BACKGROUND
typically the cpdnas of land plants contain two identical segments, the inverted repeats , separated by two single copy  sequences, the large single copy  region and the small single copy  region  <cit> .
thus four junctions, termed jla, jsa, jsb, jlb, are between the two irs and the sc regions  <cit> .
a major constraint on cpdna is its organization into large clusters of polycistronically transcribed genes  <cit> .
as a result, large structural changes in cpdna, such as segmental duplication or deletion and mutation in gene order, are relatively rare and evolutionarily useful in making phylogenetic inferences  <cit> .
in land plants, the sizes of rrna gene-containing irs are notably variable, ranging from  <dig> kb in liverworts to 20– <dig> kb in most angiosperms  <cit> , and up to  <dig> kb in pelargonium   <cit> .
successive ir expansions, either within angiosperms or between non-vascular plants and angiosperms, have led to floating of jla and jlb  <cit>  and have evolutionary significance  <cit> .
several models concerning the expansion and contraction of ir regions have been proposed to explain the possible mechanisms that result in shift of the ir-lsc junctions.
for example, the unusual triple-sized expansion of the geranium ir was hypothesized as an outcome of inversion due to recombination between homologous dispersed repeats  <cit> .
similarly, the at least  <dig> kb expansion of the ir in buckwheat  cpdna was also considered to be associated with an inversion  <cit> .
goulding et al.
<cit>  found that in most nicotiana species ir regions have both expanded and contracted with slight variations in length during the evolution of the genus.
the exception is n. acuminata, which underwent a large ir expansion of over  <dig> kb.
goulding et al.
<cit>  proposed two mechanisms of ir expansion:  gene conversion to account for the small ir expansion or movements in most species of the genus, and  a dna double-strand break  to explain the extensive incorporation of the lsc region into the ir of n. acuminata.
perry et al.
<cit>  analyzed the endpoint sequence of a large  <dig> kb rearrangement in adzuki bean  and concluded that the unusual organization was caused by a two-step process of expansion and contraction of the ir, rather than a large inversion.
recent phylogenetic studies using various molecular markers have yielded robust support for the hypothesis of either amborella alone or amborella-nymphaeales together as the basal-most clade of angiosperms  <cit> , and the genus acorus has been identified as the earliest splitting lineage in monocots.
however, the sister group of monocots is still unclear  <cit> .
monocots include about one-fourth of the world's flowering plants and represent one of the oldest angiosperm lineages  <cit> .
however, no comparative study has been conducted to investigate the diversity and evolutionary dynamics at the ir-lsc junctions of cpdnas in basal angiosperms and monocots as a whole.
goulding et al.
<cit>  found that each ir in rice and maize  contains a fully duplicated trnh-rps <dig> gene cluster.
chang et al.
<cit>  further discovered that the irs of two other remote monocot taxa, acorus and orchidaceae, also include trnh and rps <dig> , and speculated that the clustering of rps <dig> and trnh was probably duplicated before the diversification of extant monocot lineages.
as a result of expansion and contraction, the irs in the cpdna of angiosperms have been suggested as an evolutionary marker for elucidating relationships among some taxa  <cit> .
to improve understanding of the dynamics and evolution of ir-lsc junctions from basal angiosperms to the emergence and diversification of monocots , we sampled  <dig> key species and determined the sequences of the two regions spanning jla  and jlb .
a total of  <dig> representative angiosperms, including  <dig> basal angiosperms,  <dig> magnoliids,  <dig> eudicots, and  <dig> monocots , were analyzed.
three types of gene arrangements flanking the jla and jlb regions were recognized and mapped onto the angiosperm phylogeny.
in order to explain this arrangements we propose two alternative hypotheses concerning the evolutionary history of the flux of ir-lsc.
furthermore, to verify the transcriptional status of the duplicated trnh-rps <dig> gene cluster near the ira junctions the activity of two operons in asparagus densiflorus, s10a and psba, was investigated.
RESULTS
several terms used in this section are briefly explained here.
types of ir-lsc junction are based on the organization of genes flanking jlb and jla in angiosperms.
type i is found in most non-angiosperm dicots.
it refers to an intact trnh gene being located directly downstream of the rpl <dig> sequence in ira and an intact rps <dig> gene being located directly downstream of the rpl <dig> sequence in irb.
no full-length rps <dig> or trnh sequence is present in ira or irb respectively.
type ii refers to a partial sequence of rps <dig> being located directly between rpl <dig> and trnh in ira.
type ii pattern is only found in some eudicots while type iii characterizes the irs of most monocots, in which each ir contains a trnh-rps <dig> cluster.
the letters a, a', c, ... and g used in the text and in figure  <dig> refer to the ir-lsc junctions found in cpdnas of sampled angiosperms.
in non-monocot angiosperms ir-lsc junctions of irb are largely located between rpl <dig> and rps19
the winteraceae , exemplified by zygogynum pauciflorum and drimys granadensis  <cit> , were exceptional in that the organization of the genes flanking the ir-lsc junctions resembled the one found in most monocots, rather than the organization seen in other non-monocot angiosperms.
notably, each of their irs contained a trnh-rps <dig> cluster and their ir-lsc junctions were located within the 5' portion of rps <dig> .
type ii ir-lsc junctions were found in schisandra arisanensis  and some  <dig> representative eudicots .
unlike type i, the jla of type ii shifted to the 5' end of the truncated rps <dig> in ira .
irs of monocots generally contain trnh-rps <dig> clusters
in contrast to basal angiosperms and eudicots, most monocots  had trnh-rps <dig> clusters present in each of the two irs, and the ir-lsc junctions were generally at position f  or g  .
this type of gene organization was classified as type iii.
in addition, ir-lsc junctions of some monocots were located downstream of rpl <dig> , of trnh , or within rps <dig> .
when the ir-lsc junction was at position d, the rps <dig> sequence in ira was found to be partially truncated most of the times.
sequences flanking ir-lsc junctions are more variable in monocots than in non-monocot angiosperms
transcription of monocot trnh-rps <dig> of ira is regulated by both chloroplast s10a and psba promoters
among the chloroplast operons, the s <dig> ribosomal protein operon is the largest.
it contains genes encoding both small  and large  ribosomal protein subunits that are organized into a polycistronic transcription unit conserved in known cpdnas  <cit> .
in angiosperms, the 5' end of the s <dig> operon is initiated within the ir, but only in irb does the operon extend into the lsc region, and the s <dig> operon is only partially in ira .
however, a second operon in ira, the psba operon, is transcribed from lsc towards ira  <cit>  and opposite to the s10a operon.
in the winteraceae and a majority of monocots, the trnh-rps <dig> cluster of ira is included in both the s <dig> and psba operons.
therefore, this gene cluster may be regulated by two opposing promoters, the s10a and the psba .
in monocots, if the trnh in ira is indeed regulated by the above-mentioned two opposing promoters, the function of the trnh gene may be repressed because antisense-trnh rnas would be generated by both the s10a and s10b promoters.
to verify this possibility, we conducted rt-pcr assays using specific primers for a type iii representative, asparagus densiflorus, with the ir-lsc junction located at position f .
our results indicate that expression of the trnh gene in ira is regulated by both the s10a and psba promoters.
this suggests that the duplicated trnh gene located in the irb region of most monocots and in some non-monocots has different fates .
figure 3b shows that two rt-pcr products, a  <dig> bp and a  <dig> bp fragment, respectively, were generated when specific primer pairs for each were used .
the former fragment was amplified from the transcripts made by the psba promoter, and the latter by the s <dig> promoter.
this result confirms that the trnh-rps <dig> cluster of ira is regulated by two opposing promoters , indicating that the transcription machinery in irs of monocots may differ from that of basal angiosperms and eudicots.
discussion
two evolutionary hypotheses for the flux of ir-lsc junctions in monocots
as shown in figure 1a, ir-lsc junctions of the amborella + nymphaeales are mainly located at position b, but junctions of monocots are further expanded to encompass lsc genes and are located at positions f or g. since the two irs of monocots usually include the trnh-rps <dig> cluster , we hypothesize that at least two duplication events are required to explain the expansion of irs in monocots during the course of ir evolution from an amborella-like ancestor to present-day monocots.
if this hypothesis is correct, it is expected that an intermediate junction type could be traceable in the cpdnas of some early divergent monocot lineages between the two duplication events.
narayanan et al.
<cit>  have recently presented a model of gene amplification in eukaryotes that argues strongly for the involvement of hairpin-capped dsbs in the initiation.
based on this model and our observations, we propose two hypotheses to account for the evolution of ir expansions in monocots .
in hypothesis a, a dsb event  occurs first within the irb of an amborella-like ancestor, and then the free 3' end of the broken strand is repaired against the homologous sequence in ira.
the repaired sequence extends over the original ir-lsc junction and reaches the area downstream of trnh , so that duplication of a trnh gene in the newly repaired irb is achieved.
similarly, a second dsb event occurs in ira adjacent to the ira-lsc junction  so that duplication of rps <dig> at ira can be initiated, and a trnh-rps <dig> cluster nearby jlb  is created.
the newly formed irs might cover the trnh-rps <dig> cluster and extend further into the intergenic spacer between rps <dig> and rpl <dig> .
furthermore, if one additional dsb event took place within the intergenic spacer located between rps <dig> and rpl <dig> in the lsc region, a partial rpl <dig> gene would be duplicated at ira using the rpl <dig> sequence of lsc as a template, and from then on the repaired irs might have expanded towards the 5' region of the rpl <dig> .
the exceptionally long irs observed in the orchidaceae and commelinales are likely to have been generated by this process.
the same outcomes could also result if the process proceeded directly from step  <dig> to step  <dig> without step  <dig> .
hypothesis b, on the other hand, assumes that rps <dig> would be duplicated or converted prior to the duplication of trnh through a dsb event that takes place at ira first .
a second dsb event  then would take place within the irb region through a similar repair process to the one mentioned before, so that a duplicated trnh is generated at irb.
finally, the irs expand downstream of rps <dig>  in hypothesis b subsequent extension of irs is assumed to resemble step  <dig> of hypothesis a.
duplication of a partial or complete rps <dig> gene was also observed in some eudicots and schisandraceae  with their respective ir-lsc junctions located at position e or e' .
however, these duplicated rps <dig> genes  are situated between the rpl <dig> and trnh genes of the ira  rather than downstream of trnh or upstream of psba  and  of hypothesis a in figure 4).
therefore, the gene arrangement flanking the ira-lsc of type ii deviates from that of type i, suggesting that duplication of rps <dig> genes in type ii must have a distinct evolutionary history.
based on comparisons of aligned rpl2-trnh and trnh-rps <dig> intergenic spacer sequences from representatives of major monocot orders , it is apparent that these two spacer sequences are separately highly similar across the sampled monocot orders.
these data give strong support to hypothesis a that in monocots expansion and inclusion of trnh-rps <dig> gene cluster in irs might require at least two common dsbs : one occurring within irb , and the within ira .
however, we did not discover any inverted repeats that might have led to the formation of hairpins in the monocot intergenic spacers of trnh and rps <dig>  therefore, we are inclined to conclude that the expansions of monocot irs took the path depicted in hypothesis a.
ir expansion may be initiated by dsb and end in the nearby polya region in angiosperms
goulding et al.
<cit>  proposed two models to account for two kinds of ir expansion:  small and random ir expansions, caused by gene conversion ; and  large ir expansions, like those found in the nicotiana species, rice and maize, generated via dsb events.
narayanan et al.
<cit>  further demonstrated that dsbs can trigger gene amplification through a variety of mechanisms, and that breakage at the inverted repeats of chromosomes can cause gene amplification.
after a critical comparison of genes or sequences adjacent to the ir-lsc junctions in  <dig> major orders and  <dig> families of angiosperms , we hypothesize that ir expansions resulted principally from the dsb events that occurred during ir evolution from the amborella-like ancestor to monocots.
this hypothesis is founded on the following  <dig> observations:  the length of ir expansion from basal angiosperms to monocots is large ;  trnh and rps <dig> are situated downstream of ira and irb, respectively, in all sampled basal angiosperms .
this type of gene arrangement might represent the ancestral gene pattern in basal angiosperms;  irs of several basal angiosperms  and eudicots  have partially or completely duplicated trnh genes located at irb;  in comparison with other angiosperms, monocot irs have expanded further to include a duplicated rps <dig> in ira, and this expansion should have occurred before the diversification of major monocot orders; and  the irs of advanced monocots  have expanded to encompass more lsc sequences or genes .
nevertheless, the latter expansions did not apparently result from another common dsb event but from independent ones, because among sampled monocot orders the downstream regions of rps <dig> genes have low sequence similarity .
at the infra-order level of angiosperms, gene conversion might occur frequently at meiosis and cause small ir expansion or contraction during evolution, as found in apiaceae  <cit>  and nicotiana  <cit> .
studies on the ir-lsc junctions of nicotiana species  <cit>  and apiaceous plants  <cit>  have indicated that short repeats or "polya tract" sequences associated with trnas at the ir-lsc boundaries might be likely hotspots for recombination.
we also observed that polya tract sequences are commonly present near the ir-lsc junctions in all the basal angiosperms, eudicots and monocots examined , indicating that such sequences are closely linked with the dynamics of ir-lsc junctions and expansion of irs.
in this regard, we further propose that ir expansion may initiate at the dsbs and finish at the polya tract regions, where recombination may actively occur, and that the recombination mechanism in cpdna may resemble that reported for nuclear genomes by narayanan et al.
<cit> .
according to our hypothesis, dsbs within irs must have been frequent during angiosperm evolution.
however, only those which led to successful ir expansions, and have subsequently been retained in the extant taxa, are detectable.
based on our observations, it is evident that the type of ir-lsc junction appears to be informative, at least at the level of order, and is therefore useful for inferring phylogenetic relationships at this rank and above.
expansion of monocot irs is correlated with the divergence pattern of monocot phylogeny
as shown in figure 1b, ir-lsc junctions of basal monocots including acorales, pandanales and liliales are usually located at position d. this type might represent a primitive state.
in contrast, ir-lsc junctions of the derived monocots, such as asparagales and poales, have generally expanded to position f or g. this trend in ir expansion seems to correlate well with the divergence pattern of monocot lineages in the multigene tree of soltis et al.
<cit> , which shows acorales to be a sister group to other monocots.
this correlation connotes the ancient status of the order and the continuous ir expansion experienced by the more terminal and derived lineages, viz.
asparagales, commelinales, zingiberales, arecales, dasypogonaceae and poales.
it is worth mentioning that in some monocots  the ir-lsc junctions are located at position d, with a truncated rps <dig> gene at ira.
according to hypothesis a , duplication of rps <dig> at ira was due to a second dsb event in ira , followed by a sequence repair supposed to have been terminated within or downstream of the rps <dig> gene.
duplication of the rps <dig> gene will lead to a shift of the ir-lsc junction to position d or f .
however, in pandanales and liliales, the rps <dig> sequences of ira are incomplete or degraded.
we considered these common degradations likely to be secondary rather than primary, since the majority of monocot orders have the trnh-rps <dig> clusters .
moreover, among the major monocot orders  the intergenic spacer sequences within the trnh-rps <dig> cluster  have a high degree of similarity, suggesting that among the sampled monocots a common dsb event might have taken place adjacent to the trnh gene.
therefore, the irs in acorales, pandanales and liliales are likely to have contracted, causing a shift of the ir-lsc junctions from around position f to position d.
a comparison of the downstream non-coding or spacer sequences of the rps <dig> genes in monocots reveals that the sequences do not have a common origin , as they are highly variable and a reliable sequence alignment is impossible except between closely related con-ordinal taxa .
this indicates that these spacer sequences had diverse origins and are likely to have resulted from independent dsb events occurring at different points within the irs.
in contrast, it appears that expansion of ir-lsc junctions did not parallel the evolutionary diversification of basal angiosperms and eudicot lineages .
in type i , ir expansion downstream of rps <dig> is extremely rare in eudicots, with the exception of adzuki bean  and a pelargonium species .
according to our hypothesis a , the scenario of ir expansion in these two eudicots may have different origins from those of monocots and other eudicots , with irs that have expanded downstream of rps <dig> genes.
similarly, significant ir contractions in the basal angiosperm illicium oligandrum , coriander   <cit> , and cuscuta reflexa   <cit>  seem to be separate events in their respective lineages.
implications of sequences flanking ir-lsc junctions for angiosperm phylogeny
in extant angiosperms, the relationships among the remaining  <dig> lineages  are unresolved  <cit> .
to what extent the dicot lineage is a sister group of monocots remains uncertain, probably a reflection of the rapid radiation and extinction of early angiosperms soon after they originated  <cit> .
recent phylogenetic analyses based on plastid sequence data have suggested that monocots and eudicots are sister taxa , but with low bootstrap support .
in addition, several lines of evidence have indicated that ceratophyllaceae could be the sister group of monocots  <cit> .
here we present an alternative view on this issue.
as illustrated in figure  <dig>  an intact trnh is duplicated in irb of all monocots, one basal angiosperm , and two winteraceous magnoliid species   <cit> .
sequence comparison revealed that only winteraceae and monocots have highly similar spacer sequences between the rpl <dig> and trnh genes , suggesting that duplication of trnh gene in irb of the two taxa might be common or similar .
on the other hand, acorales  has its ir endpoint at position d, suggesting that those lineages with ir-lsc junctions at position b and c'  might have resulted from separate, independent contractions.
our alternative view on the relationships among monocots and their relatives is preliminary, as it is only based on comparison of genic organizations at ir-lsc junctions.
additional molecular and morphological data are required to improve our understanding of monocot phylogeny.
the presence of two anti-sense strands of trnh in monocot irs is mysterious
the presence of a trnh-rps <dig> cluster in the irs appears to be a common feature in monocots other than some alismatids , in which ir-lsc junctions are located at position b and strongly resemble those of most non-monocot angiosperms.
however, alignment of the intergenic spacers between rpl <dig> and trnh in some alismatales  and other monocots, basal angiosperms and eudicots  reveals that sequences of the alismatids are more similar to other monocots than to non-monocot angiosperms.
this implies that ir expansions in some alismatids might share evolutionary scenarios similar to those proposed for other monocots, and that the short irs  in some other alismatids are likely due to either an early termination of the repair-extension reaction after the first dsb in step  <dig> of hypothesis a , or to a contraction after this step.
in monocots, each ir usually contains a trnh gene, while in most basal angiosperms and eudicots the gene is rarely present in irb .
why is the duplicated trnh gene able to survive in irb of most monocots but is absent, degraded or truncated in most non-monocot angiosperms?
in two studied eudicots, lotus japonicus  <cit>  and spinacea oleracea  <cit> , the transcriptional activity of s10a dropped significantly because of either the high transcription levels of the psba and trnh genes or the termination of s10a proximal to jla  <cit> .
therefore, in non-monocot angiosperms, trnh-encoded mrna molecules constitute only one sense strand, transcribed solely by the psba operon rather than by the s10a operon.
because anti-sense rna molecules may interfere with the normal function of the sense rna molecules  <cit> , in monocots the mechanism by which anti-sense trnh is regulated by two s10a promoters is mysterious.
further study on the evolution and survival of the duplicated trnh gene in irb of monocots is desirable.
CONCLUSIONS
extensive comparisons of the genic organizations flanking the ir-lsc junctions in  <dig> diversified angiosperm lineages revealed that monocots and non-monocot angiosperms generally have different ir-lsc junction types.
notably, irs expanded more progressively in monocots than in non-monocot angiosperms, with more lsc genes being converted into irs.
with the exceptions of alismatales and a few acorales, the monocot ira regions either encompass a trnh-rps <dig> cluster or extend as far as the 5' portion of the rpl <dig> gene, which is typically situated at the lsc region in non-monocot angiosperms.
various expansions of irs in monocots have resulted in corresponding fluxes of ir-lsc junctions.
our results further indicate that the ir expansions in angiosperms can be explained by initiation of a dsb event and ending at a polya tract region.
we proposed two hypotheses to explain the evolutionary derivation of the trnh-rps <dig> cluster in the irs of monocots from an amborella-like ancestor .
hypothesis a proposes that a dsb event occurs first within the irb of an amborella-like ancestor, and then the free 3' end of the broken strand is repaired against the homologous sequence in ira.
the repaired sequence extends and results in the duplication of a trnh gene in the newly repaired irb.
a subsequent dsb event may occur in ira so that the rps <dig> at ira is duplicated, whereby a trnh-rps <dig> cluster is created.
hypothesis b assumes that rps <dig> is duplicated or converted before the duplication of trnh via a dsb event that occurs at ira.
it is worth noting that ir expansions in monocots appear to correlate well with the divergence pattern of monocot phylogeny.
the present study highlights the use of sequences flanking the ir-lsc junctions to address the evolutionary dynamics of irs from basal angiosperms to monocots.
taken together with the evidence from the ir-lsc junctions, we conclude that  monocots may be closely related to the winteraceae  than to other basal angiosperms or eudicots,  the shorter irs in alismatids are probably due to either an early termination of repair-extension after the first dsb, or to a contraction after this step, and  the duplicated trnh genes in the irb of most monocots and non-monocot angiosperms have distinct fates, which are likely regulated by different expression levels of s10a and s10b promoters.
further study is needed to unravel the evolutionary significance or advantage of the presence of an additional trnh in monocot irs, and of ir expansion in more recently diverged monocots.

 typically the cpdnas of land plants contain two identical segments, the inverted repeats, separated by two single copy sequences, the large single copy region and the small single copy region. thus four junctions, termed jla, jsa, jsb, jlb, are between the two irs and the sc regions. a major constraint on cpdna is its organization into large clusters of polycistronically transcribed genes. as a result, large structural changes in cpdna, such as segmental duplication or deletion and mutation in gene order, are relatively rare and evolutionarily useful in making phylogenetic inferences. in land plants, the sizes of rrna gene-containing irs are notably variable, ranging from kb in liverworts to 20– kb in most angiosperms, and up to kb in pelargonium. successive ir expansions, either within angiosperms or between non-vascular plants and angiosperms, have led to floating of jla and jlb and have evolutionary significance. several models concerning the expansion and contraction of ir regions have been proposed to explain the possible mechanisms that result in shift of the ir-lsc junctions. for example, the unusual triple-sized expansion of the geranium ir was hypothesized as an outcome of inversion due to recombination between homologous dispersed repeats. similarly, the at least kb expansion of the ir in buckwheat cpdna was also considered to be associated with an inversion. goulding et al. found that in most nicotiana species ir regions have both expanded and contracted with slight variations in length during the evolution of the genus. the exception is n. acuminata, which underwent a large ir expansion of over kb. goulding et al. proposed two mechanisms of ir expansion: gene conversion to account for the small ir expansion or movements in most species of the genus, and a dna double-strand break to explain the extensive incorporation of the lsc region into the ir of n. acuminata. perry et al. analyzed the endpoint sequence of a large kb rearrangement in adzuki bean and concluded that the unusual organization was caused by a two-step process of expansion and contraction of the ir, rather than a large inversion. recent phylogenetic studies using various molecular markers have yielded robust support for the hypothesis of either amborella alone or amborella-nymphaeales together as the basal-most clade of angiosperms, and the genus acorus has been identified as the earliest splitting lineage in monocots. however, the sister group of monocots is still unclear. monocots include about one-fourth of the world's flowering plants and represent one of the oldest angiosperm lineages. however, no comparative study has been conducted to investigate the diversity and evolutionary dynamics at the ir-lsc junctions of cpdnas in basal angiosperms and monocots as a whole. goulding et al. found that each ir in rice and maize contains a fully duplicated trnh-rps gene cluster. chang et al. further discovered that the irs of two other remote monocot taxa, acorus and orchidaceae, also include trnh and rps, and speculated that the clustering of rps and trnh was probably duplicated before the diversification of extant monocot lineages. as a result of expansion and contraction, the irs in the cpdna of angiosperms have been suggested as an evolutionary marker for elucidating relationships among some taxa. to improve understanding of the dynamics and evolution of ir-lsc junctions from basal angiosperms to the emergence and diversification of monocots, we sampled key species and determined the sequences of the two regions spanning jla and jlb. a total of representative angiosperms, including basal angiosperms, magnoliids, eudicots, and monocots, were analyzed. three types of gene arrangements flanking the jla and jlb regions were recognized and mapped onto the angiosperm phylogeny. in order to explain this arrangements we propose two alternative hypotheses concerning the evolutionary history of the flux of ir-lsc. furthermore, to verify the transcriptional status of the duplicated trnh-rps gene cluster near the ira junctions the activity of two operons in asparagus densiflorus, s10a and psba, was investigated.  several terms used in this section are briefly explained here. types of ir-lsc junction are based on the organization of genes flanking jlb and jla in angiosperms. type i is found in most non-angiosperm dicots. it refers to an intact trnh gene being located directly downstream of the rpl sequence in ira and an intact rps gene being located directly downstream of the rpl sequence in irb. no full-length rps or trnh sequence is present in ira or irb respectively. type ii refers to a partial sequence of rps being located directly between rpl and trnh in ira. type ii pattern is only found in some eudicots while type iii characterizes the irs of most monocots, in which each ir contains a trnh-rps cluster. the letters a, a', c,... and g used in the text and in figure refer to the ir-lsc junctions found in cpdnas of sampled angiosperms. in non-monocot angiosperms ir-lsc junctions of irb are largely located between rpl and rps19 the winteraceae, exemplified by zygogynum pauciflorum and drimys granadensis, were exceptional in that the organization of the genes flanking the ir-lsc junctions resembled the one found in most monocots, rather than the organization seen in other non-monocot angiosperms. notably, each of their irs contained a trnh-rps cluster and their ir-lsc junctions were located within the 5' portion of rps. type ii ir-lsc junctions were found in schisandra arisanensis and some representative eudicots. unlike type i, the jla of type ii shifted to the 5' end of the truncated rps in ira. irs of monocots generally contain trnh-rps clusters in contrast to basal angiosperms and eudicots, most monocots had trnh-rps clusters present in each of the two irs, and the ir-lsc junctions were generally at position f or g. this type of gene organization was classified as type iii. in addition, ir-lsc junctions of some monocots were located downstream of rpl, of trnh, or within rps. when the ir-lsc junction was at position d, the rps sequence in ira was found to be partially truncated most of the times. sequences flanking ir-lsc junctions are more variable in monocots than in non-monocot angiosperms transcription of monocot trnh-rps of ira is regulated by both chloroplast s10a and psba promoters among the chloroplast operons, the s ribosomal protein operon is the largest. it contains genes encoding both small and large ribosomal protein subunits that are organized into a polycistronic transcription unit conserved in known cpdnas. in angiosperms, the 5' end of the s operon is initiated within the ir, but only in irb does the operon extend into the lsc region, and the s operon is only partially in ira. however, a second operon in ira, the psba operon, is transcribed from lsc towards ira and opposite to the s10a operon. in the winteraceae and a majority of monocots, the trnh-rps cluster of ira is included in both the s and psba operons. therefore, this gene cluster may be regulated by two opposing promoters, the s10a and the psba. in monocots, if the trnh in ira is indeed regulated by the above-mentioned two opposing promoters, the function of the trnh gene may be repressed because antisense-trnh rnas would be generated by both the s10a and s10b promoters. to verify this possibility, we conducted rt-pcr assays using specific primers for a type iii representative, asparagus densiflorus, with the ir-lsc junction located at position f. our results indicate that expression of the trnh gene in ira is regulated by both the s10a and psba promoters. this suggests that the duplicated trnh gene located in the irb region of most monocots and in some non-monocots has different fates. figure 3b shows that two rt-pcr products, a bp and a bp fragment, respectively, were generated when specific primer pairs for each were used. the former fragment was amplified from the transcripts made by the psba promoter, and the latter by the s promoter. this result confirms that the trnh-rps cluster of ira is regulated by two opposing promoters, indicating that the transcription machinery in irs of monocots may differ from that of basal angiosperms and eudicots. discussion two evolutionary hypotheses for the flux of ir-lsc junctions in monocots as shown in figure 1a, ir-lsc junctions of the amborella + nymphaeales are mainly located at position b, but junctions of monocots are further expanded to encompass lsc genes and are located at positions f or g. since the two irs of monocots usually include the trnh-rps cluster, we hypothesize that at least two duplication events are required to explain the expansion of irs in monocots during the course of ir evolution from an amborella-like ancestor to present-day monocots. if this hypothesis is correct, it is expected that an intermediate junction type could be traceable in the cpdnas of some early divergent monocot lineages between the two duplication events. narayanan et al. have recently presented a model of gene amplification in eukaryotes that argues strongly for the involvement of hairpin-capped dsbs in the initiation. based on this model and our observations, we propose two hypotheses to account for the evolution of ir expansions in monocots. in hypothesis a, a dsb event occurs first within the irb of an amborella-like ancestor, and then the free 3' end of the broken strand is repaired against the homologous sequence in ira. the repaired sequence extends over the original ir-lsc junction and reaches the area downstream of trnh, so that duplication of a trnh gene in the newly repaired irb is achieved. similarly, a second dsb event occurs in ira adjacent to the ira-lsc junction so that duplication of rps at ira can be initiated, and a trnh-rps cluster nearby jlb is created. the newly formed irs might cover the trnh-rps cluster and extend further into the intergenic spacer between rps and rpl. furthermore, if one additional dsb event took place within the intergenic spacer located between rps and rpl in the lsc region, a partial rpl gene would be duplicated at ira using the rpl sequence of lsc as a template, and from then on the repaired irs might have expanded towards the 5' region of the rpl. the exceptionally long irs observed in the orchidaceae and commelinales are likely to have been generated by this process. the same outcomes could also result if the process proceeded directly from step to step without step. hypothesis b, on the other hand, assumes that rps would be duplicated or converted prior to the duplication of trnh through a dsb event that takes place at ira first. a second dsb event then would take place within the irb region through a similar repair process to the one mentioned before, so that a duplicated trnh is generated at irb. finally, the irs expand downstream of rps in hypothesis b subsequent extension of irs is assumed to resemble step of hypothesis a. duplication of a partial or complete rps gene was also observed in some eudicots and schisandraceae with their respective ir-lsc junctions located at position e or e'. however, these duplicated rps genes are situated between the rpl and trnh genes of the ira rather than downstream of trnh or upstream of psba and of hypothesis a in figure 4). therefore, the gene arrangement flanking the ira-lsc of type ii deviates from that of type i, suggesting that duplication of rps genes in type ii must have a distinct evolutionary history. based on comparisons of aligned rpl2-trnh and trnh-rps intergenic spacer sequences from representatives of major monocot orders, it is apparent that these two spacer sequences are separately highly similar across the sampled monocot orders. these data give strong support to hypothesis a that in monocots expansion and inclusion of trnh-rps gene cluster in irs might require at least two common dsbs: one occurring within irb, and the within ira. however, we did not discover any inverted repeats that might have led to the formation of hairpins in the monocot intergenic spacers of trnh and rps therefore, we are inclined to conclude that the expansions of monocot irs took the path depicted in hypothesis a. ir expansion may be initiated by dsb and end in the nearby polya region in angiosperms goulding et al. proposed two models to account for two kinds of ir expansion: small and random ir expansions, caused by gene conversion; and large ir expansions, like those found in the nicotiana species, rice and maize, generated via dsb events. narayanan et al. further demonstrated that dsbs can trigger gene amplification through a variety of mechanisms, and that breakage at the inverted repeats of chromosomes can cause gene amplification. after a critical comparison of genes or sequences adjacent to the ir-lsc junctions in major orders and families of angiosperms, we hypothesize that ir expansions resulted principally from the dsb events that occurred during ir evolution from the amborella-like ancestor to monocots. this hypothesis is founded on the following observations: the length of ir expansion from basal angiosperms to monocots is large; trnh and rps are situated downstream of ira and irb, respectively, in all sampled basal angiosperms. this type of gene arrangement might represent the ancestral gene pattern in basal angiosperms; irs of several basal angiosperms and eudicots have partially or completely duplicated trnh genes located at irb; in comparison with other angiosperms, monocot irs have expanded further to include a duplicated rps in ira, and this expansion should have occurred before the diversification of major monocot orders; and the irs of advanced monocots have expanded to encompass more lsc sequences or genes. nevertheless, the latter expansions did not apparently result from another common dsb event but from independent ones, because among sampled monocot orders the downstream regions of rps genes have low sequence similarity. at the infra-order level of angiosperms, gene conversion might occur frequently at meiosis and cause small ir expansion or contraction during evolution, as found in apiaceae and nicotiana. studies on the ir-lsc junctions of nicotiana species and apiaceous plants have indicated that short repeats or "polya tract" sequences associated with trnas at the ir-lsc boundaries might be likely hotspots for recombination. we also observed that polya tract sequences are commonly present near the ir-lsc junctions in all the basal angiosperms, eudicots and monocots examined, indicating that such sequences are closely linked with the dynamics of ir-lsc junctions and expansion of irs. in this regard, we further propose that ir expansion may initiate at the dsbs and finish at the polya tract regions, where recombination may actively occur, and that the recombination mechanism in cpdna may resemble that reported for nuclear genomes by narayanan et al.. according to our hypothesis, dsbs within irs must have been frequent during angiosperm evolution. however, only those which led to successful ir expansions, and have subsequently been retained in the extant taxa, are detectable. based on our observations, it is evident that the type of ir-lsc junction appears to be informative, at least at the level of order, and is therefore useful for inferring phylogenetic relationships at this rank and above. expansion of monocot irs is correlated with the divergence pattern of monocot phylogeny as shown in figure 1b, ir-lsc junctions of basal monocots including acorales, pandanales and liliales are usually located at position d. this type might represent a primitive state. in contrast, ir-lsc junctions of the derived monocots, such as asparagales and poales, have generally expanded to position f or g. this trend in ir expansion seems to correlate well with the divergence pattern of monocot lineages in the multigene tree of soltis et al., which shows acorales to be a sister group to other monocots. this correlation connotes the ancient status of the order and the continuous ir expansion experienced by the more terminal and derived lineages, viz. asparagales, commelinales, zingiberales, arecales, dasypogonaceae and poales. it is worth mentioning that in some monocots the ir-lsc junctions are located at position d, with a truncated rps gene at ira. according to hypothesis a, duplication of rps at ira was due to a second dsb event in ira, followed by a sequence repair supposed to have been terminated within or downstream of the rps gene. duplication of the rps gene will lead to a shift of the ir-lsc junction to position d or f. however, in pandanales and liliales, the rps sequences of ira are incomplete or degraded. we considered these common degradations likely to be secondary rather than primary, since the majority of monocot orders have the trnh-rps clusters. moreover, among the major monocot orders the intergenic spacer sequences within the trnh-rps cluster have a high degree of similarity, suggesting that among the sampled monocots a common dsb event might have taken place adjacent to the trnh gene. therefore, the irs in acorales, pandanales and liliales are likely to have contracted, causing a shift of the ir-lsc junctions from around position f to position d. a comparison of the downstream non-coding or spacer sequences of the rps genes in monocots reveals that the sequences do not have a common origin, as they are highly variable and a reliable sequence alignment is impossible except between closely related con-ordinal taxa. this indicates that these spacer sequences had diverse origins and are likely to have resulted from independent dsb events occurring at different points within the irs. in contrast, it appears that expansion of ir-lsc junctions did not parallel the evolutionary diversification of basal angiosperms and eudicot lineages. in type i, ir expansion downstream of rps is extremely rare in eudicots, with the exception of adzuki bean and a pelargonium species. according to our hypothesis a, the scenario of ir expansion in these two eudicots may have different origins from those of monocots and other eudicots, with irs that have expanded downstream of rps genes. similarly, significant ir contractions in the basal angiosperm illicium oligandrum, coriander, and cuscuta reflexa seem to be separate events in their respective lineages. implications of sequences flanking ir-lsc junctions for angiosperm phylogeny in extant angiosperms, the relationships among the remaining lineages are unresolved. to what extent the dicot lineage is a sister group of monocots remains uncertain, probably a reflection of the rapid radiation and extinction of early angiosperms soon after they originated. recent phylogenetic analyses based on plastid sequence data have suggested that monocots and eudicots are sister taxa, but with low bootstrap support. in addition, several lines of evidence have indicated that ceratophyllaceae could be the sister group of monocots. here we present an alternative view on this issue. as illustrated in figure an intact trnh is duplicated in irb of all monocots, one basal angiosperm, and two winteraceous magnoliid species. sequence comparison revealed that only winteraceae and monocots have highly similar spacer sequences between the rpl and trnh genes, suggesting that duplication of trnh gene in irb of the two taxa might be common or similar. on the other hand, acorales has its ir endpoint at position d, suggesting that those lineages with ir-lsc junctions at position b and c' might have resulted from separate, independent contractions. our alternative view on the relationships among monocots and their relatives is preliminary, as it is only based on comparison of genic organizations at ir-lsc junctions. additional molecular and morphological data are required to improve our understanding of monocot phylogeny. the presence of two anti-sense strands of trnh in monocot irs is mysterious the presence of a trnh-rps cluster in the irs appears to be a common feature in monocots other than some alismatids, in which ir-lsc junctions are located at position b and strongly resemble those of most non-monocot angiosperms. however, alignment of the intergenic spacers between rpl and trnh in some alismatales and other monocots, basal angiosperms and eudicots reveals that sequences of the alismatids are more similar to other monocots than to non-monocot angiosperms. this implies that ir expansions in some alismatids might share evolutionary scenarios similar to those proposed for other monocots, and that the short irs in some other alismatids are likely due to either an early termination of the repair-extension reaction after the first dsb in step of hypothesis a, or to a contraction after this step. in monocots, each ir usually contains a trnh gene, while in most basal angiosperms and eudicots the gene is rarely present in irb. why is the duplicated trnh gene able to survive in irb of most monocots but is absent, degraded or truncated in most non-monocot angiosperms? in two studied eudicots, lotus japonicus and spinacea oleracea, the transcriptional activity of s10a dropped significantly because of either the high transcription levels of the psba and trnh genes or the termination of s10a proximal to jla. therefore, in non-monocot angiosperms, trnh-encoded mrna molecules constitute only one sense strand, transcribed solely by the psba operon rather than by the s10a operon. because anti-sense rna molecules may interfere with the normal function of the sense rna molecules, in monocots the mechanism by which anti-sense trnh is regulated by two s10a promoters is mysterious. further study on the evolution and survival of the duplicated trnh gene in irb of monocots is desirable.  extensive comparisons of the genic organizations flanking the ir-lsc junctions in diversified angiosperm lineages revealed that monocots and non-monocot angiosperms generally have different ir-lsc junction types. notably, irs expanded more progressively in monocots than in non-monocot angiosperms, with more lsc genes being converted into irs. with the exceptions of alismatales and a few acorales, the monocot ira regions either encompass a trnh-rps cluster or extend as far as the 5' portion of the rpl gene, which is typically situated at the lsc region in non-monocot angiosperms. various expansions of irs in monocots have resulted in corresponding fluxes of ir-lsc junctions. our results further indicate that the ir expansions in angiosperms can be explained by initiation of a dsb event and ending at a polya tract region. we proposed two hypotheses to explain the evolutionary derivation of the trnh-rps cluster in the irs of monocots from an amborella-like ancestor. hypothesis a proposes that a dsb event occurs first within the irb of an amborella-like ancestor, and then the free 3' end of the broken strand is repaired against the homologous sequence in ira. the repaired sequence extends and results in the duplication of a trnh gene in the newly repaired irb. a subsequent dsb event may occur in ira so that the rps at ira is duplicated, whereby a trnh-rps cluster is created. hypothesis b assumes that rps is duplicated or converted before the duplication of trnh via a dsb event that occurs at ira. it is worth noting that ir expansions in monocots appear to correlate well with the divergence pattern of monocot phylogeny. the present study highlights the use of sequences flanking the ir-lsc junctions to address the evolutionary dynamics of irs from basal angiosperms to monocots. taken together with the evidence from the ir-lsc junctions, we conclude that monocots may be closely related to the winteraceae than to other basal angiosperms or eudicots, the shorter irs in alismatids are probably due to either an early termination of repair-extension after the first dsb, or to a contraction after this step, and the duplicated trnh genes in the irb of most monocots and non-monocot angiosperms have distinct fates, which are likely regulated by different expression levels of s10a and s10b promoters. further study is needed to unravel the evolutionary significance or advantage of the presence of an additional trnh in monocot irs, and of ir expansion in more recently diverged monocots. 
the duplicated trnh genes in the irb of most monocots and non-monocot angiosperms have distinct fates, which are likely regulated by different expression levels of s10a and s10b promoters. two hypotheses are proposed to account for the evolution of ir expansions in monocots. based on our observations, the inclusion of a trnh-rps cluster in majority of monocot irs could be reasonably explained by the hypothesis that a dsb event first occurred at irb and led to the expansion of irs to trnh, followed by a successive dsb event within ira and lead to the expansion of irs to rps or to rpl so far. further study is needed to unravel the evolutionary significance of ir expansion in more recently diverged monocots. ir-lsc junctions commonly occurred at polya tract or a-rich regions in angiosperms. therefore, we compared the organizations of genes flanking two ir-lsc junctions in angiosperm representatives to uncover the evolutionary dynamics of ir-lsc junctions in basal angiosperms and monocots. the organizations of genes flanking ir-lsc junctions in angiosperms can be classified into three types. generally each ir of monocots contains a trnh-rps gene cluster near the ir-lsc junctions, which differs from those in non-monocot angiosperms. various expansions or contractions of inverted repeats in chloroplast genomes led to fluxes in the ir-lsc junctions. previous studies revealed that some monocot irs contain a trnh-rps gene cluster, and it has been speculated that this may be an evidence of a duplication event prior to the divergence of monocot lineages. our rt-pcr assays indicate that in monocot ira the trnh-rps gene cluster is regulated by two opposing promoters, s10a and psba. moreover, irs expanded more progressively in monocots than in non-monocot angiosperms. this implies that the duplication of trnh-rps gene cluster was prior to the diversification of extant monocot lineages.
 various expansions or contractions of inverted repeats in chloroplast genomes led to fluxes in the ir-lsc junctions. previous studies revealed that some monocot irs contain a trnh-rps gene cluster, and it has been speculated that this may be an evidence of a duplication event prior to the divergence of monocot lineages. therefore, we compared the organizations of genes flanking two ir-lsc junctions in angiosperm representatives to uncover the evolutionary dynamics of ir-lsc junctions in basal angiosperms and monocots.  the organizations of genes flanking ir-lsc junctions in angiosperms can be classified into three types. generally each ir of monocots contains a trnh-rps gene cluster near the ir-lsc junctions, which differs from those in non-monocot angiosperms. moreover, irs expanded more progressively in monocots than in non-monocot angiosperms. ir-lsc junctions commonly occurred at polya tract or a-rich regions in angiosperms. our rt-pcr assays indicate that in monocot ira the trnh-rps gene cluster is regulated by two opposing promoters, s10a and psba.  two hypotheses are proposed to account for the evolution of ir expansions in monocots. based on our observations, the inclusion of a trnh-rps cluster in majority of monocot irs could be reasonably explained by the hypothesis that a dsb event first occurred at irb and led to the expansion of irs to trnh, followed by a successive dsb event within ira and lead to the expansion of irs to rps or to rpl so far. this implies that the duplication of trnh-rps gene cluster was prior to the diversification of extant monocot lineages. the duplicated trnh genes in the irb of most monocots and non-monocot angiosperms have distinct fates, which are likely regulated by different expression levels of s10a and s10b promoters. further study is needed to unravel the evolutionary significance of ir expansion in more recently diverged monocots. 