BACKGROUND
the tasmanian devil  is the largest living carnivorous australian marsupial in the order dasyuromorphia, which also includes the anteating numbat, quolls, dunnarts, phascogales, and the iconic extinct tasmanian tiger   <cit> .
populations of the tasmanian devil are currently under serious threat of being severely diminished or eradicated by devil facial tumor disease , a contagious form of cancer  <cit> .
since it was first observed in  <dig>  dftd has nearly halved the tasmanian devil population  <cit> .
without a cure for dftd, tasmanian devils may become extinct in 10– <dig> years  <cit> .
however, with the help of next generation sequencing techniques, finding a possible cure may be within reach  <cit> .
to gain a better understanding of the genetics of the disease and possibly find a cure, the genome of the tasmanian devil was recently sequenced  <cit> .
recent studies have shown that marsupial genomes have been under massive bombardment by transposable elements  <cit> , which play important roles in genomic evolution  <cit>  and are the causes of some diseases  <cit> .
in the opossum, 52% of the genome sequence consists of such elements.
this is the largest percentage of transposable elements presently identified in any vertebrate genome.
in comparison, the human genome consists of 45% and the mouse genome of 38% transposable elements  <cit> .
transposable elements are classified as retroposons, which propagate via an rna-intermediate, and dna transposons that transpose themselves directly by dna, without an intermediate  <cit> .
typically, transposons make up only a small percentage of genome sequences, while the vast majority of the transposable elements are retroposons  <cit> .
one group of retroposons, the non-autonomous sines , is propagated in the genome via the activity of the autonomous lines   <cit>  <cit> .
most sines originate from trnas and their basic structure includes a 5′ trna-related sequence, a trna unrelated body, a 3′ line-related tail sequence that sometimes is followed by a poly- stretch  <cit> .
the 3′ tail sequence is derived from lines and is used for recognition by the reverse transcriptase.
the marsupial sines have been shown to have tail sequences originating from line <dig>  line <dig> as well as rte elements  <cit> .
when a polya stretch is present in the 3′ end of the sine it is used by line <dig> for retroposition  <cit> .
a group of highly conserved sines in mammals are the core-sines.
core-sines have a specific and highly conserved 65‒nt central sequence between the trna-related part and the line derived region  <cit> .
core-sines have been found in diverse groups of animals, such as mammals, mollusks and fish  <cit> .
wsine <dig>  a characteristic sine of marsupials, is a short core-sine, consisting only of the trna-related part and a truncated 41-nt core sequence followed by a polytail  <cit> .
in placental mammals, the core-sines have been inactive fossils for at least  <dig> million years   <cit> .
however, this retroposon type has remained active in marsupial and monotreme genomes.
in monotreme genomes, the mon core-sine dominates  <cit> , and in marsupials, the core-sines have remained active and have proliferated, giving rise to many different subtypes  <cit> .
while most placental mammalian orders are currently represented by at least one genome sequence, genomic sequences of only three marsupial species, representing three of the seven orders, are currently known: the south american opossum   <cit> , the australian wallaby   <cit> , and the tasmanian devil.
molecular data show that the respective orders of the first two, didelphimorphia  and diprotodontia , are separated by 75– <dig> myr of evolution  <cit> , representing the geographically and temporally deepest divergence among marsupials.
the retroposon content in the opossum genome has been thoroughly investigated  <cit> , and here we have screened the genome of an australian marsupial to understand the dynamics of sines and non-autonomous lines.
the tasmanian devil genome sequence will be a valuable source for understanding the dynamics of retroposons in marsupial genomes.
RESULTS
full genome screening
a summary of the abundances of all transposable elements revealed in the in silico screening of sarcophilus harrisii is shown in additional file 1: table s <dig>  the most abundant marsupial specific sines and non-autonomous lines were calculated .
* a putative pseudogene
transposition in transposition
for comparative genomics and in particular evolutionary purposes, it is useful to be able to estimate the relative order of activities of different groups of integrated sines found in the genome.
tint  is an algorithm that screens the genome for nested retroposon insertions  and calculates its relative time frame of activity based on the fact that old inactive retroposons cannot insert into younger active retroposons  <cit> .
screening of the tasmanian devil genome with the tint program  <cit>  revealed  <dig>  nested insertions for the most frequent sines and non-autonomous lines  .
the most recently active element is the wsine <dig>  the tint pattern from the tasmanian devil is reasonably similar to those previously observed in the opossum and wallaby  <cit> .
wallsi elements
several wallsis  have been described in the wallaby genome  <cit> .
three types, the wallsi1/1a, wallsi <dig>  and wallsi <dig>  have highly similar tail regions derived from rte_md .
wallsi <dig> and wallsi <dig> are both core-sines.
except for wallsi <dig>  we found all previously identified wallsis in the tasmanian devil genome but in different proportions than previously described .
there are fewer copies of the 5′-truncated wallsi <dig> in the tasmanian devil   than in the wallaby  .
by contrast, the 200-nt-longer wallsi1a is present in  <dig>  copies  in the tasmanian devil genome, which is more than the  <dig>   copies in the current assembly of the wallaby genome, which might be an underestimate given the low coverage of the genome sequence.
on the other hand wallsi <dig> is present in relatively similar numbers in both genomes  , which might indicate that the activities of the two elements terminated before the two orders split 55– <dig> myr ago and the sines were passed on to the descendants.
the wallsi <dig> is more abundant in the wallaby genome  despite lower sequence coverage of the assembled genome suggesting an expansion of the element.
we did not identify any copies of wallsi <dig> in the tasmanian devil genome by the in-silico approach.
wallsi <dig> was initially believed to be restricted to kangaroos ; however, they have also been identified in other diprotodontian marsupials  <cit> , indicating that the element is not restricted to the kangaroo subfamily macropodidae, but rather is characteristic of the entire order diprotodontia.
psl7_md
another interesting feature of the marsupial genome is the p7sl_md retropseudogene  <cit> .
the cytoplasmic 7sl rna is part of a signal recognition particle, and around  <dig> myr ago it formed the alu element, which is the most abundant sine in primates  <cit>  and other related sines  in the super-order euarchontoglires  <cit> .
however, alus are imperfect dimers derived from the 7sl sequence.
in marsupials, the p7sl_md retropseudogene sequence is very similar to the 7sl rna.
it is present in nearly identical copy numbers in the genomes of opossum  <cit> , wallaby  <cit> , and tasmanian devil, where we discovered more than  <dig>  copies.
the high abundance may indicate that this pseudogene has undergone an extreme expansion in the ancient marsupial genome that has not been observed in any other mammal.
wsine1
the wsine <dig> family is the most abundant of the recently active sines in the tasmanian devil.
we identified approximately  <dig>  wsine <dig> elements, but even this may be an underestimate, as we also found nearly  <dig>  copies of fragmented core-sines that may represent ancient and diverged copies of wsine <dig>  due to the difficulty of correctly assigning these fragmented sines, they have not been included in the final estimates.
we also identified a third, novel subtype of wsine <dig> in the tasmanian devil genome that deviates from the previously described wsine1s, wsine <dig> and wsine1a  <cit> .
this new subtype, wsine1b, lacks the 11-nt insert near the poly-tail  found in wsine1a elements.
wsine1a and wsine1b both have a 7-nt insert at position  <dig>  suggesting a close relationship.
a detailed indel analysis of all wsine <dig> elements shows that there are no other commonly occurring insertions or deletions .
all three types of wsine <dig> are present in the genome of the tasmanian devil, but in different proportions.
nearly 90% belong to the new wsine1b subtype, while the wsine1a elements represent only  <dig> % of all wsine1s .
the values for tasmanian devil are based on the analysis of the full genome, isoodon on the available trace sequences , and macropus on a screening of the complete genome.
the abundances of the three variants are expressed as % with the copy number in parenthesis.
only the complete wsine1s  were used in the analysis, as many are fragmented and lack the regions needed to safely define the subfamily.
wsine <dig> distribution in australian marsupial orders
to examine the evolutionary dynamics of wsine <dig> we also investigated the distributions of the wsine <dig> subtypes in the genomes of two other marsupial orders, the peramelemorphia  and diprotodontia .
the screened trace sequences of isoodon show a pattern similar to that of the tasmanian devil, with the majority of wsines belonging to the wsine1b subtype.
by contrast, in the wallaby genome the most abundant wsine is the wsine <dig> , though wsine1b is still widespread, making up almost 20% of the wsine copies .
the age and number of the wsine <dig> subfamilies
co-segregation analysis  of  <dig>  full-length wsine <dig> elements identified  <dig> subfamilies in the tasmanian devil genome, with copy numbers ranging from  <dig> to  <dig> .
the evolutionary age of each subfamily was determined using a local nucleotide substitution rate derived from wsine <dig> .
the oldest elements were estimated to be  <dig> myr old and the youngest  <dig> myr old .
it is evident that a burst of new subfamilies appeared around 56– <dig> myr ago.
of these, only a handful continued to be active and to give rise to additional master copies.
more than  <dig> new subfamilies were created during a very short time span.
the burst of additional subfamilies occurred around the same time as the early evolutionary phases in the order dasyuromorphia .
phylogenetic relationships and divergence times of the tasmanian devil
we conducted mitochondrial genomic analyses to reconstruct the phylogenomic relationships of the tasmanian devil to other carnivorous marsupials and to estimate the divergence times of this order.
this allowed us to correlate the evolutionary age of dasyuromorphia and place their early divergences in relation to the emergence of different wsine subfamilies.
the mitogenomic analysis identified the tasmanian devil to be the sister group to the quolls .
however, only after including third codon positions, in the nucleotide analysis could the tasmanian devil be placed outside dasyurus, indicating a rapid divergence of these lineages.
as third codon positions are the fastest evolving nucleotide position only these seem to have acquired enough information during the short interval between the groups  to resolve this relationship.
it was equally difficult to estimate the relationships among the three subfamilies thylacinidae, myrmecobiidae, and dasyuridae, as sh- and au-tests did not reject alternative hypotheses, which is consistent with a rapid speciation process .
the origin of the tasmanian devil is estimated by both sets of calibration points to be 12– <dig> myr ago .
the split within dasyurus is estimated to be  <dig> myr, merely  <dig> to  <dig> million years after the origin of the genus sarcophilus .
the deepest split within dasyuridae is estimated to be  <dig> myr .
the origin of dasyuromorphia is estimated to be  <dig> myr, with a second split around  <dig> myr leading to numbat .
discussion
tasmanian devil genome-composition and activity of retroposons
in silico screening showed that transposable elements account for about  <dig> % of the tasmanian devil genome sequence , which is virtually identical to that found in the opossum  and wallaby genome   <cit> .
the sines and non-autonomous lines make up  <dig> % of the tasmanian devil sequence , which is comparable to the percentages identified in the genomes of the opossum  and wallaby   <cit> .
the relative temporal activity of sines was studied using the tint program.
when a younger retroposon integrates into another older retroposon, it is possible to estimate relative times of activity based on the information from these nested retroposition events  <cit> .
as retroposons tend to be active in distinct waves, the temporal chain of activities can be calculated based on thousands of nested retroposition events found in the genome.
the tint method is a valuable tool for screening genomic data for phylogenetically informative retroposons, as the search can be targeted towards younger or older branches of the tree depending on the choice of elements.
the method has been successfully applied to different groups of animals, such as birds  <cit> , lagomorphs  <cit> , primates  <cit> , platypus  <cit> , and marsupials  <cit> .
using the tint approach, different sines and non-autonomous lines were investigated in the tasmanian devil genome.
several of them  with relatively similar activity patterns .
based on the tint analysis, the element that had a recent expansion in the tasmanian devil genome is the core-sine of the type wsine <dig>
wsine1
the wsine1s are more abundant in the tasmanian devil genome than in the wallaby genome  and had the most recent expansion of all sines .
the wsine <dig> is a short element, consisting of a trna-derived region, a core-sequence region, and a poly‒tail.
the core-sequence region is extraordinarily conserved across mammals, which has led to speculations concerning functional properties of this region  <cit> .
additionally, the element has a poly-tail and is flanked by target site duplications and is therefore expected to be line <dig> propagated  <cit> .
in many of the marsupial core-sines the core-region is  <dig> nt long.
in placental mammalian core-sines, however, the original core‒sequence is  <dig> nt long  <cit> .
two subtypes of wsine <dig> were previously reported from studies of the wallaby genome: the wsine <dig> and the wsine1a  <cit> .
after screening and comparative sequence analysis of the full-length wsine <dig> in the tasmanian devil genome, it was obvious that the majority of wsine1s belongs to a third subfamily; 89% of the wsine1s belong to what we refer here to as wsine1b .
comparative indel analysis of  <dig>  full-length wsine1s showed that there are two over-represented insertions.
one is located at position  <dig> and the other at position  <dig> .
it has been shown that the insert at position  <dig> is most likely the result of a duplication  <cit> .
alignments of the trna-related parts from several core-sines, both young and old, indicate that insertions and deletions are very infrequent in this region.
no deletions were observed.
insertions were found in only three other core-sines  .
wsine <dig> has no inserts, and can be considered ancestral to both wsine1a  and wsine1b  .
after screening the different marsupial sines, it is evident that the distinguishing 7-nt-long insert found in wsine1a and wsine1b is also present in wallsi <dig>  <cit> .
insertions in the core-sine trna region appear to be highly infrequent, therefore the presence of a 7-nt insert in a homologous position in wallsi <dig> and wsine1a/b indicates a common evolutionary ancestry.
wsine1a and wsine1b share the 7-nt insert but 1a has received an additional 11-nt insert.
thus the acquisition of the 11-nt insert in wsine1a would suggest an origin after 1b.
it was proposed that wallsi <dig>  might have originated from a wsine <dig> , followed by the addition of a wallaby-specific tail  <cit> .
however, the tail-region of wallsi <dig> is shared by wallsi <dig> and wallsi <dig> and is therefore not wallaby-specific.
the presence of the tail-region from wallsi <dig>  as well as wsine1b in the tasmanian devil, but absence of wallsi <dig>  suggest that the recombination into wallsi <dig> must have happened after the split leading to dasyuromorphia and occured only in diprotodontia .
molecular sequence estimates place the divergence between the two orders around 55– <dig> myr ago  <cit> .
the distribution of the three subtypes of wsine1s differs among the australian marsupial orders.
in the tasmanian devil, the 1b subtype is the most widespread, making up around 90% of the wsine copies in the genome ; isoodon macrourus, which belongs to the order peramelemorphia, has a similar distribution of wsine1s, with wsine1b being dominant  and wsine1a being present in only very low copy numbers in both dasyuromorphia and peramelemorphia.
thus, the distribution of wsine1b is consistent with the close relationship between peramelemorphia and dasyuromorphia  <cit> .
wsine <dig> subfamilies and the evolution of dasyuromorphia
sines propagate using one  active master source copies, which produce copies that randomly integrate into the genome  <cit> .
over time, there have been several master copies within given sine subtypes.
in the human genome, the alu sines seem to have had at least  <dig> active master copies during the  <dig> myr that alus were propagating  <cit> .
by comparison, the platypus genome has had around  <dig> active line <dig> copies  <cit>  that seem to have experienced a more linear evolution, where one master becomes inactivated and is then followed by a new one.
to investigate the network of wsine <dig> subfamilies in the tasmanian devil genome, we applied the same strategy used to estimate the segregation of alu elements  <cit> .
in total,  <dig> different wsine <dig> subfamilies were identified from the  <dig>  copies that aligned well to the consensus sequence .
a preliminary evolutionary age estimate of the different wsine <dig> subfamilies was calculated using a local substitution rate of  <dig> /myr, a value that was derived from wsine <dig> insertions in the marsupial genome.
other substitution rates, derived from retroposons in general, l <dig> elements, and pseudogenes  <cit> , were also applied to estimate the age of the retroposons.
however, all these were too slow to estimate the age of wsine <dig>  because the resulting divergence time estimates predate the origin of mammalia, and are thus highly incompatible with wsine <dig> activity.
wsine <dig> seems to have originated around  <dig> myr ago, which corresponds on a marsupial evolutionary time scale to the split between the two south american orders paucituberculata and microbiotheria  <cit> .
although no data is currently available for paucituberculata, wsine <dig> elements were identified in the genome of microbiotheria  <cit> , but to date there is no evidence of wsine <dig> elements in the genome of didelphimorphia  <cit> .
thus, wsine1s must have evolved after the split from didelphimorphia , either in paucituberculata or microbiotheria.
it has been shown that retroposon activation is correlated with speciation  <cit> .
the network of sine subfamilies coupled with the evolutionary age estimates and by correlation suggest that there was a burst of wsine <dig> activity around the time of the origin of the order dasyuromorphia , during which numerous speciation events lead to today’s dasyuromorphian families.
the subfamily network also indicates that several wsine1s became active master copies during the early phases of the order dasyuromorphia; however, most were inactivated relatively quickly, leaving only one line of wsine <dig> that continued producing copies until the present.
core-sines in marsupials
core-sines belong to one of four super-classes of sines defined by highly conserved sequence domains  <cit> , the others being v-sines  <cit> , deusines  <cit>  and cephsines  <cit> .
the core-sines are trna-related, but their sequences have evolved beyond recognition  <cit> , making it impossible to determine from which trna the core-sine originated.
recently, a sine from the insect tobacco budworm  was found to have a highly conserved, 65-nt-long core sequence  <cit> .
due to their high degree of conservation and abundance in mammals core-sines are speculated to have played, or still play, a functional role.
evidence for this hypothesis comes from one core-sine that has been exapted into a neural enhancer  <cit> .
the core-sines have been proliferating in marsupial genomes for at least  <dig> myr.
several different core-sines, both young and old, are found in marsupials, but with different tail-regions depending on which line is responsible for their propagation  <cit> .
our genome analyses showed that the diagnostic 65-nt-long core sequence was truncated to  <dig> nt in a few of the marsupial core-sines   <cit> .
the wallsi <dig>  mar1c_mdo, wallsi <dig>  wsine <dig>  and sine1_mdo have shorter cores, while mar1a, b, mar <dig>  and all mirs have the original core sequence.
this implies that there are two types of core-sines in marsupial genomes, one with a short and one with a long core region.
the short core-sines most likely have a shared evolutionary origin, and based on the tint analysis, the oldest element with a short core is the wallsi <dig>  both wsine <dig> and sine1_mdo probably originated from the wallsi <dig> or mar1c_mdo elements.
wsine1b and wallsi <dig> both share the 7-nt-long insert in the trna-region, which indicates a close relationship between these elements, and that wallsi <dig> evolved from wsine1b.
as wsine <dig> is absent from the opossum genome, this in turn suggests that the truncation of the core must have happened independently in the two elements.
alternatively, they may have an origin in the same sine family, such as wallsi <dig> or mar1c_mdo.
in the opossum genome,  <dig>  copies of the sine1_mdo element make up the dominant sine type  <cit> , while in the wallaby, the wallsi <dig>  with about  <dig>  copies, is the most recently active sine  <cit> .
in the tasmanian devil genome, the wsine <dig> had the most recent expansion and is present in  <dig>  copies, making up  <dig> mb of the sequence.
thus, the activity and proliferation of the short core-sines in marsupials is an ongoing and successful process.
in monotremes, another core-sine, the mon, is active  <cit> .
in placental mammals, the mir and mir <dig> are the only core-sines found, and these have been inactive for  <dig> myr  <cit> .
thus, during the evolution to placental mammals, core-sines were inactivated.
the activation and inactivation of sines and lines happens frequently in genomes over evolutionary times.
CONCLUSIONS
the core-sines, a group of retroposons, is extinct in placental mammals since  <dig> myr, while still proliferating in marsupials.
we show that core-sines are the most recently active sines in the genome of the tasmanian devil, and have since at least  <dig> myr proliferated into nearly  <dig>  copies.
for the first time we have shown how an ancient speciation event in marsupials might have lead to a burst of retroposon activity that was partially silenced by the genome over time.

 the tasmanian devil is the largest living carnivorous australian marsupial in the order dasyuromorphia, which also includes the anteating numbat, quolls, dunnarts, phascogales, and the iconic extinct tasmanian tiger. populations of the tasmanian devil are currently under serious threat of being severely diminished or eradicated by devil facial tumor disease, a contagious form of cancer. since it was first observed in dftd has nearly halved the tasmanian devil population. without a cure for dftd, tasmanian devils may become extinct in 10– years. however, with the help of next generation sequencing techniques, finding a possible cure may be within reach. to gain a better understanding of the genetics of the disease and possibly find a cure, the genome of the tasmanian devil was recently sequenced. recent studies have shown that marsupial genomes have been under massive bombardment by transposable elements, which play important roles in genomic evolution and are the causes of some diseases. in the opossum, 52% of the genome sequence consists of such elements. this is the largest percentage of transposable elements presently identified in any vertebrate genome. in comparison, the human genome consists of 45% and the mouse genome of 38% transposable elements. transposable elements are classified as retroposons, which propagate via an rna-intermediate, and dna transposons that transpose themselves directly by dna, without an intermediate. typically, transposons make up only a small percentage of genome sequences, while the vast majority of the transposable elements are retroposons. one group of retroposons, the non-autonomous sines, is propagated in the genome via the activity of the autonomous lines. most sines originate from trnas and their basic structure includes a 5′ trna-related sequence, a trna unrelated body, a 3′ line-related tail sequence that sometimes is followed by a poly- stretch. the 3′ tail sequence is derived from lines and is used for recognition by the reverse transcriptase. the marsupial sines have been shown to have tail sequences originating from line line as well as rte elements. when a polya stretch is present in the 3′ end of the sine it is used by line for retroposition. a group of highly conserved sines in mammals are the core-sines. core-sines have a specific and highly conserved 65‒nt central sequence between the trna-related part and the line derived region. core-sines have been found in diverse groups of animals, such as mammals, mollusks and fish. wsine a characteristic sine of marsupials, is a short core-sine, consisting only of the trna-related part and a truncated 41-nt core sequence followed by a polytail. in placental mammals, the core-sines have been inactive fossils for at least million years. however, this retroposon type has remained active in marsupial and monotreme genomes. in monotreme genomes, the mon core-sine dominates, and in marsupials, the core-sines have remained active and have proliferated, giving rise to many different subtypes. while most placental mammalian orders are currently represented by at least one genome sequence, genomic sequences of only three marsupial species, representing three of the seven orders, are currently known: the south american opossum, the australian wallaby, and the tasmanian devil. molecular data show that the respective orders of the first two, didelphimorphia and diprotodontia, are separated by 75– myr of evolution, representing the geographically and temporally deepest divergence among marsupials. the retroposon content in the opossum genome has been thoroughly investigated, and here we have screened the genome of an australian marsupial to understand the dynamics of sines and non-autonomous lines. the tasmanian devil genome sequence will be a valuable source for understanding the dynamics of retroposons in marsupial genomes.  full genome screening a summary of the abundances of all transposable elements revealed in the in silico screening of sarcophilus harrisii is shown in additional file 1: table s the most abundant marsupial specific sines and non-autonomous lines were calculated. * a putative pseudogene transposition in transposition for comparative genomics and in particular evolutionary purposes, it is useful to be able to estimate the relative order of activities of different groups of integrated sines found in the genome. tint is an algorithm that screens the genome for nested retroposon insertions and calculates its relative time frame of activity based on the fact that old inactive retroposons cannot insert into younger active retroposons. screening of the tasmanian devil genome with the tint program revealed nested insertions for the most frequent sines and non-autonomous lines. the most recently active element is the wsine the tint pattern from the tasmanian devil is reasonably similar to those previously observed in the opossum and wallaby. wallsi elements several wallsis have been described in the wallaby genome. three types, the wallsi1/1a, wallsi and wallsi have highly similar tail regions derived from rte_md. wallsi and wallsi are both core-sines. except for wallsi we found all previously identified wallsis in the tasmanian devil genome but in different proportions than previously described. there are fewer copies of the 5′-truncated wallsi in the tasmanian devil than in the wallaby. by contrast, the 200-nt-longer wallsi1a is present in copies in the tasmanian devil genome, which is more than the copies in the current assembly of the wallaby genome, which might be an underestimate given the low coverage of the genome sequence. on the other hand wallsi is present in relatively similar numbers in both genomes, which might indicate that the activities of the two elements terminated before the two orders split 55– myr ago and the sines were passed on to the descendants. the wallsi is more abundant in the wallaby genome despite lower sequence coverage of the assembled genome suggesting an expansion of the element. we did not identify any copies of wallsi in the tasmanian devil genome by the in-silico approach. wallsi was initially believed to be restricted to kangaroos; however, they have also been identified in other diprotodontian marsupials, indicating that the element is not restricted to the kangaroo subfamily macropodidae, but rather is characteristic of the entire order diprotodontia. psl7_md another interesting feature of the marsupial genome is the p7sl_md retropseudogene. the cytoplasmic 7sl rna is part of a signal recognition particle, and around myr ago it formed the alu element, which is the most abundant sine in primates and other related sines in the super-order euarchontoglires. however, alus are imperfect dimers derived from the 7sl sequence. in marsupials, the p7sl_md retropseudogene sequence is very similar to the 7sl rna. it is present in nearly identical copy numbers in the genomes of opossum, wallaby, and tasmanian devil, where we discovered more than copies. the high abundance may indicate that this pseudogene has undergone an extreme expansion in the ancient marsupial genome that has not been observed in any other mammal. wsine1 the wsine family is the most abundant of the recently active sines in the tasmanian devil. we identified approximately wsine elements, but even this may be an underestimate, as we also found nearly copies of fragmented core-sines that may represent ancient and diverged copies of wsine due to the difficulty of correctly assigning these fragmented sines, they have not been included in the final estimates. we also identified a third, novel subtype of wsine in the tasmanian devil genome that deviates from the previously described wsine1s, wsine and wsine1a. this new subtype, wsine1b, lacks the 11-nt insert near the poly-tail found in wsine1a elements. wsine1a and wsine1b both have a 7-nt insert at position suggesting a close relationship. a detailed indel analysis of all wsine elements shows that there are no other commonly occurring insertions or deletions. all three types of wsine are present in the genome of the tasmanian devil, but in different proportions. nearly 90% belong to the new wsine1b subtype, while the wsine1a elements represent only % of all wsine1s. the values for tasmanian devil are based on the analysis of the full genome, isoodon on the available trace sequences, and macropus on a screening of the complete genome. the abundances of the three variants are expressed as % with the copy number in parenthesis. only the complete wsine1s were used in the analysis, as many are fragmented and lack the regions needed to safely define the subfamily. wsine distribution in australian marsupial orders to examine the evolutionary dynamics of wsine we also investigated the distributions of the wsine subtypes in the genomes of two other marsupial orders, the peramelemorphia and diprotodontia. the screened trace sequences of isoodon show a pattern similar to that of the tasmanian devil, with the majority of wsines belonging to the wsine1b subtype. by contrast, in the wallaby genome the most abundant wsine is the wsine, though wsine1b is still widespread, making up almost 20% of the wsine copies. the age and number of the wsine subfamilies co-segregation analysis of full-length wsine elements identified subfamilies in the tasmanian devil genome, with copy numbers ranging from to. the evolutionary age of each subfamily was determined using a local nucleotide substitution rate derived from wsine. the oldest elements were estimated to be myr old and the youngest myr old. it is evident that a burst of new subfamilies appeared around 56– myr ago. of these, only a handful continued to be active and to give rise to additional master copies. more than new subfamilies were created during a very short time span. the burst of additional subfamilies occurred around the same time as the early evolutionary phases in the order dasyuromorphia. phylogenetic relationships and divergence times of the tasmanian devil we conducted mitochondrial genomic analyses to reconstruct the phylogenomic relationships of the tasmanian devil to other carnivorous marsupials and to estimate the divergence times of this order. this allowed us to correlate the evolutionary age of dasyuromorphia and place their early divergences in relation to the emergence of different wsine subfamilies. the mitogenomic analysis identified the tasmanian devil to be the sister group to the quolls. however, only after including third codon positions, in the nucleotide analysis could the tasmanian devil be placed outside dasyurus, indicating a rapid divergence of these lineages. as third codon positions are the fastest evolving nucleotide position only these seem to have acquired enough information during the short interval between the groups to resolve this relationship. it was equally difficult to estimate the relationships among the three subfamilies thylacinidae, myrmecobiidae, and dasyuridae, as sh- and au-tests did not reject alternative hypotheses, which is consistent with a rapid speciation process. the origin of the tasmanian devil is estimated by both sets of calibration points to be 12– myr ago. the split within dasyurus is estimated to be myr, merely to million years after the origin of the genus sarcophilus. the deepest split within dasyuridae is estimated to be myr. the origin of dasyuromorphia is estimated to be myr, with a second split around myr leading to numbat. discussion tasmanian devil genome-composition and activity of retroposons in silico screening showed that transposable elements account for about % of the tasmanian devil genome sequence, which is virtually identical to that found in the opossum and wallaby genome. the sines and non-autonomous lines make up % of the tasmanian devil sequence, which is comparable to the percentages identified in the genomes of the opossum and wallaby. the relative temporal activity of sines was studied using the tint program. when a younger retroposon integrates into another older retroposon, it is possible to estimate relative times of activity based on the information from these nested retroposition events. as retroposons tend to be active in distinct waves, the temporal chain of activities can be calculated based on thousands of nested retroposition events found in the genome. the tint method is a valuable tool for screening genomic data for phylogenetically informative retroposons, as the search can be targeted towards younger or older branches of the tree depending on the choice of elements. the method has been successfully applied to different groups of animals, such as birds, lagomorphs, primates, platypus, and marsupials. using the tint approach, different sines and non-autonomous lines were investigated in the tasmanian devil genome. several of them with relatively similar activity patterns. based on the tint analysis, the element that had a recent expansion in the tasmanian devil genome is the core-sine of the type wsine wsine1 the wsine1s are more abundant in the tasmanian devil genome than in the wallaby genome and had the most recent expansion of all sines. the wsine is a short element, consisting of a trna-derived region, a core-sequence region, and a poly‒tail. the core-sequence region is extraordinarily conserved across mammals, which has led to speculations concerning functional properties of this region. additionally, the element has a poly-tail and is flanked by target site duplications and is therefore expected to be line propagated. in many of the marsupial core-sines the core-region is nt long. in placental mammalian core-sines, however, the original core‒sequence is nt long. two subtypes of wsine were previously reported from studies of the wallaby genome: the wsine and the wsine1a. after screening and comparative sequence analysis of the full-length wsine in the tasmanian devil genome, it was obvious that the majority of wsine1s belongs to a third subfamily; 89% of the wsine1s belong to what we refer here to as wsine1b. comparative indel analysis of full-length wsine1s showed that there are two over-represented insertions. one is located at position and the other at position. it has been shown that the insert at position is most likely the result of a duplication. alignments of the trna-related parts from several core-sines, both young and old, indicate that insertions and deletions are very infrequent in this region. no deletions were observed. insertions were found in only three other core-sines. wsine has no inserts, and can be considered ancestral to both wsine1a and wsine1b. after screening the different marsupial sines, it is evident that the distinguishing 7-nt-long insert found in wsine1a and wsine1b is also present in wallsi. insertions in the core-sine trna region appear to be highly infrequent, therefore the presence of a 7-nt insert in a homologous position in wallsi and wsine1a/b indicates a common evolutionary ancestry. wsine1a and wsine1b share the 7-nt insert but 1a has received an additional 11-nt insert. thus the acquisition of the 11-nt insert in wsine1a would suggest an origin after 1b. it was proposed that wallsi might have originated from a wsine, followed by the addition of a wallaby-specific tail. however, the tail-region of wallsi is shared by wallsi and wallsi and is therefore not wallaby-specific. the presence of the tail-region from wallsi as well as wsine1b in the tasmanian devil, but absence of wallsi suggest that the recombination into wallsi must have happened after the split leading to dasyuromorphia and occured only in diprotodontia. molecular sequence estimates place the divergence between the two orders around 55– myr ago. the distribution of the three subtypes of wsine1s differs among the australian marsupial orders. in the tasmanian devil, the 1b subtype is the most widespread, making up around 90% of the wsine copies in the genome; isoodon macrourus, which belongs to the order peramelemorphia, has a similar distribution of wsine1s, with wsine1b being dominant and wsine1a being present in only very low copy numbers in both dasyuromorphia and peramelemorphia. thus, the distribution of wsine1b is consistent with the close relationship between peramelemorphia and dasyuromorphia. wsine subfamilies and the evolution of dasyuromorphia sines propagate using one active master source copies, which produce copies that randomly integrate into the genome. over time, there have been several master copies within given sine subtypes. in the human genome, the alu sines seem to have had at least active master copies during the myr that alus were propagating. by comparison, the platypus genome has had around active line copies that seem to have experienced a more linear evolution, where one master becomes inactivated and is then followed by a new one. to investigate the network of wsine subfamilies in the tasmanian devil genome, we applied the same strategy used to estimate the segregation of alu elements. in total, different wsine subfamilies were identified from the copies that aligned well to the consensus sequence. a preliminary evolutionary age estimate of the different wsine subfamilies was calculated using a local substitution rate of /myr, a value that was derived from wsine insertions in the marsupial genome. other substitution rates, derived from retroposons in general, l elements, and pseudogenes, were also applied to estimate the age of the retroposons. however, all these were too slow to estimate the age of wsine because the resulting divergence time estimates predate the origin of mammalia, and are thus highly incompatible with wsine activity. wsine seems to have originated around myr ago, which corresponds on a marsupial evolutionary time scale to the split between the two south american orders paucituberculata and microbiotheria. although no data is currently available for paucituberculata, wsine elements were identified in the genome of microbiotheria, but to date there is no evidence of wsine elements in the genome of didelphimorphia. thus, wsine1s must have evolved after the split from didelphimorphia, either in paucituberculata or microbiotheria. it has been shown that retroposon activation is correlated with speciation. the network of sine subfamilies coupled with the evolutionary age estimates and by correlation suggest that there was a burst of wsine activity around the time of the origin of the order dasyuromorphia, during which numerous speciation events lead to today’s dasyuromorphian families. the subfamily network also indicates that several wsine1s became active master copies during the early phases of the order dasyuromorphia; however, most were inactivated relatively quickly, leaving only one line of wsine that continued producing copies until the present. core-sines in marsupials core-sines belong to one of four super-classes of sines defined by highly conserved sequence domains, the others being v-sines, deusines and cephsines. the core-sines are trna-related, but their sequences have evolved beyond recognition, making it impossible to determine from which trna the core-sine originated. recently, a sine from the insect tobacco budworm was found to have a highly conserved, 65-nt-long core sequence. due to their high degree of conservation and abundance in mammals core-sines are speculated to have played, or still play, a functional role. evidence for this hypothesis comes from one core-sine that has been exapted into a neural enhancer. the core-sines have been proliferating in marsupial genomes for at least myr. several different core-sines, both young and old, are found in marsupials, but with different tail-regions depending on which line is responsible for their propagation. our genome analyses showed that the diagnostic 65-nt-long core sequence was truncated to nt in a few of the marsupial core-sines. the wallsi mar1c_mdo, wallsi wsine and sine1_mdo have shorter cores, while mar1a, b, mar and all mirs have the original core sequence. this implies that there are two types of core-sines in marsupial genomes, one with a short and one with a long core region. the short core-sines most likely have a shared evolutionary origin, and based on the tint analysis, the oldest element with a short core is the wallsi both wsine and sine1_mdo probably originated from the wallsi or mar1c_mdo elements. wsine1b and wallsi both share the 7-nt-long insert in the trna-region, which indicates a close relationship between these elements, and that wallsi evolved from wsine1b. as wsine is absent from the opossum genome, this in turn suggests that the truncation of the core must have happened independently in the two elements. alternatively, they may have an origin in the same sine family, such as wallsi or mar1c_mdo. in the opossum genome, copies of the sine1_mdo element make up the dominant sine type, while in the wallaby, the wallsi with about copies, is the most recently active sine. in the tasmanian devil genome, the wsine had the most recent expansion and is present in copies, making up mb of the sequence. thus, the activity and proliferation of the short core-sines in marsupials is an ongoing and successful process. in monotremes, another core-sine, the mon, is active. in placental mammals, the mir and mir are the only core-sines found, and these have been inactive for myr. thus, during the evolution to placental mammals, core-sines were inactivated. the activation and inactivation of sines and lines happens frequently in genomes over evolutionary times.  the core-sines, a group of retroposons, is extinct in placental mammals since myr, while still proliferating in marsupials. we show that core-sines are the most recently active sines in the genome of the tasmanian devil, and have since at least myr proliferated into nearly copies. for the first time we have shown how an ancient speciation event in marsupials might have lead to a burst of retroposon activity that was partially silenced by the genome over time. 
the radiation and abundance of core-sines in marsupial genomes indicates that they may be a major player in the evolution of marsupials. a correlation between a speciation event and a major burst of retroposon activity is for the first time shown in a marsupial genome. moreover, we discovered a new subtype of wsine that comprises at least 90% of all tasmanian devil wsine1s. to better understand the tasmanian devil genome, we screened it for transposable elements and investigated the dynamics of short interspersed element retroposons. the temporal history of tasmanian devil sines, elucidated using a transposition in transposition analysis, indicates that wsine a core-sine present in around copies, is the most recently active element. it is evident that the early phases of evolution of the carnivorous marsupial order dasyuromorphia was characterized by a burst of sine activity. the frequencies of wsine subtypes differ in the genomes of two of the other australian marsupial orders. the genome of the carnivorous marsupial, the tasmanian devil, was sequenced in the hopes of finding a cure for or gaining a better understanding of the contagious devil facial tumor disease that is threatening the species’ survival. phylogenetic analyses and divergence time estimates of mitochondrial genome data indicate a rapid radiation of the tasmanian devil and the closest relative the quolls around million years ago.
 the genome of the carnivorous marsupial, the tasmanian devil, was sequenced in the hopes of finding a cure for or gaining a better understanding of the contagious devil facial tumor disease that is threatening the species’ survival. to better understand the tasmanian devil genome, we screened it for transposable elements and investigated the dynamics of short interspersed element retroposons.  the temporal history of tasmanian devil sines, elucidated using a transposition in transposition analysis, indicates that wsine a core-sine present in around copies, is the most recently active element. moreover, we discovered a new subtype of wsine that comprises at least 90% of all tasmanian devil wsine1s. the frequencies of wsine subtypes differ in the genomes of two of the other australian marsupial orders. a co-segregation analysis indicated that at least subfamilies of wsine evolved during the evolution of dasyuromorphia. using a substitution rate derived from wsine insertions, the ages of the subfamilies were estimated and correlated with a newly established phylogeny of dasyuromorphia. phylogenetic analyses and divergence time estimates of mitochondrial genome data indicate a rapid radiation of the tasmanian devil and the closest relative the quolls around million years ago.  the radiation and abundance of core-sines in marsupial genomes indicates that they may be a major player in the evolution of marsupials. it is evident that the early phases of evolution of the carnivorous marsupial order dasyuromorphia was characterized by a burst of sine activity. a correlation between a speciation event and a major burst of retroposon activity is for the first time shown in a marsupial genome. sinewsine1retroposontasmanian devilsarcophilusgenomemarsupials 