Evolution and Classification of Myosins, a Paneukaryotic Whole-Genome Approach

1The evolution of molecular motors was key to the origin and diversification of the eukaryotic cell. There are three major superfamilies of motor proteins: kinesins, dyneins, and myosins. The first two act as motors on microtubule filaments, while myosins function on actin. Myosins participate in a variety of cellular processes, including cytokinesis, organellar transport, cell polarization, transcriptional regulation, intracellular transport, and signal transduction. They bind to filamentous actin and produce physical forces by hydrolyzing ATP and converting chemical energy into mechanical force. Both activities reside in the myosin head domain (PF00063). This head domain is accompanied by a broad diversity of N-terminal and/or C-terminal domains that bind to different molecular cargos, providing the functional specificity of the protein. Some myosins, such as myosins V and II, act as dimers that contact through their C-terminal coiled-coils, while others, such as myosins I, III, VI, VII, IX, X, XV, and XIX, act as monomers.

2The identification of gene orthologs can be best accomplished by phylogenetic analyses, especially when complex architectures that are likely to undergo rearrangements are involved. Thus, myosin phylogenetic analysis is important to classify myosin paralog families and identify the ancestry of different gene architectures. Previous efforts have been made to classify the myosin family and to reconstruct its evolutionary diversification (;;, although information from some key eukaryotic groups that have recently become available were missing from all of these studies. Therefore, there is a need to revise schemes of myosin evolution using improved taxon sampling and phylogenetic methods. This is important both to update the classification of myosins diversity and also understand the origin and evolutionary history of the wider gene family. Moreover, a precise reconstruction of the ancestral eukaryotic myosin toolkit (along with that of the other motor proteins) has important implications for understanding the phylogenetic patterns and functional attributes of early eukaryotes.

3Previous analyses, using different genome datasets and different phylogenetic methods provided conflicting hypotheses on myosin classification and the reconstruction of this ancestral toolkit. For example, provided a classification of myosins based on two criteria: phylogenetic reconstruction and analysis of protein domain architecture. They inferred that the last eukaryotic common ancestor (LECA) had 3 of the 37 defined eukaryotic myosin types, including Myo_head-MYTH4/FERM, Myo_head-SMC-DIL, and Myo_head-TH1. In contrast,, in a study focused on apicomplexan myosins, defined 29 classes and did not infer an ancestral complement. Also based on phylogeny, defined 35 different myosin classes, most with an extremely restricted phylogenetic distribution. To make things more complex, different authors have used different criteria for classification, leading to inconsistencies in the classification and nomenclature between studies.

4In this article, we present a new evolutionary history and classification of eukaryotic myosins. We use a significantly expanded taxon sampling than previous studies, in which, for the first time, all major eukaryotic lineages are represented. In particular, we include data from four previously unsampled eukaryotic lineages (Apusozoa, Rhizaria, Haptophyta, and Cryptophyta) so that all the major eukaryotic supergroups are represented. Evolutionary analyses have consistently demonstrated that the evolution of parasitic phenotypes is often accompanied by large-scale gene losses. To overcome this problem, we here include free-living representatives of lineages that were previously represented only by parasitic taxa (such as Ectocarpus siliculosus and unicellular brown algae in Heterokonta/Stramenopiles and Naegleria gruberi in Excavata). Furthermore, we include data from taxa occupying phylogenetic positions that are key to understand major evolutionary transitions, including deep-branching fungi (the Chytridiomycota Spizellomyces punctatus), green algae, deeply derived plants, unicellular holozoan lineages (choanoflagellates, filastereans, and ichthyosporeans) and early-branching metazoans (ctenophores and sponges). We also use improved alignment and phylogenetic inference methods. We do not aim to infer a eukaryotic tree of life from the myosin genomic content. Convergence (discussed later), gene fission, duplication, gene loss, and horizontal gene transfer (HGT) are important phenomena in eukaryotes and, therefore, molecular markers such as the distribution pattern of gene orthologs need to be tested using gene phylogeny and updated as new genome sequences are released. We based our myosin classification exclusively on phylogenetic affinity, which allowed us to identify: gene and domain loss, paralog groups, and convergent evolution of gene domain architecture. The use of updated phylogenetic methods and improved taxon representation allowed us to analyse the classification, evolutionary history, and functional diversification of myosins in new detail.

5Myosin sequences were queried in complete genome or transcriptome sequences of 62 taxa representing all known eukaryotic supergroups. Taxon sampling included 8 animals, 10 unicellular holozoans, 12 fungi, 1 apusozoan, 3 amoebozoans, 5 plants, 4 chlorophytes, 2 rhodophytes, 5 heterokonts, 5 alveolates, 1 rhizarian, 1 haptophyte, 1 cryptophyte, and 4 excavates (supplementary table S2, Supplementary Material online). The complete proteomes of all included species were analysed using Pfamscan (a HMMER search-based algorithm; ) with the default gathering threshold. Using custom Perl scripts, the resulting output files were parsed and all proteins containing a Myosin_head (PF00063) domain were extracted.

6The sequences retrieved were aligned using the Mafft L-INS-i algorithm, optimized for local sequence homology. The alignment was then manually inspected and edited in Geneious. This resulted in a matrix containing 353 amino acid residues, belonging to the Myosin_head domain (as this is the only conserved domain across all myosin classes). This way we avoid as well any effect that convergently acquired protein domain architectures may have while inferring the phylogeny.

7Maximum likelihood (ML) phylogenetic trees were estimated by RaxML using the PROTGAMMALGI model, which uses the Le and Gascuel (LG) model of evolution and accounts for between-site rate variation with a four category discrete gamma approximation and a proportion of invariable sites (LG + Γ + I). Statistical support for bipartitions was estimated by performing 1,000-bootstrap replicates using RaxML with the same model. Bayesian inference trees were estimated with Phylobayes 3.3, using two parallel runs for 500,000 generations and sampling every 100 and with the LG + Γ + I model of evolution. Bayesian posterior probabilities (BPP) were used for assessing the statistical support of each bipartition.

8The domain architecture of all retrieved sequences was inferred with Pfamscan, using the gathering threshold as cutoff value. Then, the number of different concurrent domains (domains encoded within the same predicted open reading frame [ORF]) was calculated for each species using custom Perl scripts (excluding the myosin head domain itself). This information was further used to build Venn diagrams of shared concurrent domains between groups, using custom Bash scripts and the website: http://bioinformatics.psb.ugent.be/webtools/Venn/ (last accessed January 29, 2014).

9Our genomic survey and phylogenetic analyses defined 31 myosin classes. Figure 1 displays their distribution across eukaryotic taxonomic groups and their canonical protein domain architecture for each class and subclass. Our data corroborated previous findings and also identified a number of new families. This was somewhat expected, given that the number of myosin classes discovered has grown considerably since the pioneering studies of and. For the sake of clarity, we incorporated the nomenclature used in previous studies, except for a number of classes in which there were conflicting names (see table S1, Supplementary Material online, for a comparison of nomenclature among studies). We dismissed and/or reused class names only on those cases in which we unambiguously inferred a different phylogenetic relationship, and therefore alternative classification, to that identified in previous analyses. Thus, our new updated and integrative classification provides a useful systematic framework for myosins.

10Myosin I (bootstrap support [BS] = 64%, BPP = 1.0; see fig. 2 and supplementary fig. S1, Supplementary Material online) comprises five subclasses including myosin Ik, newly identified here (BS = 79%, BPP = 0.99). Subclasses c/h, d/g, and a/b (named according to their vertebrate co-orthologs) have a tail composed of IQ domains (PF00612) and a myosin TH1 domain (PF06017). Co-orthologs of these four subclasses are present in several eukaryotic taxa (fig. 1). Interestingly, we find orthologs of each subclass in unicellular holozoans. Myosin Ik, which is found in choanoflagellates, filastereans, ichthyosporeans, and, with weaker support, in Thecamonas trahens, was lost in metazoans, and thus the diversification of these four subclasses (Ia/b, Ic/h, Id/g, and Ik) most likely occurred in the common ancestor of Holozoa prior to the radiation of Metazoa.

11Myosin II is the second largest class of myosins, and is characterized by a myosin N-terminal domain (PF02736) and a tail containing an IQ domain and a myosin tail domain (PF01576), consisting of several coiled-coil domains. Although myosin II was previously thought to be exclusive to amorpheans (also known as unikonts) and was used as a phylogenetic marker, a myosin II homolog was recently identified in the excavate N. gruberi. Myosin II therefore probably had a deeper ancestry, although a HGT event from Amoebozoa to Excavata cannot be ruled out—especially considering the several cases of HGT that have recently been described between Heterolobosea and Amoebozoa. However, myosin proteins form numerous and specific interactions with actin filaments, plasma membrane, and numerous secondary protein complexes. Proteins with complex protein–protein interaction networks have been shown to be less likely to undergo HGT probably because integration into foreign protein interactions is limited. Therefore, our favoured explanation for aberrant taxon distribution of myosin orthologs and domain architecture patterns identified in this study (as in the case of myosin VI discussed below) are patterns of multiple secondary loss or convergence, rather than HGT. Irrespective of whether the N. gruberi myosin II is a result of HGT or not, this shows that myosin II is no longer a valid molecular synapomorphy for amorpheans.

12Interestingly, myosin II is the major motor protein involved in actomyosin contraction in metazoan muscle and nonmuscle cells, providing contractile force during cytokinesis in the latter, a function also performed by members of yeast myosin class II. Metazoans have two subclasses of myosin II, referred to here as smooth (Myo2) and striated (Myo11/zipper) muscle myosins (fig. 1), which have been shown to have architectural differences in the composition of their coiled-coil domains and to have originated most likely at the stem of Holozoa, although striated muscle myosin was later lost in unicellular holozoans. We confirm this hypothesis by showing that an extant filasterean species, Ministeria vibrans, has a striated myosin homolog (BS = 72%, BPP = 1.0) with the extra 29 aa-based coiled-coil that is typical of striated muscle myosin II (fig. 2). We therefore infer that myosin II was derived early in the radiation of the eukaryotes and diverged into two classes in the holozoan lineage (smooth and striated), the latter being secondarily lost in ichthyosporeans and choanoflagellates.

13The myosin III class is characterized by an N-terminal Protein kinase domain (PF00069) and several IQ domains (fig. 1). It is strictly metazoan-specific, although a larger group of choanoflagellate, sponge, and filasterean sequences appear to be related to it (BS = 68%, BPP = 1.0) (figs. 1, 2, and 6). This group represents a choanoflagellate-specific expansion of myosin genes, with different domain arrangements, including some members with protein kinase domains, WW domains (PF00397), SH2 domains (PF00017), PH domains (PF00169), Y-phosphatase domains (PF00102), and others (discussed later; fig. 3). The metazoan-specific myosin XVI is also related to myosin III and myosin III-like sequences. Our data demonstrate that myosin III-like originated at the stem of the Filozoa clade (i.e., Filasterea, Choanoflagellata, and Metazoa), acquiring its definitive domain configuration (with an N-terminal protein kinase domain) and leading to the birth of an additional paralog class (myosin XVI) at the base of the Metazoa.

14All myosin IV proteins have WW domains that can either be N-terminal or C-terminal to the Myosin_head domain, and a tail with a MyTH4 domain (PF00784), followed in some cases by a SH3 domain (in T. trahens and ichthyosporeans) (fig. 1). Previously considered an orphan myosin of the amoebozoan Acanthamoeba castellanii (, our results show that many other lineages have class IV myosins namely, ichthyosporeans, apusozoans, rhizarians, and heterokonts (BS = 67%, BPP = 1.0; figs. 1 and 2). Thus, despite its patchy distribution, it is likely that this myosin class was present in the LECA (fig. 4).

15Class V myosins have an N-terminal Myosin_head domain and a C-terminal tail with IQ and a globular DIL domains (PF01843) (fig. 1). Myosin V and the structurally similar plant myosin XI carry a remarkable variety of cargo, including organelles, vesicles, and protein complexes. A relationship between myosin V and plant myosin XI has long been proposed due to their similar domain architectures. Moreover, the orthology between opisthokont myosin V and amoebozoan myosin V (renamed here as myosin XXXIII) was assumed but not well-supported phylogenetically. Here, we show that all myosin V-like proteins cluster together phylogenetically with low ML nodal support in the global analysis (BS = 2%, BPP = 0.85), but maximum nodal support (BS = 100%, BPP = 1.00) if a closer outgroup is used (supplementary fig. S3, Supplementary Material online). This group includes other bikont myosins with different domain architectures. Therefore, we propose a unique ancestral origin in the LECA for the progenitor of this paralogous family (fig. 2; supplementary figs. S1–S3, Supplementary Material online). We group them in several classes, including plant myosin XI (BS = 87%, BPP = 1.0), opisthokont myosin V (BS = 73%, BPP = 1.0), amoebozoan myosin XXXIII (BS = 44%, BPP = 0.99) (formerly called myosin V, but phylogenetically not related to it), stramenopile + haptophyte myosin XXI (BS = 68%, BPP = 1.0), stramenopile + alveolate myosin XXVII (BS = 65%, BPP = 1.0), and a group of Guillardia theta orphan myosins (BS = 38%, BPP = 0.9) (these last three do not have the consensus myosin V architecture, presenting a wide variety of alternative domain architectures) (fig. 1). In the case of opisthokont myosin V, we confirm that myosin XIX is related to it (BS = 66%, BPP = 1.0), but we demonstrate that it is not a metazoan-specific class because it is also present in ichthyosporeans. Moreover, our phylogenetic trees strongly suggest that myosins V and Vp originated in the last common ancestor of opisthokonts (BS = 73%, BPP = 1.0) (supplementary fig. S3, Supplementary Material online). Myosin Vp was secondarily lost in fungi, metazoans, and choanoflagellates. Interestingly, the two filasterean species analysed have differentially lost one or the other, as Capsaspora owczarzaki has myosin Vp and M. vibrans has myosin V (fig. 1).

16The unique class VI myosins move toward the minus end of actin filaments, in contrast to all other known myosins. Myosins from this class are involved in diverse processes such as cytokinesis, transcription regulation, and endocytosis. Our phylogeny shows that homologs of this class are present in metazoans, choanoflagellates, filastereans, Corallochytrium limacisporum, and apusozoans, but not in fungi or amoebozoans (fig. 1). found putative VI-like genes in alveolates, but our analysis places them within myosin XXIII (supplementary table S1, Supplementary Material online). Yet, we identified an ortholog in the haptophyte Emiliania huxleyi (BS = 25%, BPP = 0.99). It is not clear whether this nonamorphean myosin VI represents an ancestral member that was lost in all other bikonts, or whether it derives from a HGT event. The fact that this and a T. trahens homolog share a unique C-terminal RUN domain (PF02759) that is not found in any other myosin supports the latter possibility.

17Myosins VII, IX, X, XV, XVIII, and XIX were previously considered to be unique to animals, but we demonstrate the presence of clear orthologs in unicellular holozoans as well. In mammals, myosin VII is a MyTH4-FERM myosin class found in structures based on highly ordered actin filaments, such as stereocilia and microvilli. Its members have a tail with two MyTH4 domains (PF00784), two FERM (PF00373) domains, likely the product of a partial gene tandem duplication, and addition of a SH3 domain. Myosin VII homologs are found only in metazoans, choanoflagellates and Co. limacisporum (fig. 1). Some authors described a group of amoebozoan proteins with a similar architecture, involved in chemotaxis and cell polarization, and identified them as VII myosins. Yet, our phylogenetic analysis does not place them with the Holozoan VII class and, therefore, we reclassify them as myosin XXV (discussed later).

18Myosin VII is phylogenetically related to myosins X and XV (the other MyTH4-FERM myosins found in metazoans, discussed later) and to a group of apusozoan orphan myosins, although with low nodal support in ML analysis (BS = 10%, BPP = 0.96) (fig. 2; supplementary figs. S1 and S2, Supplementary Material online). Our results therefore suggest that all three originated from a single ancestral protein in the last common ancestor of Holozoa (being differentially lost in some unicellular lineages; only the unicellular Co. limacisporum has orthologs of all three classes, XV, X, and VII). Interestingly, ctenophores have lost these three myosin classes. Myosin IX is composed of a N-terminal RA domain (PF00788) and a tail with IQ domains, a C1_1 domain (PF00130) and a RhoGAP domain (PF00620). Homologs of this class are found only in metazoans and filasterea (fig. 1).

19Myosin X and XV are MyTH4-FERM classes of crucial importance for metazoan filopodia. The tail of myosins X is composed of a variable number of IQ motifs, two PH (PF00169), one MyTH4, and one FERM domain; while those of myosins XV are composed of two MyTH4, one FERM, and one SH3 domain. Myosin XVIII often has an N-terminal PDZ domain and has a C-terminal myosin tail domain. This family is present in the filasterean C. owczarzaki and all metazoans examined (BS = 95%, BPP = 1.0) (fig. 1). Although not statistically supported, myosin XVIII could be closely related to myosin II, as previously described. Finally, myosin XIX has a variable number of IQ domains and it is only found in eumetazoans and ichthyosporeans (BS = 92%, BPP = 1.0) (fig. 1). It is closely related to myosin V (BS = 66%, BPP = 1.0) (fig. 2; supplementary figs. S1–S3, Supplementary Material online).

20Myosins VIII and XI are the only myosin classes present in plants and several chlorophytes (; fig. 1). Myosin VIII, whose monophyly is strongly supported (BS = 90%, BPP = 1.0), has a tail with IQ domains. As for myosin XI, several authors have pointed out its strong similarity to myosin class V in terms of domain architecture. Here, we show that this class is found in embryophytes and chlorophytes and is well supported (BS = 87%, BPP = 1.0; fig. 1). This class is phylogenetically related to myosin V, and is included in a major myosin cluster that we name myosin V-like (fig. 2; supplementary figs. S1–S3, Supplementary Material online).

21Myosin XIV has been shown to be involved in phagosome motility and nuclear elongation in the ciliate Tetrahymena thermophila. We find that this is an alveolate-specific class that has expanded in many species (specifically in ciliates) and that shows various domain architectures. Interestingly, the ciliate Te. thermophila has several myosin XIV homologs with MyTH4 and FERM domains, and is the only known bikont (nonamorphean) taxon with myosins that have a MyTH4-FERM protein domain combination. This configuration is very common in amorphean myosins, and was probably convergently acquired in the ciliates.

22Myosin XVII, also called chitin synthase, is a fungus transmembrane myosin with Cyt-b5 (PF00173), chitin synthase 2 (PF03142) and DEK_C (PF08766) domains in its tail, a domain combination unique to this class. Its monophyly is well supported (BS = 91%, BPP = 0.99), and it is phylogenetically related to amorphean FERM domain myosins. This chitin synthase class was thought to be specific to Fungi. Interestingly, the holozoan Co. limacisporum has a highly derived myosin that is associated with a chitin synthase domain and that is phylogenetically related to the fungal myosin XVII (fig. 3). This implies that class XVII chitin synthase precedes the appearance of the Opisthokonta and was lost in most holozoan lineages (except for Co. limacisporum) and so is not a valid synapomorphy for the fungi. Moreover, we also identified myosins with chitin synthases in annelids and molluscs (figs. 1 and 3), which are members of the XVI class. Thus, they are not orthologous to fungus chitin synthases, but rather appeared convergently in annelids and molluscs (fig. 3).

23Myosin XXII is a MyTH4-FERM domain myosin found in some opisthokonts, including the chytrid fungus S. punctatus, filastereans, choanoflagellates, poriferans, and Drosophila melanogaster. Its tail is composed of an IQ, two MyTH4 and two FERM domains, with a RA domain (PF00788) between the first MyTH4 and the first FERM domain. It was secondarily lost in Co. limacisporum, ichthyosporeans, and many metazoans (fig. 1). Myosin XXII seems to be related to amoebozoan myosin XXV (fig. 2). They may comprise a single class, although there are some architectural differences between them (discussed later).

24These three myosin classes are found in heterokonts and haptophytes, which suggests that they were secondarily lost in rhizarians and alveolates (figs. 1 and 4) as these groups are thought to branch closer to heterokonts than haptophytes. Myosin XXI homologs present diverse myosin tail architectures, including IQ, WW (PF00397), PX (PF00787), and Tub (PF01167) domains. This class has become considerably expanded in the oomycete Phytophthora infestans. Myosin XXX homologs in E. siliculosus have a C-terminal PH domain and P. infestans homologs have a PX domain. Finally, the myosin XXXI class, in which we also include the old myosin XXXIII, has a characteristic tail architecture in several heterokonts homologs, with a variable number of IQ domains, a PH domain flanked by two ankyrin domains, and a C-terminal Aida_C2 domain (PF14186).

25The myosin XXV class (BS = 62%, BPP = 1.0) comprises amoebozoan sequences that were previously considered to be myosin VII homologs. They are MyTH4-FERM myosins known to have a role in cell adhesion and filopodia formation. They show remarkable architectural similarities with both myosin XV and myosin VII (fig. 1), but seem to be phylogenetically related to myosin XXII (although they have different tail architectures and their sister-group relationship is low supported) (fig. 2; supplementary figs. S1 and S2, Supplementary Material online), and thus were classified as an independent class. Myosin XXVI (BS = 100%, BPP = 1.0) is another class of amoebozoan MyTH4-FERM myosins, which does not cluster with either myosin VII or myosin XXV. We suggest a common ancestry for a group of amorphean myosin classes that are generally characterised by the presence of MyTH4 domains. This group includes these two amoebozoan classes (XXV and XXVI; fig. 2; supplementary figs. S1, S2, and S4, Supplementary Material online), as well as myosins III, XVI, IX, XVII, XX, XXXIV, X, XV, VII, and XXII (fig. 3; supplementary fig. S4, Supplementary Material online).

26Myosin XXXIII includes the amoebozoan sequences previously considered as class V myosin, and shares the same domain architecture as plant myosin XI. Our phylogenetic analysis does not support a close relationship between myosin XXXIII and myosin V; it rather demonstrates that they are related to the myosin V-like clade (fig. 2), leading us to rename the group as myosin XXXIII (fig. 2; supplementary figs. S1–S3, Supplementary Material online).

27Phylogenetic analysis allowed us to define broader groups of myosin classes and to reconstruct the evolution of the myosin toolkit across the eukaryotes. This reconstruction is based on the favored hypothesis for the root of eukaryotes, the unikont–bikont split, that has recently been recovered in a rooted multi gene concatenated phylogeny with a modification with regards to the placement of the apusozoan T. trahens within the unikonts. Based on this root, our data suggest that the LECA had at least six myosin types, with different protein domain architectures (fig. 4 for the reconstruction of LECA and other ancestral nodes). According to our reconstruction, LECA had the following: 1) an ancestral myosin I (progenitor paralog of the myosin I a/b/c/h/d/g/k ortholog subfamilies) with an architecture consisting of a myosin head domain followed by 0 to 2 IQ repeats and a C-terminal myosin TH1 domain; 2) a myosin If, with a myosin head domain followed by a myosin TH1 domain and a C-terminal SH3 domain; 3) a myosin II, with a myosin N-terminal domain, a myosin motor domain, 0 to 1 IQ domains and a myosin tail domain; 4) a myosin IV with a myosin head domain followed by a MyTH4 domain and a characteristic WW domain (either C-terminal or N-terminal); 5) a myosin V-like myosin with a myosin head followed by variable number of IQ repeats and a C-terminal DIL domain; and 6) a myosin VI, with a myosin N-terminal domain followed by a myosin head domain.

28In figure 4, we show the diversity of the myosin complement in the LECA genome under a modified version of the unikont–bikont root. Our reconstruction indicates that LECA possessed a minimum of six paralog families all encoding different protein domain architectures. Even if alternative rooting hypothesis are taken into account the inferred number of myosin paralog families in the LECA is still high (supplementary figs. S5 and S6, Supplementary Material online). This result is consistent with the pattern observed in the kinesin gene family, which also demonstrated a diverse repertoire of paralog families present in the LECA. Together these data suggest that the LECA possessed a complex and diversified actin and tubulin cytoskeleton and that this ancestral cell possessed a large number of complex eukaryotic cellular characteristics prior to the diversification of extant and sampled eukaryotic groups. Assuming this root, these results have two implications: 1) they strongly suggest that a large quantity of protein diversification and cellular complexity evolved between the point of eukaryogenesis and LECA, and 2) indicate that gene loss and subsequent reduction in cytoskeletal systems played a significant role in the diversification of eukaryotes, a pattern that is increasingly apparent on other gene families and cellular systems.

29Our analysis reconstructed the LBikCA (Last Bikont Common Ancestor) with the same complement of myosins as the LECA (fig. 4). New classes appeared later in bikont evolution, such as myosin XIII at the stem of Kinetoplastida + Heterolobosea and myosin XXI, XXX, and XXXI at the stem of SAR + Haptophyta. Assuming the unikont–bikont root, our analyses demonstrate that many groups underwent secondary losses, with two extreme cases of complete loss of the myosin toolkit in the following: 1) metamonads (including Trichomonas vaginalis and Giardia lamblia) and 2) rhodophytes (including the unicellular Cyanidioschyzon merolae and the multicellular alga Chondrus crispus) (figs. 4 and 5).

30The LACA (Last Amorphean Common Ancestor, modified by inclusion of Apusozoa) added a new myosin type from LECA, a MyTH4-FERM myosin that includes several phylogenetically related myosin classes (supplementary figs. S1, S2, and S4, Supplementary Material online). These myosins have a complex protein domain architecture including a myosin head domain followed by 0 to 2 IQ repeats, a MyTH4 domain, a FERM domain, in some cases a SH3 domain, and an additional MyTH4 and FERM domains (fig. 4). This ancestral protein domain architecture is found in diverse myosins from extant amoebozoans (classes XXV and XXVI) to holozoans (class VII and, with some variations, classes XV and X). In any case, the putative ancestral MyTH4-FERM myosin underwent major architectural rearrangements as the family expanded during diversification of the amorpheans (figs. 3 and 4).

31The LOCA (Last Opisthokont Common Ancestor) had an even more complex myosin complement, with the addition of new myosin classes (as a consequence of the diversification of ancestral myosin types, such as myosin V-like or MyTH4-FERM myosins), including myosin V, myosin Vp, myosin XVII (a chitin synthase that is present in all fungi and in a single holozoan species, Co. limacisporum, discussed earlier), and myosin XXII. This complexity became even greater in the LHolCA (Last Holozoan Common Ancestor), which had the highest diversity of myosin types among all reconstructed ancestors (fig. 4). This diversity was further expanded during holozoan evolution, with little innovation at the stem of Metazoa.

32Our data show that there are strong phylogenetic patterns across lineages, in terms of abundance and number of classes, and the diversity of concurrent domains (i.e., domains that appear together with the myosin head domain in a given protein or ORF).

33The number of myosin genes varies markedly between lineages (fig. 5A). Holozoan genomes, as well as some amoebozoans and heterokonts, have the highest numbers of myosins of all eukaryotes. In particular, the haptophyte Em. huxleyi has the highest number of myosin genes (53), followed by the ichthyosporean Pirum gemmata (43), the filasterean M. vibrans (39), and the metazoan Homo sapiens (38). On the other hand, dikaryan fungi, plants, green algae, alveolates, and some excavates have few or no myosins.

34A comparison of the abundance of myosin proteins with the diversity of myosin classes (fig. 5A), reveals that Em. huxleyi, which has a high number of myosins, has only six myosin classes. This implies that the high number of myosin homologs found in this species is due to class-specific expansions rather than possession of a wide diversity of ancestrally derived myosin types. In contrast, many unicellular holozoans, especially choanoflagellates and filastereans, and some metazoans (such as H. sapiens and the homoscleropmorph sponge Oscarella carmela) have a high diversity of myosin classes. In general, our data reveal a marked increase in the number of myosin classes at the origin of Holozoa, although some specific taxa, such as the ctenophore Mnemiopsis leidyi and the ichthyosporeans Sphaeroforma arctica and Creolimax fragrantissima, secondarily reduced their repertoire of myosins.

35Myosin motor domains are found in a diverse collection of protein domain architectures, therefore another aspect that reflects differences in myosin diversity is the number of concurrent protein domains found associated with the motor domain (fig. 5B). The richest species in terms of protein domain diversity attached to the myosin motor domain within a putative ORF are the choanoflagellate Salpingoeca rosetta, the filastereans M. vibrans and C. owczarzaki and the metazoan H. sapiens. This implies that myosins were highly diversified prior to the origin and divergence of metazoans. Indeed, the sponge O. carmela also has a rich repertoire of concurrent domains, which corroborates (together with the fact that it has the richest range of myosin classes among analysed taxa) that the myosin repertoire was already rich and diverse in early metazoan evolution.

36Interestingly, the oomycete plant pathogen P. infestans, which has a high number of myosin genes, also shows a remarkable diversity of concurrent protein domains, a feature that has already been described for other gene families. In contrast, the myosin-rich taxon Em. huxleyi is relatively poor in both class diversity (fig. 5A) and protein domain diversity. The poorest taxa in protein domain diversity are plants, chlorophytes, excavates and alveolates. The cryptophyte G. theta represents an extreme case with no identified protein domains within the predicted ORF of any of its 11 myosins.

37An examination at the concurrent protein domain composition of myosin in different taxa (fig. 6) reveals that 14 protein domains are conserved between amorpheans and bikonts (fig. 6A) with similar levels of innovation in both clades (20 and 21 new concurrent protein domains, respectively). A comparison of the most widely studied eukaryote clades (metazoans, embryophytes, and fungi [fig. 6B]) reveals that there are no specific concurrent domains in plants (only those present in myosin XI, which are shared by metazoan and fungus myosin class V) and in fungi there are only two specific domains (those associated with myosin XVII, i.e., DEK_C and Cyt-b5). In contrast, metazoans have many specific domains associated with myosins.

38Within amorpheans (fig. 6C) there is a core of conserved domains (such as Myosin_tail_1 or Myosin_TH1) and a burst of innovation in the Holozoa. A closer look reveals that most of these domain combinations are present in unicellular holozoans, while little actual innovation occurred at the origin of metazoans (only the PDZ domain) (fig. 6D). In contrast, every single unicellular holozoan lineage has new specific associated domains: three in choanoflagellates (Mcp5_PH, SAM_2 and Y_phosphatase), two in filastereans (Rap_GAP and zf-MYND) and two in ichthyosporeans (AIP3 and LIM).

39Within bikonts (fig. 6E) there are no protein domains shared by all major lineages and little innovation in protein domain combinations is observed, except in the case of haptophytes (five domains) and particularly in the SAR clade (Stramenopiles/Heterokonta, Alveolata, and Rhizaria). A closer look at the SAR clade (fig. 6F) reveals that this diversification of protein domains is largely lineage-specific, with five new domains in alveolates and thirteen new domains in heterokonts.

40It is interesting to note that some of these shared protein domains were acquired convergently, for example the LIM domain in haptophytes and ichthyosporeans, the Mcp5_PH domain in haptophytes and choanoflagellates and the FERM_M domain in alveolates and amorpheans. This points to another source of homoplasy when considering protein domain architectures as evolutionary synapomorphies.

41Our data show several lineage-specific expansions, often accompanied by major protein domain architecture rearrangements. This is the case, for example, of myosin class XXVII, which is expanded in both the oomycete P. infestans and the alveolate Perkinsus marinus, with unique protein domain architectures. Another example is the ciliate Te. thermophila, which has 12 myosin homologs of the alveolate-specific class XIV. In addition to the consensus architecture found in most alveolates, Te. thermophila myosin XIV is the only bikont myosin with the MyTH4-FERM domain combination, a domain architecture that was convergently acquired (compared with amorphean MyTH4-FERM myosins, discussed earlier).

42The most spectacular lineage-specific expansion is that observed in choanoflagellate myosin III-like myosins (fig. 3). This phylogenetically defined group includes bona fide eumetazoan myosin III homologs, the related metazoan myosin XVI class (including annelid and mollusc chitin synthases), filasterean sequences (comprising a unique group), a single sequence of the sponge Amphimedon queenslandica, a single sequence of the sponge O. carmela, and several choanoflagellate myosins (15 from Monosiga brevicollis and 18 from Sa. rosetta). These choanoflagellate sequences have a wide diversity of protein domain rearrangements (fig. 3). Interestingly, many of these domains, like SH2 and Y-phosphatase domains, are related to tyrosine kinase signaling, a prominent feature of choanoflagellates. Sequences belonging to the myosin III-like group with a C-terminal SH2 domain were also identified in filastereans, which also have an extensive tyrosine kinase toolkit. Another interesting configuration found within this myosin III-like group is an Sa. rosetta and an O. carmela sequence with a C-terminal MH2 PF03166 domain. This domain is typically present in Smad transcription factors, where it is found at the C-terminal of the MH1 DNA-binding domain and acts as a protein binding motif that mediates cofactor interactions. Interestingly, the MH2 domain is only found in choanoflagellates and metazoans, while Smad transcription factors are exclusive to animals. The fact that the single MH2 domain found in choanoflagellates is associated with a myosin, together with that fact that the sponge O. carmela also has this configuration, suggests that MH2 initially appeared associated with myosins as a protein–protein interaction domain. Later on, early in metazoan evolution, MH2 was fused by domain shuffling to a MH1 DNA-binding domain to create the Smad transcription factors.

43Our results show that all metazoan myosin classes but one (Myosin XVI, also known as Dachs) have a premetazoan origin, many of them being holozoan innovations (fig. 6) (including myosin III-like, VII, IX, X, XV, XVIII, and XIX). Moreover, several subclass diversifications occurred in unicellular holozoans, for example in Myosin V (Myosin V and Myosin Vp), in Myosin I (Myosin I a/b, I/c/h, Id/g, and Ik) and in Myosin II (smooth and striated). In terms of number of myosins and diversity of concurrent domains (fig. 5), unicellular Holozoa have the highest counts among eukaryotes (even higher than most Metazoa). In fact, the choanoflagellate Sa. rosetta has the most diverse repertoire of myosin concurrent domains (fig. 5B), followed by another choanoflagellate (Mo. brevicollis), the filasterean C. owczarzaki and the metazoan H. sapiens. Overall, we can infer that the complexity of the myosin toolkit was extremely high before the advent of animal multicellularity and that this system is of paramount importance in extant unicellular holozoans.

44We provide a robust updated myosin classification, based on ML and Bayesian phylogenetic methods and broad genomic taxon sampling that includes, for the first time, all major eukaryotic lineages. We provide a redefinition and/or confirmation of previously defined myosin classes (with an effort to reconcile myosin nomenclature between various previous classifications), and we assess the presence/absence of myosin classes in eukaryotes. Furthermore, we reconstruct a more complex myosin complement in the LECA genome than previously proposed, with six different myosin types and six different inferred domain architectures under the modified unikont–bikont root. Notably, we find strong phylogenetic patterns related to the complexity of the myosin system. Finally, we infer an intricate evolutionary history of the myosin gene family, including multiple lineage-specific expansions (such as the myosin III-like group in the choanoflagellate lineage), domain diversifications (specially in holozoans), secondary losses (in metamonads and rhodophytes), and convergences (e.g., in the fungal and metazoan myosin–chitin synthases). Taken together our results demonstrate that myosin gene family underwent multiple large-scale expansions and contractions in paralog families combined with extensive remodelling of domain architectures. As the diversity of this gene family directly relates to the function of the actin cytoskeleton, these results tell a story of extensive remodelling of this cytoskeleton system across the eukaryotes. These results also suggest that evolutionary inference of species relationships based on myosin distribution patterns is difficult without reliable phylogenetic analysis and comprehensive sampling. As such, the expansion of available genome data will provide a more accurate inference of the relative phylogenetic age of myosin classes and types—likely expanding the repertoire of myosins, and therefore the cellular complexity, of ancestral eukaryotic forms.

45Supplementary data S1, figures S1–S6, and tables S1 and S2 are available at Genome Biology and Evolution online (http://www.gbe.oxfordjournals.org/).