
{dagger} Department of Biological Sciences, Louisiana State University

E-mail: david.ray{at}mail.wvu.edu.


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Transposable elements make up a significant fraction of many eukaryotic genomes. Although both classes of transposable elements, the DNA transposons and the retrotransposons, show substantial expansion in plants and invertebrates, the DNA transposons are thought to have become inactive in mammalian genomes long ago. Here, we report the first evidence for recent activity of DNA transposons in a mammalian lineage, the bat genus Myotis. Six recently active families of nonautonomous hobo/Activator/TAM transposons were identified in the Myotis lucifugus genome using computational tools. Low sequence divergence among the individual sequences and between individual sequences and their respective consensus sequences suggest their recent expansion in the M. lucifugus genome. Furthermore, amplification and sequencing of polymorphic insertion loci in a related taxon, M. austroriparius, confirms their recent activity. Myotis is one of the largest mammalian genera with 103 species. The discovery of DNA transposon activity in this genus may therefore influence our understanding of genome evolution and diversification in bats and in mammals in general. Furthermore, the identification of a likely autonomous element may lead to new approaches for mammalian genetic manipulation.

Key Words: mobile element • hAT • transposon • Chiroptera • Myotis


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
With 103 species and a nearly worldwide distribution, genus Myotis is a very successful group of mammals whose diversification is notably extensive. The genus appears to have been radiating for at least the last 30 Myr (Quinet 1965Go) with a majority of the diversification occurring since the late Miocene/early Pliocene, 5–6 MYA (Savage and Russel 1983Go; Ariagno 1984Go). The most recent radiation has given Myotis a nearly global distribution (being absent only from polar regions and some oceanic islands) and has included considerable ecomorphological diversification (Nowak 1991Go). For example, New World Myotis appear to have been radiating for approximately the last 4.6 Myr (2001) and accumulation of diversity within the New World clade has not been random. Rather, a number of New World forms have converged to assume very similar morphologies as forms found in the Old World (Ruedi and Mayer 2001Go). Thus, it appears that Myotis represents a relatively plastic group of species that can readily adapt to a variety of habitats. Such morphological plasticity has made phylogenetic inferences on this genus difficult (Findley 1972Go; Ruedi and Mayer 2001Go; Kawai et al. 2002Go; Hoofer and Van Den Bussche 2003Go). The inclusion of information on mobile element activity and diversity in this taxon may contribute greatly to our understanding of evolutionary relationships and processes. For example, genomic instability due to the presence of mobile element activity may represent one way to induce the genomic variation needed for the type of rapid diversification observed in this genus of bats (Kidwell and Lisch 1997Go, 2001Go).

Transposable elements, comprising both Class I retrotransposons and Class II DNA transposons, represent a substantial portion of many eukaryotic genomes. For example, ~50% of the human genome is thought to be derived from transposable element sequences (Lander et al. 2001Go). Other genomes, especially in plants, may consist of substantially higher proportions of transposable element–derived DNA (SanMiguel and Bennetzen 1998Go; Bennetzen 2000Go). In addition to these observations on genomic content, the activity of transposons and retrotransposons can substantially alter genome structure, produce novel exon combinations, and affect gene expression (reviewed in Kidwell and Lisch 2001Go). It is clear therefore that the study of these elements is important to our understanding of genomic structure and function. The focus of this study is the Class II transposable elements, the DNA transposons, and their derivatives. Although they are common and active in bacteria and many eukaryotes (Kempken and Windhofer 2001Go), the analysis of sequenced mammalian genomes suggests that Class II elements effectively became extinct long ago in mammalian lineages (Lander et al. 2001Go; Waterston et al. 2002Go).

Here, we report the discovery of 6 families of nonautonomous DNA transposons in the Myotis lucifugus genome. Remarkably, these families appear to have been active in the genome in the recent past and are likely still active today. This is illustrated by minimal observed differences among individual insertions and the consensus sequence and by the existence of polymorphic insertion sites in a closely related taxon, M. austroriparius. The discovery of an active transposon family in a mammalian lineage represents an important advance on several levels. First, the instability introduced into the Myotis genome by an active group of transposons has implications for our understanding of the evolutionary processes underlying the rapid diversification of this group. Furthermore, transposons are efficient vectors for the introduction of foreign DNA into cells. Low DNA transposon activity in mammalian cells, however, has been a stumbling block to their utilization in mammalian systems. The discovery of an active nonautonomous DNA transposon family in a mammalian lineage suggests the presence of an active autonomous partner. Identification of that partner could be an important step forward in increasing our ability to experimentally manipulate mammalian genomes.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
An initial survey for recently active mobile element families was performed by obtaining genomic sequence data for 4 chiropteran taxa from the National Institutes of Health (NIH) Comparative Vertebrate Sequencing database (http://www.nisc.nih.gov/). Data were obtained for Target 1 (human homolog—chr7:115404472–117281897) from the following taxa: Artibeus jamaicensis (DP000001 [GenBank] ; 584,809 bp), Carollia perspicillata (DP000018 [GenBank] ; 1,501,503 bp), Rhinolophus ferrumequinum (DP000028 [GenBank] ; 1,706,389 bp), and M. lucifugus (AC174829 [GenBank] , AC174830 [GenBank] , AC174831 [GenBank] , AC174832 [GenBank] , AC181995 [GenBank] , AC181998 [GenBank] , AC182002 [GenBank] , AC182003 [GenBank] , AC182004 [GenBank] , AC182774 [GenBank] , AC183324 [GenBank] , and AC184715 [GenBank] ; 2,041,025 bp). For all taxa except M. lucifugus, the data were available as complete genomic scaffolds. For M. lucifugus, data were obtained from the individual bacterial artificial chromosome (BAC) clones.

For each taxon, an initial scan for recently active mobile element families was performed using PILER (Edgar and Myers 2005Go). Minimum length for discovered repetitive families was set to 100 bp and percentage identity was set to 90. The output from PILER was organized into families (all sequences with 90% and higher similarity) and superfamilies (sequences from 2 or more families that exhibited sequence similarity). Each superfamily and family alignment was given a numerical designation (table 1). Superfamily and/or family consensus sequences were subjected to CENSOR (Jurka et al. 2005Go) searches to determine similarity to known repetitive elements.


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  		Table 1. Results of Our Initial PILER Analysis Using NIH Target 1 Sequence Data

 
Based on the results of this analysis, a more extensive analysis of M. lucifugus sequences comprising ~7.2 Mb of BAC-derived genomic sequences was conducted (AC174829 [GenBank] , AC174830 [GenBank] , AC174831 [GenBank] , AC174832 [GenBank] , AC181995 [GenBank] , AC181998 [GenBank] , AC182002 [GenBank] , AC182003 [GenBank] , AC182004 [GenBank] , AC182774 [GenBank] , AC183324 [GenBank] , AC184715 [GenBank] , AC175782 [GenBank] , AC175783 [GenBank] , AC181992 [GenBank] , AC181996 [GenBank] , AC182772 [GenBank] , AC183326 [GenBank] , AC175607 [GenBank] , AC175608 [GenBank] , AC181991 [GenBank] , AC181994 [GenBank] , AC181997 [GenBank] , AC182001 [GenBank] , AC183841 [GenBank] , AC175784 [GenBank] , AC175785 [GenBank] , AC181993 [GenBank] , AC181999 [GenBank] , AC183325 [GenBank] , AC186243 [GenBank] , AC182000 [GenBank] , AC182325 [GenBank] , AC182330 [GenBank] , AC186242 [GenBank] , AC182326 [GenBank] , AC182327 [GenBank] , AC182328 [GenBank] , AC182329 [GenBank] , AC182510 [GenBank] , and AC182773 [GenBank] ). For these data, parameters for the PILER analysis were refined to identify elements with sequence identity of 95% or higher.

Consensus sequences for each of the recovered transposon-like families (now referred to as Myotis-nhAT) were used to create a custom library for a local installation of RepeatMasker (Smit AFA, Hubley R, Green P, RepeatMasker at http://repeatmasker.org). Target 1 scaffolds from Artibeus, Carollia, and Rhinolophus were subjected to RepeatMasker analyses using this library to determine if members of the DNA transposon-like families were present. Also, as an independent estimate of the distribution and number of Myotis-nhAT elements in the genome, we subjected ~274.9 Mb of sequence (100,000 contigs with an average length of 2,749 bp; AAPE01000001–AAPE01100000) from the M. lucifugus–sequencing project to the same RepeatMasker analysis.

For analysis of population level variation at individual Myotis-nhAT loci, 10 individuals of M. austroriparius were examined. All animals were collected by mist netting over water sources in and around the Tunica Hills Wildlife Management Area located in West Feliciana Parish, Louisiana. Bats were collected between 11 March 2006 and 24 May 2006. Shortly after capture, bats were euthanized and frozen (–80 °C). Specimens were thawed and tissues (muscle, heart, liver, and kidney) were removed. Voucher specimens and tissues can be obtained by contacting the authors. DNA was isolated from organ tissue using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI).

We used Primer3 (Rozen and Skaletsky 1998Go) to design oligonucleotide primers for 27 of the suspected recent integrations (supplementary table 1, Supplementary Material online) and used them to amplify the loci in a panel of 10 DNA templates from M. austroriparius. Twenty-five–microliter polymerase chain reaction amplifications were performed under the following conditions: 10–50 ng template DNA, 7 pM of each oligonucleotide primer, 200 mM dNTPs, in 50 mM KCl, 10 mM Tris–HCl (pH 8.4), 2.0 mM MgCl2, and Taq DNA polymerase (1.25 units). An initial denaturation at 94 °C for 2 min was followed by 30–32 cycles of 94 °C for 15 s, the appropriate annealing temperature for 15 s, and 72 °C for 1 min and 10 s. A final incubation at 72 °C for 5 min prepared the fragments for potential cloning.

Six loci exhibited patterns that suggested polymorphism with regard to Myotis-nhAT presence/absence. Successful amplicons of representative filled (containing the transposon integration) and empty (without the transposon integration) loci were cloned using the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA), and inserts were sequenced using chain termination sequencing on an ABI 3130xl Genetic Analyzer. Sequences were aligned with the matching computationally derived locus from M. lucifugus. All alignments are available as supplementary data and at http://www.as.wvu.edu/~dray/pubs.html. All sequences generated for this manuscript have been deposited in GenBank under accession numbers DQ906144–DQ906155.

We also attempted to identify any candidates for the autonomous partner of these elements using the available shotgun sequences from M. lucifugus by creating a custom Blast database consisting of all available sequences from that project (GenBank accession numbers AAPE01000001–AAPE01640786, ~1.76 x 109 bases). The consensus sequences from each suggested autonomous transposon match (hobo/Activator/TAM [hAT]-4_XT, hAT1_MD, and hAT2_MD) from our original search (table 1) were used to query that database.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Supplementary Material
 Acknowledgements
 References
 
Results of our initial analyses of 4 chiropteran taxa (A. jamaicensis, C. perspicillata, R. ferrumequinum, and M. lucifugus) using Target 1 data from the NIH Comparative Vertebrate Sequencing database are presented in table 1. It was clear from the data that M. lucifugus presented by far the largest proportion of potential repetitive families, 89 sequences in 11 families. However, because of the potential for overlapping BAC clones and thus multiple examples of the same insertion being recovered, we examined both of the flanking sequences of each element for similarity with the flanking sequences of all other elements. Pairs with one or both flanking sequences exhibiting 95% identity or higher were assumed to be multiple instances of the same element, and one from each pair was removed from further analysis. Characterization of the remaining 84 elements using CENSOR led to the observation that within the ~2 Mb of sequence representing Target 1, there were 56 instances of elements with similarity to known DNA transposons spanning a range of families. Analyses of the same target in the 3 other chiropteran taxa revealed no comparable numbers of DNA transposon-like sequences.

During the subsequent analysis of 7.2 Mb of BAC-derived sequence for M. lucifugus, we identified a total of 111 elements with similarity to known DNA transposons. Multiple instances of the same element were removed using the methods described above. The remaining elements were subdivided among 6 groups based on sequence similarity. These groups have been designated Myotis-nhAT1–6. Two groups, Myotis-nhAT4 and 5, consisted of 2 subfamilies each based on sequence differences and indels. As indicated by the names, details suggest that all of the elements described are nonautonomous members of the hAT superfamily of transposons. This is supported by the observation that all of the families described share 2 distinguishing features of the superfamily. First, hAT transposons usually exhibit 8 bp target site duplications (TSD) and short (5–27 bp) terminal inverted repeats (TIR) (Kempken and Windhofer 2001Go). Each of the 6 families of Myotis-nhAT elements exhibits TIRs of 16 bp (table 2). These TIRs match well with the consensus TIRs for hAT elements described by Rubin et al. (2001)Go. Second, TSDs were identifiable for nearly all of the Myotis-nhAT elements, and consensus TSD sequences are available in table 2. The TSDs are typically 8 bp long and almost invariably contain a central TA dinucleotide (fig. 1 and table 2). Furthermore, as described in table 3, all of the Myotis-nhAT consensus sequences described here exhibit some degree of sequence similarity with known DNA transposons.


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  		Table 2. Descriptive Information for Each Myotis-nhAT Family Recovered

 

Figure 1
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  		FIG. 1.— Multiple sequence alignment of 10 randomly selected Myotis-nhAT1 elements recovered from a search of 7.2 Mb of Myotis lucifugus genomic sequence data using PILER. Twenty-two bases of flanking sequence are also included. Shaded regions indicate the 8-bp TSDs typical of hAT transposons. The central TA dinucleotide within the TSD sequences is also indicated (**).

 

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  		Table 3. Best CENSOR Matches of Myotis-nhAT Elements to Known Transposon Sequences

 
Two of the Myotis-nhAT families exhibited very high degrees of sequence similarity to previously described elements. For example, over portions of their length the Myotis-nhAT 4 and 5 subfamilies are essentially identical to the URR1, URR1B, and MarinerNA6A transposon families. One might argue that elements exhibiting high sequence similarity to already described elements should properly be referred to using those names. However, in each case the elements recovered from Myotis all share unique indels (see supplementary Myotis nhAT family alignments, Supplementary Material online) that distinguish them from the previously described elements. We argue that the taxonomic distribution and the unique sequence characteristics, whether as indels or nucleotide differences, merit their distinction as separate families.

On a secondary nomenclatural note, the Repbase entries for both URR1 and MarinerNA6A extend past the ends of the Myotis-nhAT elements by 5 bases. The final 2 bases on both ends are TA in each case. These were interpreted by their respective discoverers as TA direct repeats, a common feature of DNA transposons. We suggest instead that these 5-bp overhangs might represent incomplete and unrecognized 8-bp TSDs. If that is the case, we suggest their reclassification as hAT elements.

Calculations based on the ~274.9 Mb that were subjected to a custom RepeatMasker analysis suggest an average density of 1 Myotis-nhAT insertion every 14.3 kb. The size of the M. lucifugus genome is unknown. However, several estimates for other Myotis species are available at http://www.genomesize.com. Using the average genome size values for Myotis available, we can presume a size of ~2.4 Gb. Thus, the total number of Myotis-nhAT elements in the M. lucifugus genome may be in the neighborhood of 168,000, occupying ~1.3% of its total sequence (table 2). Of course, all of these calculations are based on the assumption that the contigs used are representative of the overall composition of the M. lucifugus genome and that our estimate of the genome's size is correct. Thus, they should be taken only as preliminary estimates.

The presence of multiple insertions with only minimal divergence from each other and from the consensus suggested that genomes of Myotis may have experienced a recent burst of DNA transposon activity. Myotis-nhAT elements from the 6 families described fit this criterion based on genetic distance estimates of the members discovered versus their respective consensus sequence (1.4–3.3%; table 2). Based on the estimated neutral mutation rate for mammals (2.2 x 10–9 [Kumar and Subramanian 2002Go]), the average age for these transposon families is between 6.4 and 15 Myr. This observation supports the idea that these elements arose in and are unique to genus Myotis.

To further test the assumption of recent activity, we hypothesized that very recent integrations, those with minimal divergence from the consensus sequence, might still be polymorphic in populations of M. lucifugus and/or related taxa. Ten DNA samples from a related taxon, M. austroriparius (divergence <6 Myr), were tested using primers for specific Myotis-nhAT loci. Samples from M. lucifugus would have been preferred but were unavailable. Of the primer pairs tested, 6 showed evidence of polymorphism for presence and/or absence among the 10 M. austroriparius samples (fig. 2). Sequence analysis of the filled and empty sites clearly demonstrates the absence of the Myotis-nhAT sequence at each locus along with only one copy of the presumed TSD (fig. 3; supplementary fig. 1, Supplementary Material online).


Figure 2
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  		FIG. 2.— Agarose gel chromatographs illustrating the 6 polymorphic Myotis-nhAT insertion sites in Myotis austroriparius. Numbers across the top are identifiers for individual specimens. Fragment sizes for filled and empty sites are indicated.

 

Figure 3
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  		FIG. 3.— Representative sequence alignment for 1 polymorphic Myotis-nhAT insertion site. The top sequence is the original sequence obtained from BAC-derived Myotis lucifugus genomic sequence. The bottom 2 sequences correspond to filled and empty sites pictured in figure 1. The underlined segments are TSDs, and the boxed sequences represent TIR. Dots represent identical bases, and dashes indicate indels.

 
A Blast search of M. lucifugus genomic sequences yielded 7 hits with high sequence similarity to hAT1_MD, a putative autonomous DNA transposon from Monodelphis domestica. No sequences with significant similarity to other queried DNA transposons were obtained. An alignment of the sequences recovered by the Blast search, including a consensus approximated from them and 1 Myotis-nhAT sequence, is presented as supplementary figure 2 (Supplementary Material online). One of the hits encompassed what appears to be a full-length element with very similar TIRs (5'-CAGTGATGGMGAACCT-3') to all of the Myotis-nhAT families (table 2) and easily recognizable 8-bp TSDs (5'-ACCTAGGG-3'). However, 3 single nucleotide indels (at positions 215, 884, and 1,741) produce frameshifts that introduce early stop codons (supplementary fig. 3, Supplementary Material online). The consensus sequence generated from all 7 hits, however, encodes a presumably functional transposase identical in size to hAT1_MD, 643 amino acids. We have named this potentially active autonomous hAT transposon Myotis-hAT1. Given the similarity in TSD sequences and TIR composition, it is likely that each Myotis-nhAT family described above utilizes the Myotis-hAT1–encoded transposase.


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Supplementary Material
 Acknowledgements
 References
 
The observations presented here suggest that much care should be taken when extrapolating results gathered from only a few model organisms to an entire taxonomic group and illustrate the value of analyzing the genomes of a broad range of mammalian species. Recent DNA transposon activity in a mammalian lineage and the identification of a potentially active DNA transposon sequence in the genome of M. lucifugus was unexpected given the lack of observed transposase activity in mammals in general and in the 2 most well studied mammals, human and mouse (Lander et al. 2001Go; Waterston et al. 2002Go)—an observation that has disappointed researchers interested in using transposons as tools for genetic manipulation in mammals (Koga et al. 2003Go). Recent work with DNA transposons from other taxa, including Sleeping Beauty, piggyBac, and Tol2, has given hope for the potential to use transposons as tools for such manipulation (Geurts et al. 2003Go; Koga et al. 2003Go; Bestor 2005Go; Ding et al. 2005Go; Dupuy et al. 2005Go). Although these are significant advances, the discovery of an active mammalian transposase could provide an alternative to these systems. We hope that the description here of the potentially active Myotis-hAT1 element will revitalize the search for mammalian transposases.

The high degree of sequence similarity among some Myotis-nhAT elements and elements in very divergent taxa raises questions regarding their origin. It is unlikely that elements with such highly similar sequences in such widely divergent taxa arose by chance alone. We believe the most likely explanation is horizontal transfer. DNA transposons are thought to be more prone to horizontal transfer than the retrotransposons (Kidwell 1992Go). We suggest the relatively recent (at least 15 MYA, see Results) introduction of an autonomous element, Myotis-hAT1, to the ancestral Myotis genome via horizontal transfer from some unknown source. After the incorporation of Myotis-hAT1 into the genome, nonautonomous versions arose via mechanisms that remain unclear (Hua-Van et al. 2005Go).

The estimated divergence values of the subfamilies described in table 2 are notably uniform. However, given the presumed mode of expansion for these elements, it is not unexpected. The relationship between age of a subfamily and the divergence of its members from the consensus is essentially linear when dealing with retrotransposons because they follow a modified master gene model of expansion. Following this model, only one or a few copies of a given subfamily are active at any given time, and the copies are generated from single or very few active master genes (Cordaux et al. 2004Go). Subfamilies of retrotransposons also have a wide range of ages depending on the period during which the various subfamilies were active. As an example, RepeatMasking Target 1 from the human genome yields raw percentage–divergence values ranging from 0.0% to 24.6% for Alu elements (a primate retrotransposon)—presumably spanning the range of potential ages from de novo insertions to insertions dating back to the origin of Alu elements ~60 MYA.

When dealing with DNA transposon families, however, the pattern would necessarily differ because they follow a different model of mobilization. As long as the transposase is active and as long as there are TIRs for the transposase to recognize and mobilize, many copies can be "active" and many loci can act as sources. Thus, the relationship that exists for retrotransposons between age and divergence from the consensus sequence likely does not apply to these elements. Instead, we would be more likely to find a uniform set of ages for each of these families given that the source of their transposition activity is the same, the autonomous transposon responsible for the mobilization of the entire group. In other words, unlike retrotransposons all of the nonautonomous families will likely be active or inactive at any given time depending on the state of their autonomous partner.

The detection of multiple nearly identical instances of these nonautonomous DNA transposons in M. lucifugus is strong evidence for their recent activity in that genome. Observing instances of insertion polymorphism adds experimental evidence to further support this hypothesis. Of course, we would have preferred to test for insertion polymorphism in a panel of taxa from that species, and it is unfortunate that we did not have access to such samples. However, the detection of polymorphic nonautonomous DNA transposon insertion sites in a panel of 10 M. austroriparius using loci first identified in M. lucifugus DNA sequence is interesting at several levels. First, such results clearly indicate the recent incorporation of Myotis-nhAT transposons at these loci in a common ancestor of M. lucifugus and M. austroriparius. The observation that some loci are heterozygous also supports the hypothesis of recent activity. No divergence estimates for M. lucifugus and M. austroriparius are available in the literature. However, given that both are New World taxa and an assumption of monophyly for New World Myotis (Ruedi and Mayer 2001Go; Hoofer and Van Den Bussche 2003Go), it is likely that they shared a common ancestor no later than the late Miocene (5–6 MYA) (Ruedi and Mayer 2001Go). Their polymorphic status in M. austroriparius indicates that the alleles are undergoing a period of lineage sorting. This is common among species that are experiencing or have experienced a rapid burst of speciation. Such insertions have been observed previously when short interspersed element (SINE) loci were examined in cichlids and charr (Hamada et al. 1998Go; Takahashi et al. 2001Go). The recent radiation suggests the possibility for shared polymorphisms among taxa, and the observation that in as few as 4.6 Myr a large portion of the ~103 species have emerged clearly makes Myotis spp. candidates for observing lineage sorting in action.

Given the transposition mechanism of DNA transposons, it is possible that the observed Myotis-nhAT polymorphisms could have a different source—excision of the elements via transposase activity. Although this is possible, we think it is unlikely given the clear absence of any telltale signatures associated with the removal of a transposon. If the elements had undergone a precise "cut and paste" mobilization, we would expect to at least see the presence of 2 TSD sequences in the empty sites. Furthermore, the typical footprint in cases of excision is a few basepairs of the transposon. The lack of any of these telltale signs strongly suggests that these empty sites were never occupied by the transposon and thus the polymorphisms result from insertion rather than excision events. Even if excision were the case, it would serve as additional evidence of the activity of the presumed autonomous element. Another possibility that must be addressed is illegitimate recombination. We do not believe that this process is playing a major role in our observations given the easily identifiable TSDs for most loci.

It is also possible that the polymorphisms we have observed are actually the result of paralogous insertions. This scenario would require the presence of duplicated regions of the M. austroriparius genome, an occurrence that is not uncommon in some mammals (Cheung et al. 2003Go; Tuzun et al. 2004Go; Cheng et al. 2005Go). One copy of the duplicated region would have to have been the site of an insertion, whereas the other remained clear. In such cases, recent activity of the Myotis-nhAT transposons is still indicated given that the paralogous insertions are not fixed in the population and are heterozygous in some individuals.

The discovery of this activity in a taxonomic group that has undergone a recent diversification suggests a possible model system to study the effects of mobile element activity on genomic diversification and speciation. Future work will be needed in several areas. First, we will need to determine the taxonomic limits of the transposon activity. Is the observed activity truly limited to Myotis? Is it limited to only the North American Myotis? Is it common for species-rich mammalian genera to have transposon activity? Four hAT2-like elements and 2 unidentified repetitive families were recovered from the R. ferrumequinum genome during the initial analysis. Rhinolophus is also a relatively species-rich genus with 77 taxa. Although it is possible that this taxon also experienced a burst of transposable element activity, they have apparently not been as active as in Myotis given the substantially smaller number of elements recovered. Alternatively, a burst of activity in Rhinolophus may have occurred in the more distant past. If that were the case, our search for very recent elements using PILER would likely not have identified them.

In conclusion, we have demonstrated that nonautonomous members of the hAT transposon superfamily have recently been active in the genomes of at least 2 bat species from genus Myotis. The presumed autonomous partner was also identified and experiments are currently being designed to determine if mobilization can be observed in mammalian cells. These observations represent the first suggestion of an active DNA transposon in a mammalian lineage, and the future utilization of the full-length autonomous element may be a stepping stone to an increased ability to manipulate mammalian genomes. The burst of activity may have influenced the ability of Myotis genomes to adapt to a wide variety of habitats and feeding strategies through its effects on genome structure and by consequence phenotypic change.


   Supplementary Material
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
Acknowledgements
 References
 
Supplementary table 1 and figures 1–3 and the sequences of the transposon loci, both filled and empty sites that have been assigned GenBank accession numbers (DQ906144–DQ906155), are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).


   Acknowledgements
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
References
 
This work was supported by the Eberly College of Arts and Sciences at West Virginia University (D.A.R.). R.D.S. was supported by National Science Foundation (Grant #DEB 0535939). Drs S. DiFazio and J. Xing and an anonymous reviewer contributed valuable comments to earlier drafts.


   Footnotes
 
Adriana Briscoe, Associate Editor


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 

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Accepted for publication November 29, 2006.


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