BACKGROUND
gene duplication is an important evolutionary force, providing the raw material for new genes that, if not lost, may be co-opted to perform novel functions  <cit> .
gene duplication has been implicated in the evolution of transcription factors that play key roles in developmental pathways and are likely involved in morphological evolution  <cit> .
in plants, for example, the mads-box and tcp gene families underwent duplication deep within angiosperm phylogeny, and these duplication events are thought to have played a role in major changes to flower morphology  <cit> .
tcp genes are less well studied than mads-box genes, but they are increasingly appreciated for their role in generating diverse floral forms.
the tcp gene family is named for the conserved helix-loop-helix  tcp domain from teosinte branched <dig>  in zea mays, cycloidea  in antirrhinum majus, and the proliferating cell factor  dna-binding proteins in oryza sativa.
in arabidopsis, the tcp gene family has  <dig> copies  <cit> , which are divided into a clade of pcf genes, which control cell growth, and a clade of cyc/tb <dig> genes.
these two clades differ in the length and sequence of the tcp domain, and some members of the cyc/tb <dig> clade have an additional conserved arginine rich "r domain"  <cit> .
within cyc/tb <dig> the "ece" clade  <cit>  contains a group of genes with a conserved short motif  between the tcp and r domains.
there are three major clades of ece genes in the core eudicots, and the duplications leading to these copies predate this major lineage  <cit> .
we refer to members of the ece clade as "cyc-like" genes.
the best studied of these genes, cyc itself, is required for the production of bilaterally symmetrical  flowers as a function of its expression in the dorsal portion of the floral meristem  <cit> .
while cyc-like genes have been primarily studied in the context of floral symmetry in single flowers, they have also been implicated in the development of the capitulum  inflorescence in asteraceae, a trait that may be associated with the evolutionary success of this group  <cit> .
a typical capitulum consists of many small flowers  packed tightly into a condensed head that can closely resemble a single large flower.
in some groups, the florets in different parts of the capitulum are morphologically distinct.
in radiate species, the outer florets  are bilaterally symmetrical while the inner florets  are radially symmetrical .
in discoid species, there is little or no variation in floret morphology and the capitulum is composed of disk florets only.
a third inflorescence type in asteraceae, characteristic of the lactuceae, consists of a capitulum with entirely bilaterally symmetrical flowers .
in asteraceae, the evolution of cyc-like genes has been studied thoroughly in the radiate species helianthus annuus , where ten copies have been recovered, which is more that in any other species to date  <cit> .
such extensive gene duplication is thought to be correlated with the evolution of ray versus disk florets  <cit> ; however, this possible connection has not been investigated in any groups outside of the asteraceae, nor has copy number been compared in clades with different inflorescence types.
dipsacaceae  is one of relatively few groups outside of asteraceae where the capitulum inflorescence occurs and that also contains species with both radiate and discoid heads .
dipsacaceae is part of the valerina clade of dipsacales  <cit>  and contains ca.
<dig> species of perennial and annual herbs that are divided into three main clades  <cit> : bassecoia , scabioseae , and the dipknautid clade , the latter two of which comprise the "core dipsacaceae" .
most dipsacaceae species have radiate capitula, with the exception of a few small lineages.
these include bassecoia, which is sister to the remaining dipsacaceae, and two groups within the dipknautid clade: dipsacus  and a small clade composed of succisa , succisella , and pseudoscabiosa .
howarth and donoghue  <cit>  investigated the evolution of cyc-like and divaricata-like  genes in dipsacales, and found additional duplications of cyc-like genes in dipsacus pilosus, one of two representatives of dipsacaceae included in the analysis, as well as additional duplications of div-like genes in sixalix atropurpurea, the sole representative of dipsacaceae included in the study of div-like genes.
given the unusual floral characteristics associated with this group , further investigation of cyc-like genes from a broader sampling of dipsacaceae is warranted.
the aim of this study is to investigate patterns of gene duplication in cyc-like genes and relate patterns of gene evolution to the evolution of radiate versus discoid inflorescences.
we also seek to compare the diversification of cyc-like genes in dipsacaceae to that reported for members of asteraceae , to test for a similar pattern of gene duplication.
this study represents a first step in eventually deciphering the involvement of cyc-like genes in determining capitulum form and differences in floral symmetry in dipsacaceae.
RESULTS
phylogenetic analysis
the combined matrix contains  <dig> sequences and  <dig> bases.
alignment of the tcp and r domains is unambiguous across taxa, however, the intervening region is difficult to align; therefore, we omitted ambiguous portions of this region from the alignment.
all datasets, as well as the full-length alignments, are available in treebase http://www.treebase.org or upon request from the first author.
to improve phylogenetic resolution within dpccyc <dig> and dpccyc2b, we analyzed these sequences separately in order to align the intervening region between the tcp and r domain.
the aligned length of the dpccyc <dig> matrix was  <dig> bases with  <dig> sequences and the dpccyc2b matrix was  <dig> bases with  <dig> sequences.
the tree topologies of the bayesian and maximum likelihood  analyses were congruent with only minor differences in branch lengths, and consensus trees are shown in figures  <dig>   <dig>   <dig>
we recovered between  <dig> and  <dig> copies for each species.
while we extensively screened  <dig> representatives of the major lineages of dipsacaceae, we did not recover all hypothesized copies from each species, which may have been the result of unsuccessful amplification or absence/loss from those genomes.
our analysis recovered the three main clades of cyc-like genes that have been reported for core eudicots and the additional duplications previously found in representatives of dipsacaceae   <cit> .
we name the three major clades of dipsacaceae cyc-like genes: dpccyc <dig>  dpccyc <dig>  and dpccyc <dig>  also consistent with previous results is the sister relationship of dpccyc <dig> and dpccyc <dig>  novel findings include copies dpccyc2a and dpccyc3a, whose orthologs had been inferred for other members of the dipsacales  <cit> , and additional duplications within dpccyc <dig>  and within dpccyc2ba and dpccyc2bb , which are unique to dipsacaceae.
the additional duplications within these clades are found only in species with radiate capitula.
dpccyc <dig> is well supported, with the sequence from b. bretschneideri resolved as sister to two clades of dpccyc <dig> in the separate analysis .
that is, the duplication within dpccyc <dig> appears to have occurred after the core dipsacaceae split from bassecoia.
dpccyc1a is recovered only from members of the dipknautid clade, including both discoid  and radiate  species, while dpccyc1b is found only in radiate species from scabioseae and the dipknautids.
within dpccyc1b, there are four clades  representing four putative copies .
the dpccyc <dig> clade is the largest and most complicated group in terms of duplication events and this clade aligns with five copies of helianthus, which has undergone a similar radiation in this gene lineage.
we found evidence for one copy of dpccyc2a in all major lineages of dipsacaceae, and within this clade, the gene phylogeny is generally consistent with the species phylogeny  <cit> .
with the separate analysis of dpccyc2b, we were able to further clarify relationships within this gene lineage .
the two major copies that were previously reported  <cit>  - dpccyc2ba and dpccyc2bb - are hereafter referred to as "2ba" and "2bb" for simplicity.
in the combined analysis of all cyc-like genes, the dpccyc2ba sequence from b. bretschneideri is sister to sequences from all remaining species in this clade, similar to the placement of the sequences from b. bretschneideri in dpccyc <dig>  in the separate analysis of dpccyc2ba, however, the position of the sequence from b. bretschneideri is unresolved but joins sequences from the other discoid species pseudoscabiosa limonifolia, dipsacus pilosus, and dipsacus inermis outside a large clade composed only of sequences from radiate species from both the scabioseae and dipknautid groups.
we inferred five putative copies within this core clade of 2ba .
similarly, within the 2bb lineage, we found only one copy in the discoid species .
sequences from these species, along with the dipknautid radiate species knautia calycina and cephalaria hirsuta, are resolved outside of a clade composed of sequences from radiate species from both the dipknautids and scabioseae.
within this core clade of 2bb, we inferred two putative copies , although support for 2bb <dig> was low .
lastly, we recovered the fewest sequences from the dpccyc <dig> clade.
within dpccyc <dig>  we found evidence for one copy of dpccyc3a for members of scabioseae, but did not recover this gene for bassecoia or members of the dipknautid clade.
dpccyc3b was recovered for four species that represent the three major lineages of dipsacaceae  and there were two copies of this gene for k. calycina.
sequence characteristics
the three major clades of dpccyc, as well as the putative copies within these clades, differ in sequence characteristics, most notably the length between the tcp and r domain .
dpccyc <dig> is the longest copy, with fewer gaps in the intervening region , followed by dpccyc2a.
duplications within both dpccyc <dig> and dpccyc <dig> appear to have been accompanied by a reduction in sequence length between the a and b copies, and the b copies of dpccyc <dig> and dpccyc <dig> have the shortest sequence lengths.
the "ece" region, the characteristic conserved domain found in the intervening region, is easily identifiable in dpccyc <dig>  dpccyc2a, and dpccyc3a, but apparently absent in all copies of both dpccyc2b and dpccyc3b .
in addition to differences in the intervening region, there are also amino acid differences in the tcp domain between the different copies.
dpccyc <dig> is the most divergent from the other copies.
there are also notable differences in sequence characteristics between the putative copies within each of the subclades, including the length of the intervening region as well as amino acid polymorphisms.
discussion
our study verifies that gene duplication events yielded five major copies of cyc-like genes prior to the origin of dipsacaceae   <cit> .
the major duplication found in dpccyc <dig>  and the additional putative copies within dpccyc1b are found in dipsacaceae but not in other dipsacales.
similarly, the major duplication in dpccyc2b  is consistent with previous studies  <cit> , but the additional duplications within these copies are unique to dipsacaceae.
these duplications do not appear to be the result of recent polyploidization events, as they do not occur across all copies.
additionally, chromosome counts indicate that there is no evidence for genome doubling in the species used in this study  <cit>  .
we feel that the newly discovered subclades within dpccyc <dig> and dpccyc2b likely represent different copies, as opposed to alleles, because they contain species from throughout the dipsacaceae phylogeny.
in other words, we would not expect allele sequences to be conserved across distant relatives.
gene trees in relation to the species tree
congruence between gene trees and the species tree within dipsacaceae is difficult to interpret.
outgroups in the linnina clade are generally resolved as sister to dipsacaceae, although phylogenetic relationships between these species tend to be poorly supported.
the two helianthus cyc1-like copies are resolved outside of the cyc1-like clade, indicating that better phylogenetic sampling is needed to clarify relationships among copies in this part of the tree.
within dpccyc <dig>  the failure to amplify dpccyc1a from members of scabioseae , combined with the additional copies in dpccyc1b, renders species relationships within this clade unclear.
the dpccyc2a tree  is more or less consistent with phylogenetic studies  <cit> , although the position of b. bretschneideri is weakly resolved  with members of the dipknautids rather than as sister to all other dipsacaceae, as in the species phylogeny.
within dpccyc2b, the presence of multiple copies precludes the inference of species relationships.
lastly, congruence between the gene trees and the species tree in both copies of dpccyc <dig> cannot be determined due to limited taxon sampling and poor phylogenetic signal in the gene trees .
based on these results, the utility of most dpccyc genes for use in phylogenetic studies would be problematical due to the presence of multiple putative copies, low amplification success for some genes , short sequences with a highly conserved tcp domain that yield low phylogenetic signal, and difficulties in aligning the intervening region.
a possible exception is dpccyc2a, which shows some promise in elucidating species relationships.
this gene may be enhanced as a phylogenetic marker by using the full-length sequence rather than the shorter region between the tcp and r domain used in this study.
strikingly, we found no additional duplications in any of the major subclades for bassecoia bretschneideri.
in the separate analysis of dpccyc <dig>  the main duplication event occurred after bassecoia split from the core dipsacaceae.
similarly, we found no evidence for additional duplications in dpccyc <dig> in dipsacus inermis, dipsacus pilosus, or pseudoscabiosa limonifolia, the other discoid species included in this study.
in the 2ba and 2bb clades, both dipsacus species and p. limonifolia occupy a position along with b. bretschneideri  outside of the core clades of these lineages that include all radiate species.
given these results and our current understanding of dipsacaceae phylogeny , it is perhaps most likely that the 2ba and 2bb genes duplicated in the lineage leading to the core dipsacaceae and were subsequently lost twice: once in the lineage leading to pseudoscabiosa and once in dipsacus.
however, we cannot rule out that by using a degenerate primer approach we failed to amplify these copies in bassecoia, pseudoscabiosa, and dipsacus.
in any case, the occurrence of the same general pattern of duplication in three different cyc-like genes  is potentially of great interest.
diversification of cyc-like genes and character evolution
the pattern of cyc-like gene diversification appears to generally correlate with inflorescence form in dipsacaceae.
groups with radiate capitula are represented in the major clades and subclades of dpccyc, while the discoid groups bassecoia, pseudoscabiosa, and dipsacus appear to have only one copy of dpccyc <dig> , 2ba, and 2bb.
while cyc-like genes have been investigated in functional studies of other asteraceae species , gene duplication has been investigated most extensively in helianthus.
the highest reported number of cyc-like genes is  <dig> for helianthus annuus  <cit> , a radiate species of asteraceae, and the pattern of diversification in radiate dipsacaceae groups is remarkably similar.
while we did not recover all copies for each species, we hypothesize that there are at least  <dig> and possibly  <dig> copies of cyc-like genes in radiate members of dipsacaceae , depending on better resolution within 2bb and whether the duplication in dpccyc3b found in knautia can be generalized to other radiate species.
for discoid groups, our study infers  <dig> copies.
the duplications in helianthus and radiate dipsacaceae occur within the same gene lineages.
helianthus has two copies of cyc1-like genes and we recovered five putative copies for radiate species of dipsacaceae.
most notably, there are five copies of helianthus in the cyc2-like clade, and seven or eight  copies in radiate species of dipsacaceae.
lastly, helianthus has three copies in the cyc3-like clade, while members of dipsacaceae appear to have at least two , although copy number was difficult to assess for this gene clade due to problems with amplification.
if we assume that most cyc-like genes that are present have been discovered, then it is possible that the independent evolution of radiate capitula in asteraceae and dipsacaceae may have been associated with independent duplication events in orthologous cyc-like genes.
however, in the absence of expression data for cyc-like genes in dipsacaceae and broader studies of gene duplication events within asteraceae, this is clearly speculative.
to date, the evolutionary history of cyc-like genes has not been investigated in discoid asteraceae , so it is unknown whether these species also diverged in copy number from their radiate relatives.
the additional duplications in cyc1-like and cyc2b-like genes are of particular interest because, within the core eudicots, they may be specific to helianthus and dipsacaceae, at least within the species that have been surveyed thus far .
in most previous studies, only a single copy of cyc1-like genes has been found  <cit> .
our finding that only members of the dipknautid clade have dpccyc1a is potentially interesting given the differences in floral traits between the main clades of dipsacaceae.
members of scabioseae have five petals while the dipknautids  have four, and the epicalyx in scabioseae is characterized by several structural modifications that are not present in bassecoia or in the dipknautids  <cit> .
however, functional information for cyc1-like genes is limited, although putative orthologs in arabidopsis are thought to be involved in branching architecture  <cit> .
tb <dig> is similar in sequence to genes in the cyc1-like clade, and is also responsible for differences in branching architecture between cultivated maize and its wild progenitor, teosinte  <cit> .
while there are no functional data for helianthus, the two copies of cyc1-like genes  were found to diverge in expression patterns, with hacyc1a expressed across all tissues except roots, and hacyc1b expressed only in the petals  <cit> .
due to potential differences in cyc1-like genes between scabioseae and the dipknautid clade and the presence of multiple copies of dpccyc1b in radiate species, cyc1-like genes are attractive candidates for future study.
previous studies have generally found more duplications in the cyc2-like genes than in the cyc1-like or cyc3-like genes; however, no more than three copies have been reported outside of helianthus and dipsacaceae  <cit> .
the cyc2-like clade includes cyc from antirrhinum, and its orthologs have been studied in several plant groups, including in members of asteraceae, where it has been suggested that cyc-like genes might be responsible for differentiating the radially symmetrical disk florets from the bilaterally symmetrical ray florets  <cit> .
in both gerbera and senecio, cyc2-like genes are expressed in ray florets and functional data suggest they are involved in differentiating the radially symmetrical disk florets from the bilaterally symmetrical ray florets  <cit> .
while no functional data exist for the helianthus orthologs of cyc2-like genes, their expression patterns were found to diverge, which may be indicative of sub and/or neofunctionalization  <cit>  of the duplicated cyc2-like copies.
while our correlational study cannot establish causation, the higher number of dpccyc2b genes in radiate versus discoid dipsacaceae suggests that they might also play a role in the development of ray florets.
expression studies and functional data are needed to address this possibility.
CONCLUSIONS
the results presented here represent the first step in assessing the role of cyc-like genes in dipsacaceae development.
we recovered the five major clades that were reported in previous studies and discovered several additional copies, most notably in the dpccyc <dig> and dpccyc2b clades.
the dpccyc2b clade in particular demonstrates a dynamic history of gene duplication.
interestingly, the pattern of cyc-like diversification in dipsacaceae is very similar to that of helianthus annuus, a species of asteraceae that has been screened for cyc-like genes.
our study also suggests that the number of dpccyc copies tends to correlate with the distribution of radiate versus discoid inflorescence form, with species bearing radiate heads having more than twice the number of cyc-like genes as discoid species.
investigations of cyc-like genes in discoid members of asteraceae are needed to assess the generality of this pattern.
capitulum inflorescences have evolved independently in a number of angiosperm lineages, but perhaps most often within the campanulidae, a clade of over  <dig>  species that includes both the asteraceae and the dipsacaceae.
many campanulids produce flat-toped inflorescences containing many small flowers, and this arrangement appears to have set the stage both for the differentiation of enlarged  flowers around the periphery of the inflorescence  and for the condensation of the inflorescence into a capitulum .
such inflorescences could have originated in each of these lineages via different pathways, deploying different sets of genes.
instead, our analyses indicate that two widely separated groups within the campanulid angiosperms - asteraceae and dipsacaceae - may have taken advantage of gene duplications within the same family of genes.

 gene duplication is an important evolutionary force, providing the raw material for new genes that, if not lost, may be co-opted to perform novel functions. gene duplication has been implicated in the evolution of transcription factors that play key roles in developmental pathways and are likely involved in morphological evolution. in plants, for example, the mads-box and tcp gene families underwent duplication deep within angiosperm phylogeny, and these duplication events are thought to have played a role in major changes to flower morphology. tcp genes are less well studied than mads-box genes, but they are increasingly appreciated for their role in generating diverse floral forms. the tcp gene family is named for the conserved helix-loop-helix tcp domain from teosinte branched in zea mays, cycloidea in antirrhinum majus, and the proliferating cell factor dna-binding proteins in oryza sativa. in arabidopsis, the tcp gene family has copies, which are divided into a clade of pcf genes, which control cell growth, and a clade of cyc/tb genes. these two clades differ in the length and sequence of the tcp domain, and some members of the cyc/tb clade have an additional conserved arginine rich "r domain". within cyc/tb the "ece" clade contains a group of genes with a conserved short motif between the tcp and r domains. there are three major clades of ece genes in the core eudicots, and the duplications leading to these copies predate this major lineage. we refer to members of the ece clade as "cyc-like" genes. the best studied of these genes, cyc itself, is required for the production of bilaterally symmetrical flowers as a function of its expression in the dorsal portion of the floral meristem. while cyc-like genes have been primarily studied in the context of floral symmetry in single flowers, they have also been implicated in the development of the capitulum inflorescence in asteraceae, a trait that may be associated with the evolutionary success of this group. a typical capitulum consists of many small flowers packed tightly into a condensed head that can closely resemble a single large flower. in some groups, the florets in different parts of the capitulum are morphologically distinct. in radiate species, the outer florets are bilaterally symmetrical while the inner florets are radially symmetrical. in discoid species, there is little or no variation in floret morphology and the capitulum is composed of disk florets only. a third inflorescence type in asteraceae, characteristic of the lactuceae, consists of a capitulum with entirely bilaterally symmetrical flowers. in asteraceae, the evolution of cyc-like genes has been studied thoroughly in the radiate species helianthus annuus, where ten copies have been recovered, which is more that in any other species to date. such extensive gene duplication is thought to be correlated with the evolution of ray versus disk florets; however, this possible connection has not been investigated in any groups outside of the asteraceae, nor has copy number been compared in clades with different inflorescence types. dipsacaceae is one of relatively few groups outside of asteraceae where the capitulum inflorescence occurs and that also contains species with both radiate and discoid heads. dipsacaceae is part of the valerina clade of dipsacales and contains ca. species of perennial and annual herbs that are divided into three main clades: bassecoia, scabioseae, and the dipknautid clade, the latter two of which comprise the "core dipsacaceae". most dipsacaceae species have radiate capitula, with the exception of a few small lineages. these include bassecoia, which is sister to the remaining dipsacaceae, and two groups within the dipknautid clade: dipsacus and a small clade composed of succisa, succisella, and pseudoscabiosa. howarth and donoghue investigated the evolution of cyc-like and divaricata-like genes in dipsacales, and found additional duplications of cyc-like genes in dipsacus pilosus, one of two representatives of dipsacaceae included in the analysis, as well as additional duplications of div-like genes in sixalix atropurpurea, the sole representative of dipsacaceae included in the study of div-like genes. given the unusual floral characteristics associated with this group, further investigation of cyc-like genes from a broader sampling of dipsacaceae is warranted. the aim of this study is to investigate patterns of gene duplication in cyc-like genes and relate patterns of gene evolution to the evolution of radiate versus discoid inflorescences. we also seek to compare the diversification of cyc-like genes in dipsacaceae to that reported for members of asteraceae, to test for a similar pattern of gene duplication. this study represents a first step in eventually deciphering the involvement of cyc-like genes in determining capitulum form and differences in floral symmetry in dipsacaceae.  phylogenetic analysis the combined matrix contains sequences and bases. alignment of the tcp and r domains is unambiguous across taxa, however, the intervening region is difficult to align; therefore, we omitted ambiguous portions of this region from the alignment. all datasets, as well as the full-length alignments, are available in treebase http://www.treebase.org or upon request from the first author. to improve phylogenetic resolution within dpccyc and dpccyc2b, we analyzed these sequences separately in order to align the intervening region between the tcp and r domain. the aligned length of the dpccyc matrix was bases with sequences and the dpccyc2b matrix was bases with sequences. the tree topologies of the bayesian and maximum likelihood analyses were congruent with only minor differences in branch lengths, and consensus trees are shown in figures we recovered between and copies for each species. while we extensively screened representatives of the major lineages of dipsacaceae, we did not recover all hypothesized copies from each species, which may have been the result of unsuccessful amplification or absence/loss from those genomes. our analysis recovered the three main clades of cyc-like genes that have been reported for core eudicots and the additional duplications previously found in representatives of dipsacaceae. we name the three major clades of dipsacaceae cyc-like genes: dpccyc dpccyc and dpccyc also consistent with previous results is the sister relationship of dpccyc and dpccyc novel findings include copies dpccyc2a and dpccyc3a, whose orthologs had been inferred for other members of the dipsacales, and additional duplications within dpccyc and within dpccyc2ba and dpccyc2bb, which are unique to dipsacaceae. the additional duplications within these clades are found only in species with radiate capitula. dpccyc is well supported, with the sequence from b. bretschneideri resolved as sister to two clades of dpccyc in the separate analysis. that is, the duplication within dpccyc appears to have occurred after the core dipsacaceae split from bassecoia. dpccyc1a is recovered only from members of the dipknautid clade, including both discoid and radiate species, while dpccyc1b is found only in radiate species from scabioseae and the dipknautids. within dpccyc1b, there are four clades representing four putative copies. the dpccyc clade is the largest and most complicated group in terms of duplication events and this clade aligns with five copies of helianthus, which has undergone a similar radiation in this gene lineage. we found evidence for one copy of dpccyc2a in all major lineages of dipsacaceae, and within this clade, the gene phylogeny is generally consistent with the species phylogeny. with the separate analysis of dpccyc2b, we were able to further clarify relationships within this gene lineage. the two major copies that were previously reported - dpccyc2ba and dpccyc2bb - are hereafter referred to as "2ba" and "2bb" for simplicity. in the combined analysis of all cyc-like genes, the dpccyc2ba sequence from b. bretschneideri is sister to sequences from all remaining species in this clade, similar to the placement of the sequences from b. bretschneideri in dpccyc in the separate analysis of dpccyc2ba, however, the position of the sequence from b. bretschneideri is unresolved but joins sequences from the other discoid species pseudoscabiosa limonifolia, dipsacus pilosus, and dipsacus inermis outside a large clade composed only of sequences from radiate species from both the scabioseae and dipknautid groups. we inferred five putative copies within this core clade of 2ba. similarly, within the 2bb lineage, we found only one copy in the discoid species. sequences from these species, along with the dipknautid radiate species knautia calycina and cephalaria hirsuta, are resolved outside of a clade composed of sequences from radiate species from both the dipknautids and scabioseae. within this core clade of 2bb, we inferred two putative copies, although support for 2bb was low. lastly, we recovered the fewest sequences from the dpccyc clade. within dpccyc we found evidence for one copy of dpccyc3a for members of scabioseae, but did not recover this gene for bassecoia or members of the dipknautid clade. dpccyc3b was recovered for four species that represent the three major lineages of dipsacaceae and there were two copies of this gene for k. calycina. sequence characteristics the three major clades of dpccyc, as well as the putative copies within these clades, differ in sequence characteristics, most notably the length between the tcp and r domain. dpccyc is the longest copy, with fewer gaps in the intervening region, followed by dpccyc2a. duplications within both dpccyc and dpccyc appear to have been accompanied by a reduction in sequence length between the a and b copies, and the b copies of dpccyc and dpccyc have the shortest sequence lengths. the "ece" region, the characteristic conserved domain found in the intervening region, is easily identifiable in dpccyc dpccyc2a, and dpccyc3a, but apparently absent in all copies of both dpccyc2b and dpccyc3b. in addition to differences in the intervening region, there are also amino acid differences in the tcp domain between the different copies. dpccyc is the most divergent from the other copies. there are also notable differences in sequence characteristics between the putative copies within each of the subclades, including the length of the intervening region as well as amino acid polymorphisms. discussion our study verifies that gene duplication events yielded five major copies of cyc-like genes prior to the origin of dipsacaceae. the major duplication found in dpccyc and the additional putative copies within dpccyc1b are found in dipsacaceae but not in other dipsacales. similarly, the major duplication in dpccyc2b is consistent with previous studies, but the additional duplications within these copies are unique to dipsacaceae. these duplications do not appear to be the result of recent polyploidization events, as they do not occur across all copies. additionally, chromosome counts indicate that there is no evidence for genome doubling in the species used in this study. we feel that the newly discovered subclades within dpccyc and dpccyc2b likely represent different copies, as opposed to alleles, because they contain species from throughout the dipsacaceae phylogeny. in other words, we would not expect allele sequences to be conserved across distant relatives. gene trees in relation to the species tree congruence between gene trees and the species tree within dipsacaceae is difficult to interpret. outgroups in the linnina clade are generally resolved as sister to dipsacaceae, although phylogenetic relationships between these species tend to be poorly supported. the two helianthus cyc1-like copies are resolved outside of the cyc1-like clade, indicating that better phylogenetic sampling is needed to clarify relationships among copies in this part of the tree. within dpccyc the failure to amplify dpccyc1a from members of scabioseae, combined with the additional copies in dpccyc1b, renders species relationships within this clade unclear. the dpccyc2a tree is more or less consistent with phylogenetic studies, although the position of b. bretschneideri is weakly resolved with members of the dipknautids rather than as sister to all other dipsacaceae, as in the species phylogeny. within dpccyc2b, the presence of multiple copies precludes the inference of species relationships. lastly, congruence between the gene trees and the species tree in both copies of dpccyc cannot be determined due to limited taxon sampling and poor phylogenetic signal in the gene trees. based on these results, the utility of most dpccyc genes for use in phylogenetic studies would be problematical due to the presence of multiple putative copies, low amplification success for some genes, short sequences with a highly conserved tcp domain that yield low phylogenetic signal, and difficulties in aligning the intervening region. a possible exception is dpccyc2a, which shows some promise in elucidating species relationships. this gene may be enhanced as a phylogenetic marker by using the full-length sequence rather than the shorter region between the tcp and r domain used in this study. strikingly, we found no additional duplications in any of the major subclades for bassecoia bretschneideri. in the separate analysis of dpccyc the main duplication event occurred after bassecoia split from the core dipsacaceae. similarly, we found no evidence for additional duplications in dpccyc in dipsacus inermis, dipsacus pilosus, or pseudoscabiosa limonifolia, the other discoid species included in this study. in the 2ba and 2bb clades, both dipsacus species and p. limonifolia occupy a position along with b. bretschneideri outside of the core clades of these lineages that include all radiate species. given these results and our current understanding of dipsacaceae phylogeny, it is perhaps most likely that the 2ba and 2bb genes duplicated in the lineage leading to the core dipsacaceae and were subsequently lost twice: once in the lineage leading to pseudoscabiosa and once in dipsacus. however, we cannot rule out that by using a degenerate primer approach we failed to amplify these copies in bassecoia, pseudoscabiosa, and dipsacus. in any case, the occurrence of the same general pattern of duplication in three different cyc-like genes is potentially of great interest. diversification of cyc-like genes and character evolution the pattern of cyc-like gene diversification appears to generally correlate with inflorescence form in dipsacaceae. groups with radiate capitula are represented in the major clades and subclades of dpccyc, while the discoid groups bassecoia, pseudoscabiosa, and dipsacus appear to have only one copy of dpccyc, 2ba, and 2bb. while cyc-like genes have been investigated in functional studies of other asteraceae species, gene duplication has been investigated most extensively in helianthus. the highest reported number of cyc-like genes is for helianthus annuus, a radiate species of asteraceae, and the pattern of diversification in radiate dipsacaceae groups is remarkably similar. while we did not recover all copies for each species, we hypothesize that there are at least and possibly copies of cyc-like genes in radiate members of dipsacaceae, depending on better resolution within 2bb and whether the duplication in dpccyc3b found in knautia can be generalized to other radiate species. for discoid groups, our study infers copies. the duplications in helianthus and radiate dipsacaceae occur within the same gene lineages. helianthus has two copies of cyc1-like genes and we recovered five putative copies for radiate species of dipsacaceae. most notably, there are five copies of helianthus in the cyc2-like clade, and seven or eight copies in radiate species of dipsacaceae. lastly, helianthus has three copies in the cyc3-like clade, while members of dipsacaceae appear to have at least two, although copy number was difficult to assess for this gene clade due to problems with amplification. if we assume that most cyc-like genes that are present have been discovered, then it is possible that the independent evolution of radiate capitula in asteraceae and dipsacaceae may have been associated with independent duplication events in orthologous cyc-like genes. however, in the absence of expression data for cyc-like genes in dipsacaceae and broader studies of gene duplication events within asteraceae, this is clearly speculative. to date, the evolutionary history of cyc-like genes has not been investigated in discoid asteraceae, so it is unknown whether these species also diverged in copy number from their radiate relatives. the additional duplications in cyc1-like and cyc2b-like genes are of particular interest because, within the core eudicots, they may be specific to helianthus and dipsacaceae, at least within the species that have been surveyed thus far. in most previous studies, only a single copy of cyc1-like genes has been found. our finding that only members of the dipknautid clade have dpccyc1a is potentially interesting given the differences in floral traits between the main clades of dipsacaceae. members of scabioseae have five petals while the dipknautids have four, and the epicalyx in scabioseae is characterized by several structural modifications that are not present in bassecoia or in the dipknautids. however, functional information for cyc1-like genes is limited, although putative orthologs in arabidopsis are thought to be involved in branching architecture. tb is similar in sequence to genes in the cyc1-like clade, and is also responsible for differences in branching architecture between cultivated maize and its wild progenitor, teosinte. while there are no functional data for helianthus, the two copies of cyc1-like genes were found to diverge in expression patterns, with hacyc1a expressed across all tissues except roots, and hacyc1b expressed only in the petals. due to potential differences in cyc1-like genes between scabioseae and the dipknautid clade and the presence of multiple copies of dpccyc1b in radiate species, cyc1-like genes are attractive candidates for future study. previous studies have generally found more duplications in the cyc2-like genes than in the cyc1-like or cyc3-like genes; however, no more than three copies have been reported outside of helianthus and dipsacaceae. the cyc2-like clade includes cyc from antirrhinum, and its orthologs have been studied in several plant groups, including in members of asteraceae, where it has been suggested that cyc-like genes might be responsible for differentiating the radially symmetrical disk florets from the bilaterally symmetrical ray florets. in both gerbera and senecio, cyc2-like genes are expressed in ray florets and functional data suggest they are involved in differentiating the radially symmetrical disk florets from the bilaterally symmetrical ray florets. while no functional data exist for the helianthus orthologs of cyc2-like genes, their expression patterns were found to diverge, which may be indicative of sub and/or neofunctionalization of the duplicated cyc2-like copies. while our correlational study cannot establish causation, the higher number of dpccyc2b genes in radiate versus discoid dipsacaceae suggests that they might also play a role in the development of ray florets. expression studies and functional data are needed to address this possibility.  the results presented here represent the first step in assessing the role of cyc-like genes in dipsacaceae development. we recovered the five major clades that were reported in previous studies and discovered several additional copies, most notably in the dpccyc and dpccyc2b clades. the dpccyc2b clade in particular demonstrates a dynamic history of gene duplication. interestingly, the pattern of cyc-like diversification in dipsacaceae is very similar to that of helianthus annuus, a species of asteraceae that has been screened for cyc-like genes. our study also suggests that the number of dpccyc copies tends to correlate with the distribution of radiate versus discoid inflorescence form, with species bearing radiate heads having more than twice the number of cyc-like genes as discoid species. investigations of cyc-like genes in discoid members of asteraceae are needed to assess the generality of this pattern. capitulum inflorescences have evolved independently in a number of angiosperm lineages, but perhaps most often within the campanulidae, a clade of over species that includes both the asteraceae and the dipsacaceae. many campanulids produce flat-toped inflorescences containing many small flowers, and this arrangement appears to have set the stage both for the differentiation of enlarged flowers around the periphery of the inflorescence and for the condensation of the inflorescence into a capitulum. such inflorescences could have originated in each of these lineages via different pathways, deploying different sets of genes. instead, our analyses indicate that two widely separated groups within the campanulid angiosperms - asteraceae and dipsacaceae - may have taken advantage of gene duplications within the same family of genes. 
the similarity in cyc-like gene diversification seen in dipsacaceae and some members of the asteraceae sets the stage to investigate whether the convergent evolution of capitulum inflorescences in both groups may have been underlain by convergent evolution in the same gene family. we found that the number of cyc-like genes in dipsacaceae is similar to that reported for members of asteraceae and that the same gene lineages have duplicated in a similar fashion independently in both groups. two main inflorescence types are present in dipsacaceae: radiate species contain two kinds of floret within the flowering head, and discoid species contain only disk florets. cyc-like genes have undergone extensive duplication in dipsacaceae, with radiate species having more copies than discoid species, suggesting a potential role for these genes in the evolution of disk and ray florets. cycloidea -like genes have been implicated in the development of capitulum inflorescences in asteraceae, where many small flowers are packed tightly into an inflorescence that resembles a single flower. to test whether a dynamic pattern of gene duplication, similar to that documented in asteraceae, is present in dipsacaceae, and whether these patterns are correlated with different inflorescence types, we inferred a cyc-like gene phylogeny for dipsacaceae based on representative species from the major lineages. we recovered within dipsacaceae the three major forms of cyc-like genes that have been found in most core eudicots, and identified several additional duplications within each of these clades. the number of cyc-like genes recovered for radiate versus discoid species differed, with discoid species having fewer copies of cyc1-like and cyc2b-like genes. we investigated the evolution of cyc-like genes in dipsacaceae, a plant clade in which capitulum inflorescences originated independently of asteraceae. several rounds of duplication of cyc-like genes have occurred in asteraceae, and this is hypothesized to be correlated with the evolution of the capitulum, which in turn has been implicated in the evolutionary success of the group.
 cycloidea -like genes have been implicated in the development of capitulum inflorescences in asteraceae, where many small flowers are packed tightly into an inflorescence that resembles a single flower. several rounds of duplication of cyc-like genes have occurred in asteraceae, and this is hypothesized to be correlated with the evolution of the capitulum, which in turn has been implicated in the evolutionary success of the group. we investigated the evolution of cyc-like genes in dipsacaceae, a plant clade in which capitulum inflorescences originated independently of asteraceae. two main inflorescence types are present in dipsacaceae: radiate species contain two kinds of floret within the flowering head, and discoid species contain only disk florets. to test whether a dynamic pattern of gene duplication, similar to that documented in asteraceae, is present in dipsacaceae, and whether these patterns are correlated with different inflorescence types, we inferred a cyc-like gene phylogeny for dipsacaceae based on representative species from the major lineages.  we recovered within dipsacaceae the three major forms of cyc-like genes that have been found in most core eudicots, and identified several additional duplications within each of these clades. we found that the number of cyc-like genes in dipsacaceae is similar to that reported for members of asteraceae and that the same gene lineages have duplicated in a similar fashion independently in both groups. the number of cyc-like genes recovered for radiate versus discoid species differed, with discoid species having fewer copies of cyc1-like and cyc2b-like genes.  cyc-like genes have undergone extensive duplication in dipsacaceae, with radiate species having more copies than discoid species, suggesting a potential role for these genes in the evolution of disk and ray florets. the similarity in cyc-like gene diversification seen in dipsacaceae and some members of the asteraceae sets the stage to investigate whether the convergent evolution of capitulum inflorescences in both groups may have been underlain by convergent evolution in the same gene family. 