BACKGROUND
cyclin-dependent kinases  are serine and threonine kinases whose actions are dependent on the binding of regulatory subunits known as cyclins  <cit> .
various cyclins are synthesized and destroyed at specific times during the cell cycle, thus regulating cdk activity in a timely manner  <cit> .
cdk and cyclin families function in a variety of cellular processes, including cell cycle regulation, transcription, rna processing, translation, neurogenesis, and apoptosis  <cit> .
the evolution of metazoans from protozoans is a major milestone in the history of life.
this transition has been generally marked by increases in the number of genes involved in cell differentiation, cell-cell communication, and cell adhesion  <cit> .
the evolutionary histories of transcription factors   <cit> , cell-cell communication-related genes   <cit> , and cell adhesion genes   <cit>  during the unicellular to metazoan transition have been extensively investigated.
in addition to cell communication and cell adhesion proteins, other proteins may be linked with metazoan emergence.
based on a comprehensive phylogenetic analysis of sponge amphimedon queenslandica proteins, it has been proposed that the emergence of metazoan multicellularity may have been related to the evolution of various genes functioning in cell cycling and growth, programmed cell death, cell-cell and cell-matrix adhesion, developmental signaling and gene regulation, allorecognition and innate immunity, and cell type specialization  <cit> .
as implied by the aforementioned study, investigation of the evolutionary history of cell cycle control genes could enhance our understanding of metazoan emergence from single-celled ancestors.
at present, however, comprehensive evolutionary analyses have been carried out only for a few cell cycle control genes, such as p <dig>  rb, and e2f families  <cit> .
the core machinery of the animal cell cycle can generally be traced back to early eukaryotes  <cit> .
it was previously proposed that the eukaryotic cell cycle was controlled by the dna damage checkpoint kinase chk1p at early stages of evolution, and duplications of kinase genes occurred during subsequent evolution.
gradually, eukaryotic kinases were added to the cell cycle control system, with cdks being among the last major additions  <cit> .
however, cyclin-dependent kinases  in yeast and animal are thought to be the cornerstone in cell cycle control  <cit> .
according to recent reports,  <dig> cdk and approximately  <dig> cyclin genes are present in humans  <cit> .
the evolution of cdk and cyclin families has been studied previously.
an analysis of the cdk family in yeasts and animals divided the cdk family into seven subfamilies   <cit> , while another analysis examined  <dig> cdk family members from animals, plants, yeasts, and four protists  <cit> .
with respect to the cyclin family, one phylogenetic analysis covered a-, b-, d-, and e-type cyclin proteins in animals and fungi  <cit> ; another analysis included fungal, plant, and protist cyclins, and successfully divided all cyclins in three groups  <cit> .
these analyses only incorporated a relatively limited number of organisms, however, with several representative organisms occupying key positions in the transition from unicellular to metazoan organisms not analyzed.
taking advantage of the increasing number of sequenced genomes, in this study we conducted a comprehensive evolutionary analysis of  <dig> cdk and  <dig> cyclin genes from  <dig> representative organisms.
our analysis incorporated several organisms important to the study of metazoan emergence, such as the closest known metazoan relative, the choanoflagellate monosiga brevicollis <cit> ; the oldest surviving metazoan, amphimedon queenslandica <cit> ; the earliest eumetazoan, trichoplax adhaerens <cit> ; and the cnidarian nematostella vectensis <cit> .
we also included several unicellular organisms, such as the choanoflagellate salpingoeca rosetta and the filasterean capsaspora owczarzaki, that are recognized as close relatives of metazoans based on data from the origins of multicellularity project  <cit> .
our results revealed detailed evolutionary information regarding cdk and cyclin proteins in metazoan organisms and their unicellular relatives, and provided evidence for simultaneous cdk4/6-cyclin d complex and eumetazoan emergence.
methods
database searching and identification of cdk and cyclin sequences
for cdk proteins, we performed psi-blast searches using human cdk <dig> and cdk <dig> protein sequences as queries  <cit>  against the ncbi non-redundant protein database  for  <dig> organisms: homo sapiens, ciona intestinalis , strongylocentrotus purpuratus , branchiostoma floridae, drosophila melanogaster , n. vectensis, t. adhaerens, a. queenslandica, monosiga brevicollis, s. rosetta, c. owczarzaki, schizosaccharomyces pombe , saccharomyces cerevisiae , coprinopsis cinerea , and dictyostelium discoideum .
the search results were used as new queries in a second round of blast searching, which was continued until no new sequences were returned.
we also performed a similar blastp search against the broad institute database  <cit>   to collect cdk sequences from three unicellular organisms: sphaeroforma arctica , spizellomyces punctatus , and thecamonas trahens , as these sequences are not available in the ncbi database.
for cdk genes, only the longest protein sequence encoded by each gene was retained.
we also carried out a preliminary phylogenetic analysis on all putative cdk family proteins collected from blast searching.
proteins clustering with human cdks were used in subsequent analyses, whereas those clustering with other human protein kinases, such as map kinases, were discarded.
using human cyclin b, cyclin c, and cyclin y proteins as queries, similar blast searches were carried out to identify cyclin proteins from related organisms in the ncbi and broad institute databases.
because cyclin proteins are greatly diverged, an hmm search  against non-redundant proteins from genbank was also carried out  <cit>  using pfam profile pf <dig>  which corresponds to the cyclin-n domain—the most highly conserved cyclin protein domain  <cit> .
for three unicellular organisms , cyclin-n domain-containing proteins were also collected from the broad institute database  <cit> .
only the longest protein sequence associated with each cyclin gene was retained.
we verified the putatively identified cyclin proteins by searching against pfam  and smart  databases  <cit> .
proteins lacking cyclin-n domains were discarded.
similar to a previous analysis  <cit> , non-cyclin proteins possessing cyclin-n domains  were identified by reciprocal blast searching and removed.
protein alignment and phylogenetic analyses
after evaluating several multiple alignment programs, we used msaprobs  <cit>  for multiple alignment of most full-length proteins.
alignments that included cyclin sequences from t. trahens and d. discoideum were carried out using promals  <cit> , a program more suitable for alignment of distantly related proteins  <cit> .
poorly aligned positions in these alignments were removed, with only the conserved region—the cdk domain for the cdk family, and cyclin-n and –c domains for the cyclin family—used for further phylogenetic analyses.
alignments used for phylogenetic analyses are found in additional file 1: file s <dig>  phylogenetic analyses were performed using maximum likelihood  and bayesian methods, with optimum substitution models determined for each alignment based on the akaike information criterion using prottest  <dig>   <cit> .
ml trees were constructed using raxml  <dig> .
<dig>  <cit>  as implemented in the cipres science gateway v.  <dig>   <cit>  with  <dig> bootstrap resamplings.
bayesian phylogenetic analyses were carried out under an lg substitution model using phylobayes v.  <dig>   <cit> , with markov chain monte carlo runs terminated when maxdiff <  <dig> .
multiple sequence alignments and phylogenetic tree files were deposited in labarchives .
tree files were viewed using the dendroscope program  <cit> , and phylogenetic networks were constructed with splitstree v. <dig>  <cit> .
ortholog identification
as suggested in a recent review  <cit> , ortholog identification of different cdk and cyclin subfamilies was mainly based on results of the phylogenetic analyses; however, results from reciprocal blast search methods   <cit>  were also referenced for some distantly related proteins.
in general, a protein was identified as an ortholog of a representative cdk or cyclin subfamily if it clustered with that subfamily in the ml phylogenetic tree with greater than 50% bootstrap support.
for proteins clustering with less than 50% ml bootstrap support within a subfamily, reciprocal blast results were consulted: only proteins in the initial blast query for which e-values returned for members of a representative cdk or cyclin subfamily were five orders of magnitude better  than those of the next best-scoring cdk or cyclin subfamily were considered to be orthologs of that subfamily.
such a “five-orders criterion” has recently been described and used to identify arf gap orthologs  <cit> .
cyclin proteins placed with low bootstrap support into a representative cyclin subfamily and not meeting the five-orders criterion were designated as unclassified cyclin proteins.
RESULTS
as summarized in table  <dig>  we identified  <dig> cdk and  <dig> cyclin proteins from  <dig> representative organisms.
detailed information regarding these cdks and cyclins may be found in additional file 2: file s <dig> and additional file 3: file s <dig>  respectively.
the evolutionary history of cdk family
we attempted to perform global phylogenetic analyses using ml and bayesian methods on all  <dig> cdk proteins from  <dig> organisms.
a robust and reliable phylogenetic tree could not be obtained by either method, however, possibly because of the large number of sequences.
we therefore carried out separate phylogenetic analyses on subsets of the  <dig> organisms.
we first analyzed cdk sequences from h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta <dig> as one group .
these six organisms, except for h. sapiens, are all located in key positions with respect to metazoan emergence.
given that lineage-specific gene duplication and loss may have occurred in some of these organisms, any conclusions drawn from this subset may not be adequate to fully understand the evolutionary history of the cdk family during metazoan emergence.
consequently, analyses of cdk sequences from other organisms were also carried out; one analyzed subset comprised c. intestinalis, b. floridae, s. purpuratus, d. melanogaster, t. adhaerens, and h. sapiens , while another group consisted of c. owczarzaki, s. arctica, s. cerevisiae, s. pombe, s. punctatus, c. cinerea, t. trahens, d. discoideum, t. adhaerens, and h. sapiens .
results of these phylogenetic analyses are summarized in figure  <dig> and additional file 6: table s <dig>  and are described in detail below.
based on our analyses, the entire cdk family can be divided into eight subfamilies: cdk <dig>  cdk <dig>  cdk8/ <dig> , cdk <dig> , cdk10/ <dig> , cdk <dig> , cdk4/ <dig> , and cdk <dig>  .
although these results are generally consistent with previous reports  <cit> , our analysis revealed more detailed information and included human cdk <dig> and its orthologs.
we found that orthologs of seven cdk subfamilies  are present in basal metazoans t. adhaerens and a. queenslandica, the choanoflagellate m. brevicollis, and/or s. rosetta .
in further analysis, orthologs of  <dig> cdk subfamilies  could be identified in fungi, t. trahens, and d. discoideum.
a cdk <dig> representative is present in the fungus s. punctatus , but is absent in s. cerevisiae, s. pombe, t. trahens, and d. discoideum .
compared with other cdk subfamilies, the cdk4/ <dig> subfamily is unique, as it is only found in eumetazoan organisms such as t. adhaerens  and n. vectensis , and is absent in the basal metazoan a. queenslandica, choanoflagellates, and other investigated unicellular organisms .
the different functions of these cdk subfamilies have been described in an excellent review  <cit> .
the cdk4/ <dig> subfamily is generally recognized as animal-specific, but previous phylogenetic analyses supporting this conclusion have only included a relatively small number of organisms  <cit> .
in the tree shown in figure  <dig>  subfamilies cdk4/ <dig>  cdk1/2/ <dig>  and cdk <dig> generally cluster together.
as phylogenetic networks are useful for describing complex evolutionary scenarios such as horizontal gene transfer and recombination  <cit> , we carried out a phylogenetic network analysis for cdk4/ <dig>  cdk1/2/ <dig>  and cdk <dig> subfamily proteins from h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta .
the results of that analysis were generally consistent with our phylogenetic tree topology, cdk4/ <dig> subfamily is located between cdk1/2/ <dig> subfamily and cdk <dig> subfamily.
the detail evolutionary information among cdk4/ <dig> subfamily, cdk1/2/ <dig> subfamily and cdk <dig> subfamily are still requiring further study.
anyway, our analysis is the first to map the detailed evolutionary history of the cdk4/ <dig> subfamily in representative organisms occupying key positions along the transition from unicellular organisms to metazoans.
our results indicate that the cdk4/ <dig> subfamily is linked simultaneously with eumetazoan appearance.
subfamilies cdk <dig>  cdk10/ <dig>  cdk1/2/ <dig>  and cdk <dig> all contains more than one cdk members in metazoan organisms and every subfamily could be divided into two or three clades.
our analysis provided some detailed information about how and when these clades were formed in different subfamilies.
the cdk <dig> subfamily consists of two clades, cdk <dig> and cdk12/ <dig>  basal metazoan organisms t. adhaerens and a. queenslandica have representative members in both clades .
in addition, consistent with previous reports  <cit> , s. pombe lsk <dig> and s. cerevisiae ctk1p belong to the cdk12/ <dig> clade, while s. cerevisiae sgv1p and s. pombe cdk <dig> are members of the cdk <dig> clade .
these results imply that the cdk <dig> subfamily split into two clades  before the divergence of metazoans and fungi.
in humans, cdk <dig> is reported to regulate transcription by phosphorylating the c-terminal domain of rna polymerase ii  <cit> .
the cdk10/ <dig> subfamily comprises clades cdk <dig> and cdk <dig> .
basal metazoans a. queenslandica and t. adhaerens have members in both clades; similarly, unicellular m. brevicollis is represented in both cdk <dig>  and cdk <dig> .
in further analysis, we found that one s. punctatus protein  was classified into the cdk <dig> clade, and that one s. pombe protein  and one c. cinerea protein  were placed in the cdk <dig> clade .
proteins from d. discoideum  and t. trahens  were only grouped into the cdk10/ <dig> subfamily, and were not found in clades cdk <dig> and cdk <dig>  in addition to their roles in regulating transcription, cdk <dig> and cdk <dig> display distinct functions during the g2/m transition  <cit> .
the cdk1/2/ <dig> subfamily can be divided into cdk <dig> and cdk2/ <dig> clades in metazoans.
bona fide cdk <dig> clade members are found in a. queenslandica  and t. adhaerens , and similarly cdk2/ <dig> clade members are present in a. queenslandica  and t. adhaerens  .
cdk1/2/ <dig> subfamily members from m. brevicollis , s. rosetta, fungi, t. trahens, and d. discoideum, however, are not placed into cdk <dig> or cdk2/ <dig> clades .
our data indicate that genes of the ancient cdk1/2/ <dig> subfamily arose in the common ancestor of amoebozoans and fungi, and then diverged via gene duplication into clades cdk1and cdk2/ <dig> in metazoans.
in the tree in figure  <dig>  the cdk <dig> subfamily is divided into clades cdk <dig>  cdk16/17/ <dig>  and cdk14/ <dig>  the placozoan t. adhaerens possesses three cdk <dig> subfamily members, which are classified into cdk <dig> , cdk16/17/ <dig> , and cdk14/ <dig>  clades .
we found that one m. brevicollis protein  is clustered into cdk <dig>  whereas another  is placed into cdk16/17/ <dig> .
in the comprehensive analysis, some fungal cdk <dig> subfamily members  were classified into the cdk <dig> clade, while others  were placed into clades cdk16/17/ <dig> and cdk14/ <dig> .
these data indicate that the cdk <dig> subfamily originated in single-celled ancestors, and subsequently split into three clades before or during eumetazoan emergence.
in humans, cdk <dig> is essential for neuronal cell cycle arrest and differentiation  <cit> .
the evolutionary history of cyclin family
based on the same reasons as in the cdk family, we carried out separate phylogenetic analyses on subsets of cyclin family members.
as for the cdks, we performed analyses of cyclin proteins from the group h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta , and then from a subset consisting of c. intestinalis, b. floridae, s. purpuratus, d. melanogaster, t. adhaerens, and h. sapiens .
because their cyclin sequences diverged greatly, we failed to obtain a reliable cyclin phylogenetic tree from eight organisms: c. owczarzaki, s. arctica, s. cerevisiae, s. pombe, s. punctatus, c. cinerea, t. trahens, d. discoideum, t. adhaerens, and h. sapiens.
we thus divided these organisms into smaller subsets for analysis: one group for filasterean and ichthyosporean organisms , one group for fungi  , and one group for apusozoa and amoebozoa  .
these subgroups were analyzed, and orthologs of different cyclin subfamilies were classified  based on the phylogenetic results and those of reciprocal blast analysis.
according to our phylogenetic analyses, the metazoan cyclin family could be divided into  <dig> subfamilies , and fungi organisms owns three fungi specific subfamily  .
a recent cyclin family analysis indicated that the cyclin family could be divided three groups   <cit> , and our analysis confirmed it .
in this manuscript, we will refer group i as cyclin b like group , group ii as cyclin y like group , and group iii as cyclin c like group .
though our results is general consistent with previous analysis  <cit> , some new information was revealed by our analysis.
for example, our analysis successfully identificated cyclin c ortholog  and cyclin j ortholog  in c. intestinalis, the previous analysis  <cit>  which also included c. intestinalis missed this information.
most subfamilies in the cyclin c-like group are conserved in metazoans, choanoflagellates, fungi, and d. discoideum.
we found that cyclins c, h, and l are all conserved in metazoans, choanoflagellates, fungi, t. trahens, and d. discoideum .
orthologs of cyclins t and k were found in metazoans ; in fungi and d. discoideum, however, homologous proteins of cyclin t and/or cyclin k subfamily could only be identified as the common ancestor of the two subfamilies .
our data indicate that the common ancestor of cyclins k and t originated early in the course of evolution, and then diverged no later than during the period of metazoan emergence.
fam <dig> is generally conserved only in metazoans , although an ortholog is also found in s. arctica .
we found that the cyclin y subfamily is conserved in metazoans, choanoflagellates, and d. discoideum .
interestingly, one fungal protein in s. punctatus  was identified as an ortholog of cyclin y .
pcl subfamily members are found in s. cerevisiae and c. cinerea .
in fact, cyclin y and pcl subfamilies cluster together tightly in the phylogenetic tree .
cyclins y and pcl are binding partners of cdk <dig> subfamilies in metazoans and fungi, respectively  <cit> ; although we list them as two separate subfamilies, as previously reported  <cit> , we believe they share a common ancestor.
evolutionary conservation varied greatly among different subfamilies in the cyclin b-like group.
these subfamilies are described in detail as follows.
the cyclin b subfamily is conserved in metazoans, choanoflagellates, fungi, t. trahens, and d. discoideum .
the fungus-specific subfamily clb is related to the cyclin b family, with blast e-values as low as approximately 1 × 10− <dig>  cyclins b and clb are binding partners of cdk <dig> subfamilies in metazoans and fungi, respectively  <cit> .
consequently, although we treat cyclins b and clb as two subfamilies, as previously reported  <cit> , they appear to share a common ancestor in early eukaryotic lineages, as suggested by previous analyses  <cit> .
alignments of representative cyclin b subfamily proteins from metazoan organisms h. sapiens and t. adhaerens and unicellular organisms s. rosetta, c. owczarzaki, t. trahens, and d. discoideum are shown in figure  <dig>
our analysis results indicate that the cyclin a subfamily is conserved in metazoans, unicellular choanoflagellates, and c. owczarzaki, but is absent in fungi, t. trahens, and d. discoideum .
alignments of representative cyclin a subfamily proteins from metazoans h. sapiens, t. adhaerens, and unicellular organisms m. brevicollis, s. rosetta, and c. owczarzaki are given in figure  <dig>
the fungus-specific subfamily cln, which functions in cell cycle regulation, is the binding partner of fungus cdk <dig>  results of blast analysis revealed similar genetic distances between the cln subfamily and the metazoan cyclin a subfamily, and between cln and the metazoan cyclin b subfamily.
the cyclin e subfamily is not only conserved in metazoans, but is also present in several unicellular organisms such as choanoflagellate s. rosetta, c. owczarzaki, and t. trahens .
this result conflicts with previous studies indicating that cyclin e is animal-specific  <cit> .
this inconsistency may be due to the failure of previous analyses to incorporate several important unicellular organisms, such as s. rosetta and c. owczarzaki.
alignments of representative cyclin e subfamily proteins from metazoans h. sapiens and t. adhaerens and unicellular organisms s. rosetta, c. owczarzaki, and t. trahens are shown in figure  <dig>
we found that orthologs of cyclin d are present in t. adhaerens  and n. vectensis , but are absent in m. brevicollis, s. rosetta, and a. queenslandica .
comprehensive analysis unexpectedly revealed that cyclin d orthologs are present in t. trahens  , although absent in fungi and d. discoideum .
although plant d-type cyclin is generally considered to be homologous to animal cyclin d  <cit> , plants do not possess cdk4/ <dig> orthologs; instead, plant d-type cyclin functions together with plant cdka, a cdk <dig> homolog, in the g <dig> phase  <cit> .
possibly because of low sequence similarity , plant d-type cyclin did not cluster together with animal cyclin d in a previous phylogenetic analysis  <cit> .
our phylogenetic results are the first to reveal the presence of a bona fide ortholog of the animal cyclin d subfamily in a non-opisthokont, t. trahens.
the blast e-value for this protein against human cyclin d is approximately 1 × 10− <dig>  this result indicates that the cyclin d subfamily arose in early eukaryotes, and that the absence of cyclin d in fungi and many other unicellular organisms may be due to lineage-specific gene loss in these organisms.
similar to plant organisms, the non-opisthokont t. trahens does not possess an ortholog of the cdk4/ <dig> subfamily.
our alignment of representative cyclin d subfamily proteins from h. sapiens, n. vectensis, t. adhaerens, and t. trahens is displayed in figure  <dig>
in our analyses, cyclins i and g always clustered together.
cyclins i and g collectively have representative members in unicellular organisms c. owczarzaki, s. arctica, a. queenslandica, and n. vectensis .
these data indicate that cyclin subfamilies i and g are derived from a common ancestral gene that was present in unicellular organisms, with this common ancestor differentiating into cyclins i and g after the emergence of n. vectensis.
in addition to the above phylogenetic analyses, we conducted a phylogenetic network analysis of cyclin b-like group proteins from h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta .
it was found that the cyclin d subfamily is located between subfamily e and subfamily g/i.
the detail evolutionary information among cyclin d subfamily, cyclin e, and cyclin g/i will be an interesting topic for further study.
cell cycle related cdk/cyclin evolutionary histories during animal emergence
information is limited regarding evolution of cell cycle regulation in eukaryotes.
it is generally believed, however, that early eukaryotes already possessed complex cell cycle regulation, with key cell cycle regulators having subsequently undergone divergent functional specializations in different organisms  <cit> .
for example, the rb-e2f pathway, which functions in cell cycle regulation, is conserved in animals and plants, but has been lost in fungi  <cit> .
the eukaryotic cell cycle is controlled by a complicated regulatory network  <cit> .
cdk-cyclin complexes, as key regulators of the cell cycle, phosphorylate a variety of substrates during the cell cycle  <cit> .
in humans, for example, cdk4/cyclin d phosphorylates prb during the g <dig> phase  <cit> , and cdk1-cyclin b phosphorylates cdc25c and wee1a during the m phase  <cit> .
a recent structural study  <cit>  revealed that the conformation of t cdk4/ cyclin d <dig> diverges from that of previously known cdk-cyclin binary complexes, and cdk <dig> might have a unique regulation and activation mechanism compared with that of cdk2-cyclin a  <cit> .
another study has also found that the structural mechanism of cdk4-cyclin d <dig> activation differs markedly from that of previously studied cdk2-cyclin a complexes  <cit> .
our analysis has provided detailed evolutionary information on cdk and cyclin subfamilies in metazoans and related organisms.
our data are the first to reveal that cyclin d orthologs are present in a non-opisthokont , but have generally been lost in fungi and most other unicellular opisthokonts, such as m. brevicollis, s. rosetta, c. owczarzaki, and s. arctica.
our analysis also found that cyclin e is not restricted to animals, but is present in several unicellular organisms.
investigations of cell cycle regulation have primarily been carried out in animals  and yeasts .
in animals, cdk4/ <dig> and cyclin d have been determined to function in the g <dig> phase, human cdk <dig> and cyclin a/e in s and g <dig> phases, and cdk <dig> and cyclin b in the m phase  <cit> .
in yeasts, s. cerevisiae cdk <dig>  functions in g <dig>  s, g <dig>  and m phases with different cyclins  <cit> .
given the large evolutionary distance between yeasts and animals, it has proved difficult to elucidate the evolutionary history of cell cycle regulation and its relationship to the emergence of metazoans from their single-celled ancestors.
in this study, we analyzed cell cycle-related cdks  and cyclins  in several representative organisms, such as m. brevicollis <cit> , a. queenslandica <cit> , and t. adhaerens <cit> , which occupy key positions for metazoans origination from their single-celled ancestors organisms .
we discovered that the number of cell cycle-related cdk and cyclin proteins has gradually increased from m. brevicollis and a. queenslandica to t. adhaerens: m. brevicollis possesses orthologs for cdk <dig>  cyclin b, cyclin a, and cyclin e, a. queenslandica has orthologs for cdk <dig>  cdk <dig>  cyclin b, cyclin a, and cyclin e, and t. adhaerens features orthologs for cdk <dig>  cdk <dig>  cdk <dig>  cyclin b, cyclin a, cyclin e, and cyclin d .
based on the evolutionary information uncovered for these cdk and cyclin proteins, we are able to propose different scenarios for the function of cdk and cyclin proteins in cell cycle control in representative organisms m. brevicollis, a. queenslandica, and t. adhaerens .
because m. brevicollis does not possess cdk4/ <dig> and cyclin d orthologs, we speculate that the ortholog of cdk <dig> in m. brevicollis  may function throughout the cell cycle with different cyclins, similar to s. cerevisiae cdk <dig>  .
as indicated in figure  <dig>  we have inferred that the cdk4/6-cyclin d complex appeared at the same time as the emergence of the eumetazoan t. adhaerens.
it is interesting that the cdk4/6-cyclin d complex first emerged in t. adhaerens, as t. adhaerens has four morphologically identifiable somatic cell types and is thought to represent the earliest eumetazoan lineage  <cit> .
the cdk4/6-cyclin d complex functions in the g <dig> phase, the first phase within interphase  <cit> .
the duration of the g <dig> phase is highly variable among different cells in animals, and is affected by limiting growth factors, nutrient supply, temperature, and additional inhibiting factors  <cit> .
for example, human embryonic stem cells are characterized by an abbreviated g <dig> phase and lack the classical restriction  point that normally controls commitment for progression into the s phase  <cit> .
in contrast, somatic cell proliferation is linked to growth factor-dependent passage through the r point in the g <dig> phase  <cit> .
in fission yeast, a single oscillation of p34cdc <dig> kinase activity provided by a single b-type cyclin can promote ordered progression into both dna replication and mitosis  <cit> .
the function of cdk4-cyclin complexes in animals has been extensively studied and reviewed  <cit> .
with respect to the cyclin d family, mice lacking cyclin d <dig>  d <dig>  or d <dig> exhibit different developmental anomalies  <cit> .
mice expressing cyclin d <dig>  but not d <dig> and d <dig>  have been observed to die before embryonic day   <dig>   <cit> , while mice lacking all three cyclins die before e <dig>   <cit> .
in regard to cdk <dig> and cdk <dig>  mice lacking cdk <dig> or cdk <dig> also exhibit different developmental anomalies  <cit> , with mice lacking both cdk <dig> and cdk <dig> displaying progressive embryonic lethality from e <dig>  onward, and the few live pups dying shortly after birth  <cit> .
these data clearly indicate that the cdk4-cyclin d complex plays critical roles during mouse early embryonic development.
it would be interesting to study the function of cdk4-cyclin d in early eumetazoan organisms such as n. vectensis and t. adhaerens.
based on a comparative analysis of cell cycle regulatory networks in animals, yeasts, and plants, harashima et al.
<cit>  have recently suggested that the cycd/cyce clade has undergone lineage-specific expansion and specialization in both metazoans and plants.
they further speculate that this expansion and specialization of cell cycle protein families has occurred to meet the challenges of a complex multicellular lifestyle.
the comprehensive evolutionary histories of cdks and cyclins outlined in our study provided new evidence for their hypotheses.
we believe that the emergence of the cdk4/6-cyclin d complex may have contributed to the formation of eumetazoan-specific g <dig> phase regulation, and may represent a key step in the development of cell cycle regulation during eumetazoan evolution.
CONCLUSIONS
in this study, we conducted a comprehensive evolutionary analysis of cdk and cyclin proteins in metazoans and their unicellular relatives.
our results indicated that cdk family could be divided into eight subfamilies.
seven subfamilies  are conserved in metazoans and fungi, with cdk4/ <dig> subfamily found only in eumetazoans.
as to cyclins, cyclin c, h, l, y subfamilies, and cyclin k and t as a whole subfamily, are conserved in animal, fungi, and amoeba dictyostelium discoideum.
the cyclin b subfamily is conserved in d. discoideum, fungi, and animals, whereas cyclin a and e subfamilies are both present in animals and their unicellular relatives such as choanoflagellate monosiga brevicollis and filasterean capsaspora owczarzaki, but are absent in fungi and d. discoideum.
cyclin d subfamily orthologs can be found in the early-emerging, non-opisthokont apusozoan thecamonas trahens.
within opisthokonta, the cyclin d subfamily is conserved only in eumetazoans, and is absent in fungi, choanoflagellates, and the basal metazoan amphimedon queenslandica.
our data indicate that the cdk4/ <dig> subfamily and eumetazoans emerged simultaneously, with the evolutionary conservation of the cyclin d subfamily also tightly linked with eumetazoan appearance.
we speculated that establishment of the cdk4/6-cyclin d complex may have been the key step in the evolution of cell cycle control during eumetazoan emergence.
abbreviations
h. sapiens: homo sapiens; c. intestinalis: ciona intestinalis; s. purpuratus: strongylocentrotus purpuratus; b. floridae: branchiostoma floridae; d. melanogaster: drosophila melanogaster; n. vectensis: nematostella vectensis; t. adhaerens: trichoplax adhaerens; a. queenslandica: amphimedon queenslandica; m. brevicollis: monosiga brevicollis; s. rosetta: salpingoeca rosetta; c. owczarzaki: capsaspora owczarzaki; s. arctica: sphaeroforma arctica; s. pombe: schizosaccharomyces pombe; s. cerevisiae: saccharomyces cerevisiae; c. cinerea: coprinopsis cinerea; s. punctatus: spizellomyces punctatus; t. trahens: thecamonas trahens; d. discoideum: dictyostelium discoideum.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
lc and ly conceived the study.
lc, fc, jc, and jx collected the data.
lc and fc performed the phylogenetic analyses, and jx conducted the reciprocal blast analysis.
lc and xy wrote the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1: file s1
all multiple alignments of cdk or cyclin proteins which were used for phylogenetic analysis.
multiple alignments of full-length proteins were mainly carried out using msaprobs program  <cit> , however, the protein alignment which include the cyclin sequence from t. trahens and d. discoideum was carried out using promals program  <cit> , then the poorly aligned positions in these alignments were removed, only the conserved region  in these alignments were used for further phylogenetic analysis.
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additional file 2: file s2
cdk sequences from  <dig> organisms.
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additional file 3: file s3
cyclin sequences from  <dig> organisms.
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additional file 4: figure s1
phylogenetic analysis of cdk family proteins in h. sapiens, t. adhaerens, c. intestinalis, b. floridae, s. purpuratus and d. melanogaster.
maximum likelihood analysis was conducted using raxml program, and bayesian analysis was carried out using phylobayes  <dig> .
both methods produced trees with nearly identical topologies.
the first numbers above branches indicate bayesian posterior probabilities , and the second numbers above branches indicate ml bootstrap percentages.
the scale bar shows the number of substitutions per site.
the sequences of hsa-gsk3alpha, hsa-mak, and hsa-hcdkl <dig> were used as outgroup.
all proteins are labeled with their accession numbers and their specie name as prefix.
abbreviations: hsa: h. sapiens; tad: t. adhaerens; cin: c. intestinalis; bfl: b. floridae; spu: s. purpuratus; dme: d. melanogaster.
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additional file 5: figure s2
phylogenetic analysis of cdk family proteins in h. sapiens, t. adhaerens, c.owczarzaki , s. arctica, s.cerevisiae, s.pombe, c. cinerea, s. punctatus, t. trahens and d. discoideum.
maximum likelihood analysis was conducted using raxml program, and bayesian analysis was carried out using phylobayes  <dig> .
both methods produced trees with nearly identical topologies.
the first numbers above branches indicate bayesian posterior probabilities , and the second numbers above branches indicate ml bootstrap percentages.
the scale bar shows the number of substitutions per site.
the sequences of hsa-gsk3alpha, hsa-mak, and hsa-hcdkl <dig> were used as outgroup.
all proteins are labeled with their accession numbers and their specie name as prefix.
abbreviations: hsa: h. sapiens; tad: t. adhaerens; cow: c.owczarzaki; sar: s. arctica; sce:s.cerevisiae; spo:s.pombe; cci:c. cinerea; spu:s. punctatus; ttr:t. trahens; ddi:d. discoideum.
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additional file 6: table s1
evolutionary relationship of cdk family proteins from  <dig> representative organisms.
summary of the evolutionary relationship of cdk family proteins based on the results of phylogenetic analyses of cdk family proteins .
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additional file 7: figure s3
phylogenetic network analysis for cdk4/ <dig>  cdk1/2/ <dig>  and cdk <dig> subfamily proteins from h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta.
neighbor-net analysis was conducted using splitstree v. <dig> program  <cit>  with  <dig> bootstrap resamplings.
all proteins are labeled with their accession numbers preceded by their species names.
species abbreviations are as follows: hsa, h. sapiens; nve, n. vectensis; tad, t. adhaerens; aqe, a. queenslandica; mbr, m. brevicollis.
the alignment used for this analysis is found in additional file 1: file s <dig>
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additional file 8: figure s4
phylogenetic analysis of cyclin family proteins in h. sapiens, t. adhaerens, c. intestinalis, b. floridae, s. purpuratus and d. melanogaster.
maximum likelihood analysis was conducted using raxml program, and bayesian analyses were carried out using phylobayes  <dig> .
both methods produced trees with nearly identical topologies.
the first numbers above branches indicate bayesian posterior probabilities , and the second numbers above branches indicate ml bootstrap percentages.
the scale bar shows the number of substitutions per site.
the sequences of hsa-cables <dig> and hsa-cables <dig> were used as the outgroup.
all proteins are labeled with their accession numbers and their specie name as prefix.
abbreviations: hsa: h. sapiens; tad: t. adhaerens; cin: c. intestinalis; bfl: b. floridae; spu: s. purpuratus; dme: d. melanogaster.
click here for file
additional file 9: figure s5
phylogenetic analysis of cyclin family proteins in h. sapiens, t. adhaerens, c.owczarzaki , and s. arctica.
maximum likelihood analysis was conducted using raxml program, and bayesian analyses were carried out using phylobayes  <dig> .
both methods produced trees with nearly identical topologies.
the first numbers above branches indicate bayesian posterior probabilities , and the second numbers above branches indicate ml bootstrap percentages.
the scale bar shows the number of substitutions per site.
the sequences of hsa-cables <dig> and hsa-cables <dig> were used as the outgroup.
all proteins are labeled with their accession numbers and their specie name as prefix.
abbreviations: hsa: h. sapiens; tad: t. adhaerens; cow: c.owczarzaki; sar: s. arctica.
click here for file
additional file 10: figure s6
phylogenetic analysis of cyclin family proteins in h. sapiens, t. adhaerens, s.cerevisiae, s.pombe, c. cinerea, and punctatus.
maximum likelihood analysis was conducted using raxml program, and bayesian analyses were carried out using phylobayes  <dig> .
both methods produced trees with nearly identical topologies.
the first numbers above branches indicate bayesian posterior probabilities , and the second numbers above branches indicate ml bootstrap percentages.
the scale bar shows the number of substitutions per site.
the sequences of hsa-cables <dig> and hsa-cables <dig> were used as the outgroup.
all proteins are labeled with their accession numbers and their specie name as prefix.
abbreviations: hsa: h. sapiens; tad: t. adhaerens; sce: s.cerevisiae; spo: s.pombe; cci: c. cinerea; spu: s. punctatus.
click here for file
additional file 11: figure s7
phylogenetic analysis of cyclin family proteins in h. sapiens, t. adhaerens, t. trahens and d. discoideum.
maximum likelihood analysis was conducted using raxml program, and bayesian analyses were carried out using phylobayes  <dig> .
both methods produced trees with nearly identical topologies.
the first numbers above branches indicate bayesian posterior probabilities , and the second numbers above branches indicate ml bootstrap percentages.
the scale bar shows the number of substitutions per site.
the sequences of hsa-cables <dig> and hsa-cables <dig> were used as the outgroup.
all proteins are labeled with their accession numbers and their specie name as prefix.
abbreviations: hsa: h. sapiens; tad: t. adhaerens; ttr:t. trahens; ddi:d. discoideum.
click here for file
additional file 12: table s2
evolutionary relationship of cyclin family proteins from  <dig> representative organisms.
summary of the evolutionary relationship of cyclin family proteins based on the results of phylogenetic analyses of cyclin family proteins, and also refer the reciprocal blast search results .
click here for file
additional file 13: figure s8
phylogenetic network analysis for cyclin b like group proteins from h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta.
neighbor-net analysis was conducted using splitstree v. <dig> program  <cit>  with  <dig> bootstrap resamplings.
all proteins are labeled with their accession numbers preceded by their species names.
species abbreviations are as follows: hsa, h. sapiens; nve, n. vectensis; tad, t. adhaerens; aqe, a. queenslandica; mbr, m. brevicollis.
the alignment used for this analysis is found in additional file 1: file s <dig>
click here for file
acknowledgments
all protein sequences from sphaeroforma arctica, spizellomyces punctatus, and thecamonas trahens were obtained from the origins of multicellularity sequencing project, broad institute of harvard and mit .
this work was supported by the national natural science foundation of china .

 cyclin-dependent kinases are serine and threonine kinases whose actions are dependent on the binding of regulatory subunits known as cyclins. various cyclins are synthesized and destroyed at specific times during the cell cycle, thus regulating cdk activity in a timely manner. cdk and cyclin families function in a variety of cellular processes, including cell cycle regulation, transcription, rna processing, translation, neurogenesis, and apoptosis. the evolution of metazoans from protozoans is a major milestone in the history of life. this transition has been generally marked by increases in the number of genes involved in cell differentiation, cell-cell communication, and cell adhesion. the evolutionary histories of transcription factors, cell-cell communication-related genes, and cell adhesion genes during the unicellular to metazoan transition have been extensively investigated. in addition to cell communication and cell adhesion proteins, other proteins may be linked with metazoan emergence. based on a comprehensive phylogenetic analysis of sponge amphimedon queenslandica proteins, it has been proposed that the emergence of metazoan multicellularity may have been related to the evolution of various genes functioning in cell cycling and growth, programmed cell death, cell-cell and cell-matrix adhesion, developmental signaling and gene regulation, allorecognition and innate immunity, and cell type specialization. as implied by the aforementioned study, investigation of the evolutionary history of cell cycle control genes could enhance our understanding of metazoan emergence from single-celled ancestors. at present, however, comprehensive evolutionary analyses have been carried out only for a few cell cycle control genes, such as p rb, and e2f families. the core machinery of the animal cell cycle can generally be traced back to early eukaryotes. it was previously proposed that the eukaryotic cell cycle was controlled by the dna damage checkpoint kinase chk1p at early stages of evolution, and duplications of kinase genes occurred during subsequent evolution. gradually, eukaryotic kinases were added to the cell cycle control system, with cdks being among the last major additions. however, cyclin-dependent kinases in yeast and animal are thought to be the cornerstone in cell cycle control. according to recent reports, cdk and approximately cyclin genes are present in humans. the evolution of cdk and cyclin families has been studied previously. an analysis of the cdk family in yeasts and animals divided the cdk family into seven subfamilies, while another analysis examined cdk family members from animals, plants, yeasts, and four protists. with respect to the cyclin family, one phylogenetic analysis covered a-, b-, d-, and e-type cyclin proteins in animals and fungi; another analysis included fungal, plant, and protist cyclins, and successfully divided all cyclins in three groups. these analyses only incorporated a relatively limited number of organisms, however, with several representative organisms occupying key positions in the transition from unicellular to metazoan organisms not analyzed. taking advantage of the increasing number of sequenced genomes, in this study we conducted a comprehensive evolutionary analysis of cdk and cyclin genes from representative organisms. our analysis incorporated several organisms important to the study of metazoan emergence, such as the closest known metazoan relative, the choanoflagellate monosiga brevicollis; the oldest surviving metazoan, amphimedon queenslandica; the earliest eumetazoan, trichoplax adhaerens; and the cnidarian nematostella vectensis. we also included several unicellular organisms, such as the choanoflagellate salpingoeca rosetta and the filasterean capsaspora owczarzaki, that are recognized as close relatives of metazoans based on data from the origins of multicellularity project. our results revealed detailed evolutionary information regarding cdk and cyclin proteins in metazoan organisms and their unicellular relatives, and provided evidence for simultaneous cdk4/6-cyclin d complex and eumetazoan emergence. methods database searching and identification of cdk and cyclin sequences for cdk proteins, we performed psi-blast searches using human cdk and cdk protein sequences as queries against the ncbi non-redundant protein database for organisms: homo sapiens, ciona intestinalis, strongylocentrotus purpuratus, branchiostoma floridae, drosophila melanogaster, n. vectensis, t. adhaerens, a. queenslandica, monosiga brevicollis, s. rosetta, c. owczarzaki, schizosaccharomyces pombe, saccharomyces cerevisiae, coprinopsis cinerea, and dictyostelium discoideum. the search results were used as new queries in a second round of blast searching, which was continued until no new sequences were returned. we also performed a similar blastp search against the broad institute database to collect cdk sequences from three unicellular organisms: sphaeroforma arctica, spizellomyces punctatus, and thecamonas trahens, as these sequences are not available in the ncbi database. for cdk genes, only the longest protein sequence encoded by each gene was retained. we also carried out a preliminary phylogenetic analysis on all putative cdk family proteins collected from blast searching. proteins clustering with human cdks were used in subsequent analyses, whereas those clustering with other human protein kinases, such as map kinases, were discarded. using human cyclin b, cyclin c, and cyclin y proteins as queries, similar blast searches were carried out to identify cyclin proteins from related organisms in the ncbi and broad institute databases. because cyclin proteins are greatly diverged, an hmm search against non-redundant proteins from genbank was also carried out using pfam profile pf which corresponds to the cyclin-n domain—the most highly conserved cyclin protein domain. for three unicellular organisms, cyclin-n domain-containing proteins were also collected from the broad institute database. only the longest protein sequence associated with each cyclin gene was retained. we verified the putatively identified cyclin proteins by searching against pfam and smart databases. proteins lacking cyclin-n domains were discarded. similar to a previous analysis, non-cyclin proteins possessing cyclin-n domains were identified by reciprocal blast searching and removed. protein alignment and phylogenetic analyses after evaluating several multiple alignment programs, we used msaprobs for multiple alignment of most full-length proteins. alignments that included cyclin sequences from t. trahens and d. discoideum were carried out using promals, a program more suitable for alignment of distantly related proteins. poorly aligned positions in these alignments were removed, with only the conserved region—the cdk domain for the cdk family, and cyclin-n and –c domains for the cyclin family—used for further phylogenetic analyses. alignments used for phylogenetic analyses are found in additional file 1: file s phylogenetic analyses were performed using maximum likelihood and bayesian methods, with optimum substitution models determined for each alignment based on the akaike information criterion using prottest. ml trees were constructed using raxml. as implemented in the cipres science gateway v. with bootstrap resamplings. bayesian phylogenetic analyses were carried out under an lg substitution model using phylobayes v., with markov chain monte carlo runs terminated when maxdiff < . multiple sequence alignments and phylogenetic tree files were deposited in labarchives. tree files were viewed using the dendroscope program, and phylogenetic networks were constructed with splitstree v.. ortholog identification as suggested in a recent review, ortholog identification of different cdk and cyclin subfamilies was mainly based on results of the phylogenetic analyses; however, results from reciprocal blast search methods were also referenced for some distantly related proteins. in general, a protein was identified as an ortholog of a representative cdk or cyclin subfamily if it clustered with that subfamily in the ml phylogenetic tree with greater than 50% bootstrap support. for proteins clustering with less than 50% ml bootstrap support within a subfamily, reciprocal blast results were consulted: only proteins in the initial blast query for which e-values returned for members of a representative cdk or cyclin subfamily were five orders of magnitude better than those of the next best-scoring cdk or cyclin subfamily were considered to be orthologs of that subfamily. such a “five-orders criterion” has recently been described and used to identify arf gap orthologs. cyclin proteins placed with low bootstrap support into a representative cyclin subfamily and not meeting the five-orders criterion were designated as unclassified cyclin proteins.  as summarized in table  we identified cdk and cyclin proteins from representative organisms. detailed information regarding these cdks and cyclins may be found in additional file 2: file s and additional file 3: file s respectively. the evolutionary history of cdk family we attempted to perform global phylogenetic analyses using ml and bayesian methods on all cdk proteins from organisms. a robust and reliable phylogenetic tree could not be obtained by either method, however, possibly because of the large number of sequences. we therefore carried out separate phylogenetic analyses on subsets of the organisms. we first analyzed cdk sequences from h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta as one group. these six organisms, except for h. sapiens, are all located in key positions with respect to metazoan emergence. given that lineage-specific gene duplication and loss may have occurred in some of these organisms, any conclusions drawn from this subset may not be adequate to fully understand the evolutionary history of the cdk family during metazoan emergence. consequently, analyses of cdk sequences from other organisms were also carried out; one analyzed subset comprised c. intestinalis, b. floridae, s. purpuratus, d. melanogaster, t. adhaerens, and h. sapiens, while another group consisted of c. owczarzaki, s. arctica, s. cerevisiae, s. pombe, s. punctatus, c. cinerea, t. trahens, d. discoideum, t. adhaerens, and h. sapiens. results of these phylogenetic analyses are summarized in figure  and additional file 6: table s and are described in detail below. based on our analyses, the entire cdk family can be divided into eight subfamilies: cdk cdk cdk8/, cdk, cdk10/, cdk, cdk4/, and cdk. although these results are generally consistent with previous reports, our analysis revealed more detailed information and included human cdk and its orthologs. we found that orthologs of seven cdk subfamilies are present in basal metazoans t. adhaerens and a. queenslandica, the choanoflagellate m. brevicollis, and/or s. rosetta. in further analysis, orthologs of cdk subfamilies could be identified in fungi, t. trahens, and d. discoideum. a cdk representative is present in the fungus s. punctatus, but is absent in s. cerevisiae, s. pombe, t. trahens, and d. discoideum. compared with other cdk subfamilies, the cdk4/ subfamily is unique, as it is only found in eumetazoan organisms such as t. adhaerens and n. vectensis, and is absent in the basal metazoan a. queenslandica, choanoflagellates, and other investigated unicellular organisms. the different functions of these cdk subfamilies have been described in an excellent review. the cdk4/ subfamily is generally recognized as animal-specific, but previous phylogenetic analyses supporting this conclusion have only included a relatively small number of organisms. in the tree shown in figure  subfamilies cdk4/ cdk1/2/ and cdk generally cluster together. as phylogenetic networks are useful for describing complex evolutionary scenarios such as horizontal gene transfer and recombination, we carried out a phylogenetic network analysis for cdk4/ cdk1/2/ and cdk subfamily proteins from h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta. the results of that analysis were generally consistent with our phylogenetic tree topology, cdk4/ subfamily is located between cdk1/2/ subfamily and cdk subfamily. the detail evolutionary information among cdk4/ subfamily, cdk1/2/ subfamily and cdk subfamily are still requiring further study. anyway, our analysis is the first to map the detailed evolutionary history of the cdk4/ subfamily in representative organisms occupying key positions along the transition from unicellular organisms to metazoans. our results indicate that the cdk4/ subfamily is linked simultaneously with eumetazoan appearance. subfamilies cdk cdk10/ cdk1/2/ and cdk all contains more than one cdk members in metazoan organisms and every subfamily could be divided into two or three clades. our analysis provided some detailed information about how and when these clades were formed in different subfamilies. the cdk subfamily consists of two clades, cdk and cdk12/ basal metazoan organisms t. adhaerens and a. queenslandica have representative members in both clades. in addition, consistent with previous reports, s. pombe lsk and s. cerevisiae ctk1p belong to the cdk12/ clade, while s. cerevisiae sgv1p and s. pombe cdk are members of the cdk clade. these results imply that the cdk subfamily split into two clades before the divergence of metazoans and fungi. in humans, cdk is reported to regulate transcription by phosphorylating the c-terminal domain of rna polymerase ii. the cdk10/ subfamily comprises clades cdk and cdk. basal metazoans a. queenslandica and t. adhaerens have members in both clades; similarly, unicellular m. brevicollis is represented in both cdk and cdk. in further analysis, we found that one s. punctatus protein was classified into the cdk clade, and that one s. pombe protein and one c. cinerea protein were placed in the cdk clade. proteins from d. discoideum and t. trahens were only grouped into the cdk10/ subfamily, and were not found in clades cdk and cdk in addition to their roles in regulating transcription, cdk and cdk display distinct functions during the g2/m transition. the cdk1/2/ subfamily can be divided into cdk and cdk2/ clades in metazoans. bona fide cdk clade members are found in a. queenslandica and t. adhaerens, and similarly cdk2/ clade members are present in a. queenslandica and t. adhaerens. cdk1/2/ subfamily members from m. brevicollis, s. rosetta, fungi, t. trahens, and d. discoideum, however, are not placed into cdk or cdk2/ clades. our data indicate that genes of the ancient cdk1/2/ subfamily arose in the common ancestor of amoebozoans and fungi, and then diverged via gene duplication into clades cdk1and cdk2/ in metazoans. in the tree in figure  the cdk subfamily is divided into clades cdk cdk16/17/ and cdk14/ the placozoan t. adhaerens possesses three cdk subfamily members, which are classified into cdk, cdk16/17/, and cdk14/ clades. we found that one m. brevicollis protein is clustered into cdk whereas another is placed into cdk16/17/. in the comprehensive analysis, some fungal cdk subfamily members were classified into the cdk clade, while others were placed into clades cdk16/17/ and cdk14/. these data indicate that the cdk subfamily originated in single-celled ancestors, and subsequently split into three clades before or during eumetazoan emergence. in humans, cdk is essential for neuronal cell cycle arrest and differentiation. the evolutionary history of cyclin family based on the same reasons as in the cdk family, we carried out separate phylogenetic analyses on subsets of cyclin family members. as for the cdks, we performed analyses of cyclin proteins from the group h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta, and then from a subset consisting of c. intestinalis, b. floridae, s. purpuratus, d. melanogaster, t. adhaerens, and h. sapiens. because their cyclin sequences diverged greatly, we failed to obtain a reliable cyclin phylogenetic tree from eight organisms: c. owczarzaki, s. arctica, s. cerevisiae, s. pombe, s. punctatus, c. cinerea, t. trahens, d. discoideum, t. adhaerens, and h. sapiens. we thus divided these organisms into smaller subsets for analysis: one group for filasterean and ichthyosporean organisms, one group for fungi, and one group for apusozoa and amoebozoa. these subgroups were analyzed, and orthologs of different cyclin subfamilies were classified based on the phylogenetic results and those of reciprocal blast analysis. according to our phylogenetic analyses, the metazoan cyclin family could be divided into subfamilies, and fungi organisms owns three fungi specific subfamily. a recent cyclin family analysis indicated that the cyclin family could be divided three groups, and our analysis confirmed it. in this manuscript, we will refer group i as cyclin b like group, group ii as cyclin y like group, and group iii as cyclin c like group. though our results is general consistent with previous analysis, some new information was revealed by our analysis. for example, our analysis successfully identificated cyclin c ortholog and cyclin j ortholog in c. intestinalis, the previous analysis which also included c. intestinalis missed this information. most subfamilies in the cyclin c-like group are conserved in metazoans, choanoflagellates, fungi, and d. discoideum. we found that cyclins c, h, and l are all conserved in metazoans, choanoflagellates, fungi, t. trahens, and d. discoideum. orthologs of cyclins t and k were found in metazoans; in fungi and d. discoideum, however, homologous proteins of cyclin t and/or cyclin k subfamily could only be identified as the common ancestor of the two subfamilies. our data indicate that the common ancestor of cyclins k and t originated early in the course of evolution, and then diverged no later than during the period of metazoan emergence. fam is generally conserved only in metazoans, although an ortholog is also found in s. arctica. we found that the cyclin y subfamily is conserved in metazoans, choanoflagellates, and d. discoideum. interestingly, one fungal protein in s. punctatus was identified as an ortholog of cyclin y. pcl subfamily members are found in s. cerevisiae and c. cinerea. in fact, cyclin y and pcl subfamilies cluster together tightly in the phylogenetic tree. cyclins y and pcl are binding partners of cdk subfamilies in metazoans and fungi, respectively; although we list them as two separate subfamilies, as previously reported, we believe they share a common ancestor. evolutionary conservation varied greatly among different subfamilies in the cyclin b-like group. these subfamilies are described in detail as follows. the cyclin b subfamily is conserved in metazoans, choanoflagellates, fungi, t. trahens, and d. discoideum. the fungus-specific subfamily clb is related to the cyclin b family, with blast e-values as low as approximately 1 × 10− cyclins b and clb are binding partners of cdk subfamilies in metazoans and fungi, respectively. consequently, although we treat cyclins b and clb as two subfamilies, as previously reported, they appear to share a common ancestor in early eukaryotic lineages, as suggested by previous analyses. alignments of representative cyclin b subfamily proteins from metazoan organisms h. sapiens and t. adhaerens and unicellular organisms s. rosetta, c. owczarzaki, t. trahens, and d. discoideum are shown in figure  our analysis results indicate that the cyclin a subfamily is conserved in metazoans, unicellular choanoflagellates, and c. owczarzaki, but is absent in fungi, t. trahens, and d. discoideum. alignments of representative cyclin a subfamily proteins from metazoans h. sapiens, t. adhaerens, and unicellular organisms m. brevicollis, s. rosetta, and c. owczarzaki are given in figure  the fungus-specific subfamily cln, which functions in cell cycle regulation, is the binding partner of fungus cdk results of blast analysis revealed similar genetic distances between the cln subfamily and the metazoan cyclin a subfamily, and between cln and the metazoan cyclin b subfamily. the cyclin e subfamily is not only conserved in metazoans, but is also present in several unicellular organisms such as choanoflagellate s. rosetta, c. owczarzaki, and t. trahens. this result conflicts with previous studies indicating that cyclin e is animal-specific. this inconsistency may be due to the failure of previous analyses to incorporate several important unicellular organisms, such as s. rosetta and c. owczarzaki. alignments of representative cyclin e subfamily proteins from metazoans h. sapiens and t. adhaerens and unicellular organisms s. rosetta, c. owczarzaki, and t. trahens are shown in figure  we found that orthologs of cyclin d are present in t. adhaerens and n. vectensis, but are absent in m. brevicollis, s. rosetta, and a. queenslandica. comprehensive analysis unexpectedly revealed that cyclin d orthologs are present in t. trahens, although absent in fungi and d. discoideum. although plant d-type cyclin is generally considered to be homologous to animal cyclin d, plants do not possess cdk4/ orthologs; instead, plant d-type cyclin functions together with plant cdka, a cdk homolog, in the g phase. possibly because of low sequence similarity, plant d-type cyclin did not cluster together with animal cyclin d in a previous phylogenetic analysis. our phylogenetic results are the first to reveal the presence of a bona fide ortholog of the animal cyclin d subfamily in a non-opisthokont, t. trahens. the blast e-value for this protein against human cyclin d is approximately 1 × 10− this result indicates that the cyclin d subfamily arose in early eukaryotes, and that the absence of cyclin d in fungi and many other unicellular organisms may be due to lineage-specific gene loss in these organisms. similar to plant organisms, the non-opisthokont t. trahens does not possess an ortholog of the cdk4/ subfamily. our alignment of representative cyclin d subfamily proteins from h. sapiens, n. vectensis, t. adhaerens, and t. trahens is displayed in figure  in our analyses, cyclins i and g always clustered together. cyclins i and g collectively have representative members in unicellular organisms c. owczarzaki, s. arctica, a. queenslandica, and n. vectensis. these data indicate that cyclin subfamilies i and g are derived from a common ancestral gene that was present in unicellular organisms, with this common ancestor differentiating into cyclins i and g after the emergence of n. vectensis. in addition to the above phylogenetic analyses, we conducted a phylogenetic network analysis of cyclin b-like group proteins from h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta. it was found that the cyclin d subfamily is located between subfamily e and subfamily g/i. the detail evolutionary information among cyclin d subfamily, cyclin e, and cyclin g/i will be an interesting topic for further study. cell cycle related cdk/cyclin evolutionary histories during animal emergence information is limited regarding evolution of cell cycle regulation in eukaryotes. it is generally believed, however, that early eukaryotes already possessed complex cell cycle regulation, with key cell cycle regulators having subsequently undergone divergent functional specializations in different organisms. for example, the rb-e2f pathway, which functions in cell cycle regulation, is conserved in animals and plants, but has been lost in fungi. the eukaryotic cell cycle is controlled by a complicated regulatory network. cdk-cyclin complexes, as key regulators of the cell cycle, phosphorylate a variety of substrates during the cell cycle. in humans, for example, cdk4/cyclin d phosphorylates prb during the g phase, and cdk1-cyclin b phosphorylates cdc25c and wee1a during the m phase. a recent structural study revealed that the conformation of t cdk4/ cyclin d diverges from that of previously known cdk-cyclin binary complexes, and cdk might have a unique regulation and activation mechanism compared with that of cdk2-cyclin a. another study has also found that the structural mechanism of cdk4-cyclin d activation differs markedly from that of previously studied cdk2-cyclin a complexes. our analysis has provided detailed evolutionary information on cdk and cyclin subfamilies in metazoans and related organisms. our data are the first to reveal that cyclin d orthologs are present in a non-opisthokont, but have generally been lost in fungi and most other unicellular opisthokonts, such as m. brevicollis, s. rosetta, c. owczarzaki, and s. arctica. our analysis also found that cyclin e is not restricted to animals, but is present in several unicellular organisms. investigations of cell cycle regulation have primarily been carried out in animals and yeasts. in animals, cdk4/ and cyclin d have been determined to function in the g phase, human cdk and cyclin a/e in s and g phases, and cdk and cyclin b in the m phase. in yeasts, s. cerevisiae cdk functions in g s, g and m phases with different cyclins. given the large evolutionary distance between yeasts and animals, it has proved difficult to elucidate the evolutionary history of cell cycle regulation and its relationship to the emergence of metazoans from their single-celled ancestors. in this study, we analyzed cell cycle-related cdks and cyclins in several representative organisms, such as m. brevicollis, a. queenslandica, and t. adhaerens, which occupy key positions for metazoans origination from their single-celled ancestors organisms. we discovered that the number of cell cycle-related cdk and cyclin proteins has gradually increased from m. brevicollis and a. queenslandica to t. adhaerens: m. brevicollis possesses orthologs for cdk cyclin b, cyclin a, and cyclin e, a. queenslandica has orthologs for cdk cdk cyclin b, cyclin a, and cyclin e, and t. adhaerens features orthologs for cdk cdk cdk cyclin b, cyclin a, cyclin e, and cyclin d. based on the evolutionary information uncovered for these cdk and cyclin proteins, we are able to propose different scenarios for the function of cdk and cyclin proteins in cell cycle control in representative organisms m. brevicollis, a. queenslandica, and t. adhaerens. because m. brevicollis does not possess cdk4/ and cyclin d orthologs, we speculate that the ortholog of cdk in m. brevicollis may function throughout the cell cycle with different cyclins, similar to s. cerevisiae cdk. as indicated in figure  we have inferred that the cdk4/6-cyclin d complex appeared at the same time as the emergence of the eumetazoan t. adhaerens. it is interesting that the cdk4/6-cyclin d complex first emerged in t. adhaerens, as t. adhaerens has four morphologically identifiable somatic cell types and is thought to represent the earliest eumetazoan lineage. the cdk4/6-cyclin d complex functions in the g phase, the first phase within interphase. the duration of the g phase is highly variable among different cells in animals, and is affected by limiting growth factors, nutrient supply, temperature, and additional inhibiting factors. for example, human embryonic stem cells are characterized by an abbreviated g phase and lack the classical restriction point that normally controls commitment for progression into the s phase. in contrast, somatic cell proliferation is linked to growth factor-dependent passage through the r point in the g phase. in fission yeast, a single oscillation of p34cdc kinase activity provided by a single b-type cyclin can promote ordered progression into both dna replication and mitosis. the function of cdk4-cyclin complexes in animals has been extensively studied and reviewed. with respect to the cyclin d family, mice lacking cyclin d d or d exhibit different developmental anomalies. mice expressing cyclin d but not d and d have been observed to die before embryonic day, while mice lacking all three cyclins die before e. in regard to cdk and cdk mice lacking cdk or cdk also exhibit different developmental anomalies, with mice lacking both cdk and cdk displaying progressive embryonic lethality from e onward, and the few live pups dying shortly after birth. these data clearly indicate that the cdk4-cyclin d complex plays critical roles during mouse early embryonic development. it would be interesting to study the function of cdk4-cyclin d in early eumetazoan organisms such as n. vectensis and t. adhaerens. based on a comparative analysis of cell cycle regulatory networks in animals, yeasts, and plants, harashima et al. have recently suggested that the cycd/cyce clade has undergone lineage-specific expansion and specialization in both metazoans and plants. they further speculate that this expansion and specialization of cell cycle protein families has occurred to meet the challenges of a complex multicellular lifestyle. the comprehensive evolutionary histories of cdks and cyclins outlined in our study provided new evidence for their hypotheses. we believe that the emergence of the cdk4/6-cyclin d complex may have contributed to the formation of eumetazoan-specific g phase regulation, and may represent a key step in the development of cell cycle regulation during eumetazoan evolution.  in this study, we conducted a comprehensive evolutionary analysis of cdk and cyclin proteins in metazoans and their unicellular relatives. our results indicated that cdk family could be divided into eight subfamilies. seven subfamilies are conserved in metazoans and fungi, with cdk4/ subfamily found only in eumetazoans. as to cyclins, cyclin c, h, l, y subfamilies, and cyclin k and t as a whole subfamily, are conserved in animal, fungi, and amoeba dictyostelium discoideum. the cyclin b subfamily is conserved in d. discoideum, fungi, and animals, whereas cyclin a and e subfamilies are both present in animals and their unicellular relatives such as choanoflagellate monosiga brevicollis and filasterean capsaspora owczarzaki, but are absent in fungi and d. discoideum. cyclin d subfamily orthologs can be found in the early-emerging, non-opisthokont apusozoan thecamonas trahens. within opisthokonta, the cyclin d subfamily is conserved only in eumetazoans, and is absent in fungi, choanoflagellates, and the basal metazoan amphimedon queenslandica. our data indicate that the cdk4/ subfamily and eumetazoans emerged simultaneously, with the evolutionary conservation of the cyclin d subfamily also tightly linked with eumetazoan appearance. we speculated that establishment of the cdk4/6-cyclin d complex may have been the key step in the evolution of cell cycle control during eumetazoan emergence. abbreviations h. sapiens: homo sapiens; c. intestinalis: ciona intestinalis; s. purpuratus: strongylocentrotus purpuratus; b. floridae: branchiostoma floridae; d. melanogaster: drosophila melanogaster; n. vectensis: nematostella vectensis; t. adhaerens: trichoplax adhaerens; a. queenslandica: amphimedon queenslandica; m. brevicollis: monosiga brevicollis; s. rosetta: salpingoeca rosetta; c. owczarzaki: capsaspora owczarzaki; s. arctica: sphaeroforma arctica; s. pombe: schizosaccharomyces pombe; s. cerevisiae: saccharomyces cerevisiae; c. cinerea: coprinopsis cinerea; s. punctatus: spizellomyces punctatus; t. trahens: thecamonas trahens; d. discoideum: dictyostelium discoideum. competing interests the authors declare that they have no competing interests. authors’ contributions lc and ly conceived the study. lc, fc, jc, and jx collected the data. lc and fc performed the phylogenetic analyses, and jx conducted the reciprocal blast analysis. lc and xy wrote the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1: file s1 all multiple alignments of cdk or cyclin proteins which were used for phylogenetic analysis. multiple alignments of full-length proteins were mainly carried out using msaprobs program, however, the protein alignment which include the cyclin sequence from t. trahens and d. discoideum was carried out using promals program, then the poorly aligned positions in these alignments were removed, only the conserved region in these alignments were used for further phylogenetic analysis. click here for file additional file 2: file s2 cdk sequences from organisms. click here for file additional file 3: file s3 cyclin sequences from organisms. click here for file additional file 4: figure s1 phylogenetic analysis of cdk family proteins in h. sapiens, t. adhaerens, c. intestinalis, b. floridae, s. purpuratus and d. melanogaster. maximum likelihood analysis was conducted using raxml program, and bayesian analysis was carried out using phylobayes. both methods produced trees with nearly identical topologies. the first numbers above branches indicate bayesian posterior probabilities, and the second numbers above branches indicate ml bootstrap percentages. the scale bar shows the number of substitutions per site. the sequences of hsa-gsk3alpha, hsa-mak, and hsa-hcdkl were used as outgroup. all proteins are labeled with their accession numbers and their specie name as prefix. abbreviations: hsa: h. sapiens; tad: t. adhaerens; cin: c. intestinalis; bfl: b. floridae; spu: s. purpuratus; dme: d. melanogaster. click here for file additional file 5: figure s2 phylogenetic analysis of cdk family proteins in h. sapiens, t. adhaerens, c.owczarzaki, s. arctica, s.cerevisiae, s.pombe, c. cinerea, s. punctatus, t. trahens and d. discoideum. maximum likelihood analysis was conducted using raxml program, and bayesian analysis was carried out using phylobayes. both methods produced trees with nearly identical topologies. the first numbers above branches indicate bayesian posterior probabilities, and the second numbers above branches indicate ml bootstrap percentages. the scale bar shows the number of substitutions per site. the sequences of hsa-gsk3alpha, hsa-mak, and hsa-hcdkl were used as outgroup. all proteins are labeled with their accession numbers and their specie name as prefix. abbreviations: hsa: h. sapiens; tad: t. adhaerens; cow: c.owczarzaki; sar: s. arctica; sce:s.cerevisiae; spo:s.pombe; cci:c. cinerea; spu:s. punctatus; ttr:t. trahens; ddi:d. discoideum. click here for file additional file 6: table s1 evolutionary relationship of cdk family proteins from representative organisms. summary of the evolutionary relationship of cdk family proteins based on the results of phylogenetic analyses of cdk family proteins. click here for file additional file 7: figure s3 phylogenetic network analysis for cdk4/ cdk1/2/ and cdk subfamily proteins from h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta. neighbor-net analysis was conducted using splitstree v. program with bootstrap resamplings. all proteins are labeled with their accession numbers preceded by their species names. species abbreviations are as follows: hsa, h. sapiens; nve, n. vectensis; tad, t. adhaerens; aqe, a. queenslandica; mbr, m. brevicollis. the alignment used for this analysis is found in additional file 1: file s click here for file additional file 8: figure s4 phylogenetic analysis of cyclin family proteins in h. sapiens, t. adhaerens, c. intestinalis, b. floridae, s. purpuratus and d. melanogaster. maximum likelihood analysis was conducted using raxml program, and bayesian analyses were carried out using phylobayes. both methods produced trees with nearly identical topologies. the first numbers above branches indicate bayesian posterior probabilities, and the second numbers above branches indicate ml bootstrap percentages. the scale bar shows the number of substitutions per site. the sequences of hsa-cables and hsa-cables were used as the outgroup. all proteins are labeled with their accession numbers and their specie name as prefix. abbreviations: hsa: h. sapiens; tad: t. adhaerens; cin: c. intestinalis; bfl: b. floridae; spu: s. purpuratus; dme: d. melanogaster. click here for file additional file 9: figure s5 phylogenetic analysis of cyclin family proteins in h. sapiens, t. adhaerens, c.owczarzaki, and s. arctica. maximum likelihood analysis was conducted using raxml program, and bayesian analyses were carried out using phylobayes. both methods produced trees with nearly identical topologies. the first numbers above branches indicate bayesian posterior probabilities, and the second numbers above branches indicate ml bootstrap percentages. the scale bar shows the number of substitutions per site. the sequences of hsa-cables and hsa-cables were used as the outgroup. all proteins are labeled with their accession numbers and their specie name as prefix. abbreviations: hsa: h. sapiens; tad: t. adhaerens; cow: c.owczarzaki; sar: s. arctica. click here for file additional file 10: figure s6 phylogenetic analysis of cyclin family proteins in h. sapiens, t. adhaerens, s.cerevisiae, s.pombe, c. cinerea, and punctatus. maximum likelihood analysis was conducted using raxml program, and bayesian analyses were carried out using phylobayes. both methods produced trees with nearly identical topologies. the first numbers above branches indicate bayesian posterior probabilities, and the second numbers above branches indicate ml bootstrap percentages. the scale bar shows the number of substitutions per site. the sequences of hsa-cables and hsa-cables were used as the outgroup. all proteins are labeled with their accession numbers and their specie name as prefix. abbreviations: hsa: h. sapiens; tad: t. adhaerens; sce: s.cerevisiae; spo: s.pombe; cci: c. cinerea; spu: s. punctatus. click here for file additional file 11: figure s7 phylogenetic analysis of cyclin family proteins in h. sapiens, t. adhaerens, t. trahens and d. discoideum. maximum likelihood analysis was conducted using raxml program, and bayesian analyses were carried out using phylobayes. both methods produced trees with nearly identical topologies. the first numbers above branches indicate bayesian posterior probabilities, and the second numbers above branches indicate ml bootstrap percentages. the scale bar shows the number of substitutions per site. the sequences of hsa-cables and hsa-cables were used as the outgroup. all proteins are labeled with their accession numbers and their specie name as prefix. abbreviations: hsa: h. sapiens; tad: t. adhaerens; ttr:t. trahens; ddi:d. discoideum. click here for file additional file 12: table s2 evolutionary relationship of cyclin family proteins from representative organisms. summary of the evolutionary relationship of cyclin family proteins based on the results of phylogenetic analyses of cyclin family proteins, and also refer the reciprocal blast search results. click here for file additional file 13: figure s8 phylogenetic network analysis for cyclin b like group proteins from h. sapiens, n. vectensis, t. adhaerens, a. queenslandica, m. brevicollis, and s. rosetta. neighbor-net analysis was conducted using splitstree v. program with bootstrap resamplings. all proteins are labeled with their accession numbers preceded by their species names. species abbreviations are as follows: hsa, h. sapiens; nve, n. vectensis; tad, t. adhaerens; aqe, a. queenslandica; mbr, m. brevicollis. the alignment used for this analysis is found in additional file 1: file s click here for file acknowledgments all protein sequences from sphaeroforma arctica, spizellomyces punctatus, and thecamonas trahens were obtained from the origins of multicellularity sequencing project, broad institute of harvard and mit. this work was supported by the national natural science foundation of china.
in contrast, cyclin subfamilies b, a, e, and d, which are cell cycle-related, have distinct evolutionary histories. the cyclin b subfamily is generally conserved in d. discoideum, fungi, and animals, whereas cyclin a and e subfamilies are both present in animals and their unicellular relatives such as choanoflagellate monosiga brevicollis and filasterean capsaspora owczarzaki, but are absent in fungi and d. discoideum. establishment of the cdk4/6-cyclin d complex may have been the key step in the evolution of cell cycle control during eumetazoan emergence. in this study, we conducted a comprehensive evolutionary analysis of cdk and cyclin proteins in metazoans and their unicellular relatives. the molecular history of animal evolution from single-celled ancestors remains a major question in biology, and little is known regarding the evolution of cell cycle regulation during animal emergence. with respect to cyclins, cyclin c, h, l, y subfamilies, and cyclin k and t as a whole subfamily, are generally conserved in animal, fungi, and amoeba dictyostelium discoideum. although absent in fungi and d. discoideum, cyclin d subfamily orthologs can be found in the early-emerging, non-opisthokont apusozoan thecamonas trahens. within opisthokonta, the cyclin d subfamily is conserved only in eumetazoans, and is absent in fungi, choanoflagellates, and the basal metazoan amphimedon queenslandica. our data indicate that the cdk4/ subfamily and eumetazoans emerged simultaneously, with the evolutionary conservation of the cyclin d subfamily also tightly linked with eumetazoan appearance. our analysis divided the cdk family into eight subfamilies.
 the molecular history of animal evolution from single-celled ancestors remains a major question in biology, and little is known regarding the evolution of cell cycle regulation during animal emergence. in this study, we conducted a comprehensive evolutionary analysis of cdk and cyclin proteins in metazoans and their unicellular relatives.  our analysis divided the cdk family into eight subfamilies. seven subfamilies are conserved in metazoans and fungi, with the remaining subfamily, cdk4/ found only in eumetazoans. with respect to cyclins, cyclin c, h, l, y subfamilies, and cyclin k and t as a whole subfamily, are generally conserved in animal, fungi, and amoeba dictyostelium discoideum. in contrast, cyclin subfamilies b, a, e, and d, which are cell cycle-related, have distinct evolutionary histories. the cyclin b subfamily is generally conserved in d. discoideum, fungi, and animals, whereas cyclin a and e subfamilies are both present in animals and their unicellular relatives such as choanoflagellate monosiga brevicollis and filasterean capsaspora owczarzaki, but are absent in fungi and d. discoideum. although absent in fungi and d. discoideum, cyclin d subfamily orthologs can be found in the early-emerging, non-opisthokont apusozoan thecamonas trahens. within opisthokonta, the cyclin d subfamily is conserved only in eumetazoans, and is absent in fungi, choanoflagellates, and the basal metazoan amphimedon queenslandica.  our data indicate that the cdk4/ subfamily and eumetazoans emerged simultaneously, with the evolutionary conservation of the cyclin d subfamily also tightly linked with eumetazoan appearance. establishment of the cdk4/6-cyclin d complex may have been the key step in the evolution of cell cycle control during eumetazoan emergence. cell cycleevolutionmetazoan emergencecdkcyclin 