BACKGROUND
intracellular compartmentalization, via membrane-delimited organelles, is a fundamental feature of the eukaryotic cell and membrane trafficking between organelles became vital for these cells.
these mechanisms are typically regulated by rab proteins, which form by far the biggest family among the small gtpases, with more than  <dig> members in humans  <cit> .
these proteins play a crucial role in the regulation of cellular membrane trafficking in all eukaryotes  <cit> .
this role is orchestrated mainly by the switching between the gtp/gdp-bound states of these proteins, controlled by the guanine nucleotide exchange factors  and gtpase activating proteins .
most rab gtpases consist of  <dig> amino acids and are roughly 24 kda  <cit> .
rab proteins possess some conserved domains: the p-loop, a well-known nucleotide binding motif, fundamental for the switching between gtp/gdp-bound states; switch i and switch ii that are necessary for the binding of guanine nucleotides  <cit> .
each rab has a distinct subcellular localization and regulates a specific transport step.
evolutionary studies suggest that twenty rab proteins, divided into six supergroups, were already present in the last eukaryotic common ancestor   <cit> .
the number of rabs in metazoans is extremely variable due to the occurrence of several gene gains and losses  <cit> .
the genomes of all eukaryotes encode multiple members of the rab family, from 10– <dig> in most unicellular eukaryotes  to over  <dig> genes in multicellular organisms  <cit> .
the high number of rab expansions and secondary losses in several eukaryotic lineages suggest a complex evolutionary history of this family  <cit> .
among all membrane-enclosed organelles, melanosomes represent an excellent model for studying the biogenesis and motility of these structures  <cit> .
melanosomes, lysosome-related organelles , are the cellular site for synthesis, storage and transport of melanin granules that provide colour to tissues and are involved in photoprotection.
melanosomes are present in mammalian skin melanocytes, in choroidal melanocytes, in retinal pigment epithelial  cells of the eye, and in melanophores of teleosts and amphibians  <cit> .
specific rab proteins belonging to supergroup iii, rab <dig> and rab <dig>  play a key role in melanosome biogenesis  <cit> .
this process has been well described in mammals where melanosome formation is conventionally divided into four steps.
rab <dig> and rab <dig>  together with effector proteins like ap- <dig>  ap- <dig>  and bloc- <dig>  mediate the transport of enzymes fundamental for pigmentation, such as metazoan tyrosinase  and tyrosinase-related protein   <cit> , from trans-golgi network endosomes to maturing melanosomes   <cit> .
it has been shown that a point mutation in the conserved gtp/gdp-interacting domain  of the rab <dig> gene causes the chocolate  autosomal recessive mutation, which arose spontaneously in some mice strains  <cit> .
the cht mice showed an oculocutaneous albinism phenotype and a weakly diluted coat colour.
rab <dig> and the closely related rab <dig> work redundantly in melanocytes, as demonstrated by depletion of rab <dig> in in vitro cultured cht epidermal melanocytes, which severely impairs the transport of tyr and tyrp <dig> to melanosomes, resulting in severe hypopigmentation  <cit> .
mouse rab <dig> and rab <dig> are paralogues, sharing 67 % amino acid identity and are considered to have originated from the vertebrate whole genome duplication  that occurred before the gnathostomata radiation .
furthermore, it has been shown that the rab <dig> gene is mutated in ruby rats  <cit> , a strain characterized by hypopigmentation and platelet storage pool deficiency related to hermansky-pudlak syndrome   <cit> .
this pathology in humans causes oculocutaneous albinism, easy bleedings, abnormal lysosomal ceroid lipofuscin and pulmonary fibrosis in 40– <dig> years-old patients  <cit> .
finally, frog  melanophores are characterized by a strong expression of rab <dig>  suggesting an involvement in pigment formation  <cit> .
much less is known regarding invertebrate rab32/ <dig> gene functioning: in the ascidian ciona intestinalis, the closest living relative of vertebrates  <cit> , the unique rab32/ <dig> gene is expressed in four cells belonging to the pigment cell lineage  <cit> .
among these cells the otolith and ocellus, the sole pigmented sensory organs of ascidian larvae, will form  <cit> .
functional studies demonstrated that rab32/ <dig> is essential for proper pigmentation of otolith and ocellus pigmented cells  <cit> .
in the fruit fly drosophila melanogaster rab-rp <dig>  is expressed in eye lysosomes and lipid droplets of adipose tissue  <cit> , and its mutation causes eye hypopigmentation   <cit> .
to gain insight into deuterostome evolution and the possible function of the rab <dig> and rab <dig> genes, we investigated the molecular evolution and spatio-temporal localization during development in two key animal models: the cephalochordate branchiostoma lanceolatum and the teleost danio rerio.
cephalochordates represent an early-branching chordate group, their genomic, anatomical and morphological features, however, make them the best available stand-in for the chordate ancestor.
amphioxus has an unduplicated genome, but possesses representatives of all vertebrate gene families  <cit> .
amphioxus photoreceptive system is formed by  <dig> different structures: joseph cells, the lamellar body, dorsal ocelli  and the frontal eye  <cit> .
hesse cells are traditionally considered homologue of vertebrate eye photoreceptors, whereas the frontal eye pigment cells are thought to be homologue of vertebrate rpe cells, as demonstrated by mitf, otx and pax2/5/ <dig> tissue-specific expression  <cit> .
tyr, tyrp1/2a and tyrp1/2b are co-expressed at the neurula stage in the neural tube where the first primary pigment spot will form  <cit> .
among vertebrates, the teleosts  show an incredible variety of pigmentation patterns, decisive for their complex behaviour.
intriguingly, the skin pigmentation pattern of teleosts is not only due to the black/brown melanocytes, yellow/red xanthophores and reflecting iridophores, common to all vertebrates, but also to whitish leucophores and blue cyanophores that are lineage-specific novelties  <cit> .
teleosts are able to adapt perfectly to highly different environments, through the α-melanophore-stimulating hormone  secreted by the pituitary, which is influenced by neurotransmitters such as norepinephrine  <cit> .
teleost genomes have undergone a third round of whole genome duplication that probably provided the raw genetic material for the teleost radiation .
this event occurred only in the teleostean lineage, amongst actynopterigians  <cit> , and it was therefore named “teleost-specific genome duplication”   <cit> .
it has been suggested that a strong relationship exists between these genome duplications and the extraordinary pigmentation variety in teleosts: in fact teleost genomes show a dramatically wide repertoire of genes involved in pigmentation, produced both by tsgd and small-scale gene duplications  <cit> .
many pigmentation genes in teleosts gained other roles, distributing ancestral functions to duplicated genes  or acquiring new one after the split   <cit> .
in this paper, we studied all the rab <dig> and rab <dig> genes present in the genomes of the amphioxus b. lanceolatum and the zebrafish d. rerio to shed light on the evolution of this subfamily of deuterostomes and gain insight into their role during chordate development.
RESULTS
evolutionary history of rab <dig> and rab <dig> in deuterostomes
to study the evolutionary history of the rab <dig> and rab <dig> we performed a phylogenetic reconstruction starting from a database of manually curated supergroup rab iii sequences .
we included sequences from vertebrates , urochordates , cephalochordates , hemichordates  and echinoderms .
since rabs are very similar to other small gtpase proteins, we only included sequences giving the higher blast scores with known rabs.
to obtain the best phylogenetic resolution we included representative members of rab supergroup iii: rab <dig>  rab <dig>  rab <dig>  rab7l <dig>  rab <dig>  rab <dig> .
regarding rab <dig> and rab <dig>  our analysis allowed to identify two genes in amphioxus and five in zebrafish genomes.
phylogenetic analysis revealed four strongly supported clades: rab <dig>  rab <dig>  rab <dig> and a clade formed by rab7l <dig>  rab32lo, rab <dig> and rab <dig>  among these, rab <dig>  rab <dig> and rab <dig> represent phylogenetically robust monophyletic groups , while the rab7l <dig>  results as sister group of rab32lo  and rab32/rab <dig> .fig.
<dig> evolution of the rab <dig> and rab <dig> subfamily in deuterostomes.
numbers at the branches indicate replicates obtained using the maximum likelihood estimation method.
<dig> sites were used for tree inference.
colored boxes highlight three classes of proteins: rab7l <dig> present in invertebrates and vertebrates , rab32lo present in protostomes, echinoderms and cephalochordates , the rab <dig> and rab <dig> cluster present in all the deuterostomes .
black arrow indicates the acquisition of falk amino acid stretch at the stem of rab7l <dig>  rab32lo, rab32 and rab <dig> evolution.
triangles indicate monophyletic clades belonging to rab supergroup iii
rab32los, previously named rab32b  <cit> , were described as already present in choanoflagellates , sponges  and cnidarians , and therefore considered as leca gene.
moreover, we found rab32lo in protostomes  and in deuterostomes .
strikingly, our study highlighted its loss in olfactores   <cit>  prompting us to name it rab32lo .
unfortunately, our phylogenetic survey did not clarify the relationships among the vertebrate rab <dig> and rab <dig>  and invertebrate rab32/ <dig> genes  due to the high degree of sequence similarity.
the phylogenetic tree  shows that amphioxus possesses a rab32lo and a unique rab32/ <dig> member.
zebrafish presents three rab <dig> , and two rab <dig> genes .
strikingly, the position of rab32b in the tree is inconsistent with species phylogeny, probably due to its fast evolutionary rate .
a vertebrate-specific phylogenetic analysis was performed to elucidate the relationship existing between rab <dig> and rab <dig> proteins in this group .
with the aim to distinguish between rab <dig> and rab <dig> proteins, we compared the three conserved rab domains across several deuterostome species, using the human rab6a as reference  <cit> : the p-loop  located at amino acids 20– <dig>  the switch i  at amino acid positions 38– <dig> and the switch ii  at amino acids 69– <dig> .
to better understand the conservation of protein domains, we also added all human proteins of rab supergroup iii and one representative of other five rab supergroups.
the alignment shows a high degree of conservation for three known domains in deuterostomes.
the general core sequence of the p-loop is gxxxxgkt in all subfamilies.
we found that the 2nd residue is diagnostic for rab classification in vertebrates .
switch i  and switch ii  are highly conserved across deuterostomes, showing sequence divergence only in zebrafish rab32b.
moreover, we identified for the first time the existence of an ultra-conserved stretch of four amino acids  located at the end of switch i that is present only in rab <dig>  rab <dig>  rab32lo and rab7l <dig> subfamily members  among rab supergroup iii.
the presence of two conserved histidine residues exclusively in rab32lo switch ii  confirms the fact that rab32lo is a distinct class within supergroup iii, supporting our phylogenetic data.
in order to clarify the poorly resolved relationship among invertebrate rab32/ <dig> and vertebrate rab <dig> and rab <dig> we searched for an evolutionary signature of conserved introns, analysing the gene structure of human rab <dig> and rab <dig> in comparison with branchiostoma, ciona and lottia rab32/ <dig> genes.
strikingly, we found two introns that show conserved position and phase, suggesting a common ancestral origin .fig.
<dig> alignment of rab supergroup iii domains.
the alignment shows the amino acid conservation of three rab domains: the p-loop, involved in trafficking , the switch i  and the switch ii  important for supporting the binding with other molecules.
downstream of switch i, four ultra-conserved amino acids are highlighted, named falk .
the white background indicates changes in amino acid composition during evolution.
at the bottom, human representatives of other rab supergroups are reported to show the absence of falk
rab <dig> and rab <dig> synteny conservation
to further improve knowledge on the evolutionary history of the rab <dig> and rab <dig> subfamilies, we analysed their chromosomal neighbourhood genes in available deuterostome genomes .
we searched in genomes of invertebrates  and, among vertebrates, we selected the lamprey p. marinus for the agnathans, the elephant shark c. milii  <cit>  and spotted gar l. oculatus as representatives of non-teleost fish  <cit> , d. rerio for teleosts, the lizard a. carolinensis in the sauropsida clade, and h. sapiens and m. musculus among mammals.
we did not find chromosomal conservation in lamprey.
on the other hand, we discovered a high degree of synteny conservation within both gnathostome rab32s loci  and rab38s loci .
nevertheless, the genes flanking rab <dig> are different from those surrounding rab <dig>  to demonstrate the orthology of syntenic genes we performed ad hoc phylogenies of tab, nox, fzd and stxbp genes .fig.
<dig>
rab <dig> and rab <dig> synteny conservation in gnathostomes.
the rab <dig>  and rab <dig>  loci harbour several genes  conserved across evolution; in blue boxes rab genes we studied, in red boxes rab <dig> genes that are physically linked to tyrosinase .
during the gnathostome evolution, the rab <dig> is always linked to grm <dig>  while rab <dig> is linked to grm <dig>  the scheme 3b
’ shows the functional relationship between rab <dig> and tyr during mammalian melanosome biogenesis .
the position of the genes above or below the chromosome  indicates their transcriptional orientation on positive or negative strand, respectively
the existence of a chromosomal region conserved between h. sapiens and some teleosts, containing rab <dig>  grm <dig> and tyr genes has been already described in a comparative study  <cit> .
our syntenic survey across the vertebrates extended the conservation to the holocephalan elephant shark, the non-teleost actinopterygian spotted gar, the anole lizard and mice.
moreover, we also showed that rab <dig> genes are always linked to a grm family member  .
collectively, our data suggest an ancestral chromosomal linkage between a rab32/ <dig> and grm1/ <dig> gene, already present in the vertebrate’s common ancestor .
we carried out the phylogeny of the grm family to demonstrate the orthology among vertebrate grm <dig> genes and grm <dig> genes, respectively , further supporting our hypothesis of an ancient chromosomal linkage.
in more detail, we found one rab <dig> always coupled with the melanoma oncogene grm <dig>  <cit> , while one rab <dig> is always close to grm <dig>  a gene playing a fundamental role in many human disorders such as schizophrenia  <cit>  and autism  <cit> .
among zebrafish rab <dig> paralogues we found a higher degree of syntenic conservation in the rab38a locus  than in the rab38b and rab38c loci .
for instance, we found that only the zebrafish rab38a maintained the linkage with the tyr gene, as is the case in the unique tetrapod rab <dig> paralogue.
similarly, in elephant shark and spotted gar this linkage is retained in only one of the two rab <dig> paralogons .
additionally, in tetrapod fohl1a and fohl1b, and the coiled-coil domain containing proteins , belonging to a big family whose role is completely unknown, are duplicated on the same chromosomal stretch .
the mammalian rab <dig> loci show an interesting peculiarity, they present several insertions of receptors belonging to the same family derived from tandem gene duplication events .
on mouse chromosome  <dig>  beside the two fohl1s, there are fifteen vomeronasal  <dig> receptors , known for their crucial role in mouse ultrasensitive chemodetection  <cit>  and fourteen olfactory receptors   <cit> , belonging to a very large family in mice, explaining their extraordinary odour sensitivity and ability to discriminate scents  <cit> .
interestingly, in the orthologous position, the human chromosome  <dig> harbours seven tripartite motif proteins , which are involved in a plethora of biological processes, in particular the immune response  <cit>  .
on the other hand, we did not find any synteny conservation in rab loci of invertebrate deuterostomes.
the unique trace of microsynteny was found in the sea urchin and amphioxus gene scaffolds, between rab32lo and tim <dig>  an evolutionarily conserved transporter involved in metabolite import by mammalian mitochondria  <cit>  .
rab <dig> and rab <dig> gene expression patterns in amphioxus and zebrafish
to investigate the possible role of rab genes in the amphioxus b. lanceolatum, we cloned the rab32lo and rab32/ <dig> genes and studied their expression pattern by whole mount in situ hybridization  in embryos at different developmental stages .
unfortunately, rab32lo was not detectable by in situ hybridization at any analysed stage, confirmed by the low levels of expression detected by real-time rt-pcr experiments .
rab32/ <dig> was first observed at the gastrula stage in presumptive notochord territories , and later during development, at the neurula stage, it appears clearly in the notochord, from the most rostral part along the length of the embryo without reaching the caudal part .
the rab32/ <dig> expression in the notochord has been confirmed by the transverse section .
at the pre-mouth larval stage of development, the rab32/ <dig> expression in the notochord turns off and a novel territory of expression is visible in the pharynx region .fig.
<dig>
rab32/ <dig> expression pattern during amphioxus embryogenesis.
rab32/ <dig> has been observed in notochord presumptive territories at the gastrula stage , while later in development at the neurula stage it is expressed in notochord, mainly in the rostral part .
d is a vibratome section  of the neurula specimen showed in b-c, at the level of the vertical dashed line.
at the pre-mouth larval stage, rab32/ <dig> expression in the notochord turns off while a positive signal appears in the pharynx region
the same approach was used to investigate the expression pattern of rab <dig> and rab <dig> during zebrafish embryogenesis.
it was impossible to amplify rab32b from embryonic and adult tissues , suggesting that this gene is either expressed at very low levels or only under specific environmental conditions.
rab32a expression has been detected in the presumptive posterior axial mesoderm starting from 6 h post-fertlization  , as it was already described by thisse and collaborators  <cit>  .
during embryo elongation , the rab32a positive cells were localized near the animal pole and form a longitudinal band in the dorsal midline, the developing notochord that includes the tail bud region .
moreover, at this stage, a signal is detectable in the kupffer’s vesicle , a small but distinctive epithelial sac, situated posteriorly near the yolk  <cit> .
during segmentation , the expression of rab32a becomes weaker in the notochord , while it starts to be expressed in the eyes, as well as in cells that appear to be migrating melanoblasts according to their position .
starting from the long-pec stage , the expression in migrating melanoblasts disappears and is retained only in the retinal pigmented epithelium of the eye and in the notochord .
at this stage, a strong signal becomes visible  in the swim bladder, the organ that adult teleosts use for buoyancy and breathing, and persists up to the protruding-mouth larval stage .fig.
<dig>
rab32a expression pattern during zebrafish embryogenesis.
rab32a has been observed in the presumptive posterior axial mesoderm  at  <dig> hpf  and in the developing notochord  and kupffer’s vesicle  at  <dig> hpf .
at  <dig> hpf, it is present in rpe , notochord and migrating neural crest cells  .
at  <dig> hpf the signal disappears in neural crest cells, but persists in rpe and notochord   and appears in the swim bladder .
the expression in the swim bladder persists at  <dig> hpf larvae
regarding rab <dig> paralogues, rab38a is expressed across the pharyngula embryonic period  .
embryos at  <dig> hpf show a faint signal in rpe and a strong one in migrating neural crest cells, presumably migrating melanoblasts .
at this stage, a low level of expression is detectable in the mid-ventral region of brain as well .
later in development  expression is only observed in the rpe .
rab38b is expressed only at late developmental stages ; at  <dig> hpf it is expressed in a small region of the pharyngeal arch and in the developing swim bladder , while at  <dig> hpf only the signal in the swim bladder persists .
the gene rab38c is expressed from  <dig> to  <dig> hpf with a strong signal in the head .fig.
<dig>
rab38s expression pattern during zebrafish embryogenesis.
rab38a is expressed across the pharyngula embryonic period  : at  <dig> hpf there is a faint signal in rpe  and a strong one in migrating melanoblasts  and a low level of expression is detectable in the mid-ventral region of brain ; at  <dig> hpf the expression is visible only in the rpe  .
rab38b is expressed only at late developmental stages : at  <dig> hpf in a small region of the pharyngeal arches  and in the developing swim bladder , while at  <dig> hpf only in swim bladder .
the gene rab38c gene is strongly expressed in the head region .
lateral view in all images  except d and f that are ventral
discussion
rab <dig> and rab <dig> toolkit in animal evolution
our phylogeny evidences three distinct branches: rab7l <dig>  rab32lo and rab32/rab <dig>  which share the highly conserved falk stretch downstream of the switch i domain and emerged after splitting from rab <dig>  rab <dig> and rab <dig> groups .
although the falk function is still unknown, its high degree of sequence conservation in all analysed proteins suggests that it could be responsible for important functional properties .
it has been suggested that the rab32/ <dig>  already described as rab32a and rab32lo, previously named rab32b, present in the last eukaryotic common ancestor were already implicated in late endosomal or lysosomal trafficking  <cit> .
rab32lo is present in unicellular eukaryotes to chordates  <cit> , and here we showed its loss in the lineage of olfactores.
this event could be related to the loss of ancestral functions during chordate evolution, although the biological functions of these proteins should be further investigated in the future.
detailed analysis of the conserved rab functional domains allowed to identify few diagnostic residues of vertebrate rab <dig> and rab <dig> .
similarly, two histidines are distinctive residues of the rab32lo switch ii region.
our analyses suggest that the phylogenetic signal is weak for the heterogeneous evolutionary rate of this group of proteins, leaving the issue of vertebrate rab <dig> and rab <dig> origin unresolved.
a possible hypothesis is that vertebrate rab <dig> and rab <dig> arose from a prevertebrate rab32/ <dig> through a vertebrate genome duplication.
the study of intron conservation, considered a diagnostic tool for tracing back the evolutionary history of genes  <cit> , showed the existence of a conserved intron code supporting a common origin for all the subfamily members .
furthermore, the parallel evolutionary history of rab32/ <dig> and grm1/ <dig> genes is consistent with our hypothesis of a duplication event in vertebrates.
in fact, in invertebrates, similarly to the rab32/ <dig>  a single pre-duplicative grm1/ <dig> exists as demonstrated by the dedicated phylogeny .
interestingly, the chromosomal proximity of rab <dig> to grm <dig> and rab <dig> to grm <dig> indicates a duplication of an ancestral chromosomal region comprising rab32/ <dig> and grm1/ <dig> .
the retained microsynteny that we found from elephant shark to humans hints at a functional correlation between these two gene duplets.
it would be challenging to find out whether the integrity of these linkages is necessary for their functioning.
collectively our phylogeny, intron and synteny conservation analyses suggest that rab <dig> and rab <dig> derived from a vertebrate genome duplication, therefore they are both ohnologs of the pre-vertebrate rab32/ <dig> gene.
we found a very high degree of chromosomal conservation among vertebrates .
in particular the high similarity between c. milii rab38a and tetrapod rab <dig> loci confirms that cartilaginous fishes mimic the tetrapod genomic organization better than teleosts  <cit> .
surprisingly, a sharp lack of synteny involving only one side of the rab38a of spotted gar  and zebrafish  loci suggests a rearrangement event in the actinopterygii .
vertebrates have been affected by several rounds of genome duplication that have increased the repertoire of pigmentation genes mainly in teleosts  <cit> .
the present survey highlights the expansion of the rab <dig> and rab <dig> driven by a mixture of gene and genome duplications that allowed the emergence of novel functions  <cit> .
in zebrafish the rab <dig> and rab <dig> reached a total of five members through the tsgd, plus single gene duplication in the rab <dig> lineage.
the role of tsgd in the evolution of teleost pigmentation diversity is well-known resulting in 30 % more pigmentation genes than in tetrapods.
in fact, other genes involved in pigmentation are also duplicated such as mitf, kitl, tyr and tyrp  <cit>  explaining the extremely diversified body colour patterning of teleosts  <cit> .
intriguingly, we noticed in gnathostomes the existence of a specific linkage among two well-known players of the pigmentation process; the tyr and rab <dig>  retained in all analysed tetrapods and in the elephant shark, spotted gar and zebrafish rab38a paralogons .
it has been demonstrated that the delivery of tyr, tyrp <dig> and tyrp <dig> to the maturing melanosomes is required to initiate pigmentation.
rab <dig> and rab <dig> have been shown to mediate the transport of tyr and tyrp <dig> by interacting with the ubiquitous trafficking machinery  <cit>  .
taking into consideration that rab38a is the sole rab <dig> member to be expressed in zebrafish pigment-producing cells and its chromosomal vicinity to tyr, we hypothesize a bystander gene regulation modality during vertebrate melanosomes biogenesis by means of a locus control region, as previously demonstrated for developmental genes  <cit> .
expression profile in zebrafish and amphioxus
in mammals, melanocytes, platelets and mast cells, rich in lros, exhibit a high level of rab <dig>  <cit> , whereas rab <dig> is restricted to melanocytes and lung epithelial cells  <cit> .
in zebrafish, we have identified two rab <dig> and three rab <dig> genes .
rab32a is expressed in the embryonic pigmentary lineage, including both melanocytes and rpe, as described in other vertebrates.
moreover, it is expressed during notochord development, a feature that seems to be specific of zebrafish .
recently it has been proposed that rab32a function is required for correct formation of the notochord vacuoles in zebrafish  <cit> .
finally, rab32a is expressed in the natatory vesicle of zebrafish , also known as the swim bladder, an organ whose relationship with the tetrapod lung is still debated  <cit> .
considering that invertebrate rab32/ <dig> has an important role in melanogenesis, as demonstrated by a functional study in c. intestinalis  <cit> , and that this role is maintained in vertebrate rab <dig>  we can assume rab <dig> function in melanogenesis as ancestral.
in zebrafish this gene has acquired new roles in the formation of other lysosome related organelles.
zebrafish shows a varied scenario of rab32s and rab38s expressions with a high spatio-temporal diversification due to genomic events that occurred during the evolution of vertebrates.
the three zebrafish rab38s are expressed in distinct embryonic territories , including pigmented cells, swim bladder and nervous system, unravelling a functional paralogous diversification.
distinct types of duplications have permitted the functional specialization in pigmentation of one of the paralogues  <cit> .
the tsgd provided further raw genetic material for the evolution of teleost pigmentation patterns  <cit> .
the expression data of the three zebrafish rab38s are an example of ancestral function distribution among duplicates .
finally, the amphioxus rab32/ <dig> expression territories in the notochord and pharynx  are consistent with transient zebrafish rab32a expression in the notochord and rab38b in pharyngeal arches.
this findings suggest possible ancestral roles of the prechordate rab32/ <dig>  which seem to be lost in tetrapods.
CONCLUSIONS
our study focused on genes belonging to the rab family known to be involved in melanosome formation in mammals: rab <dig> and rab <dig>  phylogenetic, intron conservation and synteny analyses of these genes in deuterostomes point towards an evolutionary scenario based on a duplication of a single invertebrate rab32/ <dig> gene giving rise to vertebrate ohnologs rab <dig> and rab <dig>  additional duplicates arose in bony fishes by teleost-specific genome duplication and expression pattern of rab <dig> paralogues in zebrafish evokes sub-functionalization event.
finally, the discovery of a chromosomal linkage between the rab <dig> or rab <dig> gene with a grm <dig> or grm <dig> opens new perspectives on possible conserved bystander gene regulation across the vertebrate evolution.
however, future studies are necessary to investigate the presence of shared enhancer and to test functionally its regulative properties.

 intracellular compartmentalization, via membrane-delimited organelles, is a fundamental feature of the eukaryotic cell and membrane trafficking between organelles became vital for these cells. these mechanisms are typically regulated by rab proteins, which form by far the biggest family among the small gtpases, with more than members in humans. these proteins play a crucial role in the regulation of cellular membrane trafficking in all eukaryotes. this role is orchestrated mainly by the switching between the gtp/gdp-bound states of these proteins, controlled by the guanine nucleotide exchange factors and gtpase activating proteins. most rab gtpases consist of amino acids and are roughly 24 kda. rab proteins possess some conserved domains: the p-loop, a well-known nucleotide binding motif, fundamental for the switching between gtp/gdp-bound states; switch i and switch ii that are necessary for the binding of guanine nucleotides. each rab has a distinct subcellular localization and regulates a specific transport step. evolutionary studies suggest that twenty rab proteins, divided into six supergroups, were already present in the last eukaryotic common ancestor. the number of rabs in metazoans is extremely variable due to the occurrence of several gene gains and losses. the genomes of all eukaryotes encode multiple members of the rab family, from 10– in most unicellular eukaryotes to over genes in multicellular organisms. the high number of rab expansions and secondary losses in several eukaryotic lineages suggest a complex evolutionary history of this family. among all membrane-enclosed organelles, melanosomes represent an excellent model for studying the biogenesis and motility of these structures. melanosomes, lysosome-related organelles, are the cellular site for synthesis, storage and transport of melanin granules that provide colour to tissues and are involved in photoprotection. melanosomes are present in mammalian skin melanocytes, in choroidal melanocytes, in retinal pigment epithelial cells of the eye, and in melanophores of teleosts and amphibians. specific rab proteins belonging to supergroup iii, rab and rab play a key role in melanosome biogenesis. this process has been well described in mammals where melanosome formation is conventionally divided into four steps. rab and rab together with effector proteins like ap- ap- and bloc- mediate the transport of enzymes fundamental for pigmentation, such as metazoan tyrosinase and tyrosinase-related protein, from trans-golgi network endosomes to maturing melanosomes. it has been shown that a point mutation in the conserved gtp/gdp-interacting domain of the rab gene causes the chocolate autosomal recessive mutation, which arose spontaneously in some mice strains. the cht mice showed an oculocutaneous albinism phenotype and a weakly diluted coat colour. rab and the closely related rab work redundantly in melanocytes, as demonstrated by depletion of rab in in vitro cultured cht epidermal melanocytes, which severely impairs the transport of tyr and tyrp to melanosomes, resulting in severe hypopigmentation. mouse rab and rab are paralogues, sharing 67 % amino acid identity and are considered to have originated from the vertebrate whole genome duplication that occurred before the gnathostomata radiation. furthermore, it has been shown that the rab gene is mutated in ruby rats, a strain characterized by hypopigmentation and platelet storage pool deficiency related to hermansky-pudlak syndrome. this pathology in humans causes oculocutaneous albinism, easy bleedings, abnormal lysosomal ceroid lipofuscin and pulmonary fibrosis in 40– years-old patients. finally, frog melanophores are characterized by a strong expression of rab suggesting an involvement in pigment formation. much less is known regarding invertebrate rab32/ gene functioning: in the ascidian ciona intestinalis, the closest living relative of vertebrates, the unique rab32/ gene is expressed in four cells belonging to the pigment cell lineage. among these cells the otolith and ocellus, the sole pigmented sensory organs of ascidian larvae, will form. functional studies demonstrated that rab32/ is essential for proper pigmentation of otolith and ocellus pigmented cells. in the fruit fly drosophila melanogaster rab-rp is expressed in eye lysosomes and lipid droplets of adipose tissue, and its mutation causes eye hypopigmentation. to gain insight into deuterostome evolution and the possible function of the rab and rab genes, we investigated the molecular evolution and spatio-temporal localization during development in two key animal models: the cephalochordate branchiostoma lanceolatum and the teleost danio rerio. cephalochordates represent an early-branching chordate group, their genomic, anatomical and morphological features, however, make them the best available stand-in for the chordate ancestor. amphioxus has an unduplicated genome, but possesses representatives of all vertebrate gene families. amphioxus photoreceptive system is formed by different structures: joseph cells, the lamellar body, dorsal ocelli and the frontal eye. hesse cells are traditionally considered homologue of vertebrate eye photoreceptors, whereas the frontal eye pigment cells are thought to be homologue of vertebrate rpe cells, as demonstrated by mitf, otx and pax2/5/ tissue-specific expression. tyr, tyrp1/2a and tyrp1/2b are co-expressed at the neurula stage in the neural tube where the first primary pigment spot will form. among vertebrates, the teleosts show an incredible variety of pigmentation patterns, decisive for their complex behaviour. intriguingly, the skin pigmentation pattern of teleosts is not only due to the black/brown melanocytes, yellow/red xanthophores and reflecting iridophores, common to all vertebrates, but also to whitish leucophores and blue cyanophores that are lineage-specific novelties. teleosts are able to adapt perfectly to highly different environments, through the α-melanophore-stimulating hormone secreted by the pituitary, which is influenced by neurotransmitters such as norepinephrine. teleost genomes have undergone a third round of whole genome duplication that probably provided the raw genetic material for the teleost radiation. this event occurred only in the teleostean lineage, amongst actynopterigians, and it was therefore named “teleost-specific genome duplication”. it has been suggested that a strong relationship exists between these genome duplications and the extraordinary pigmentation variety in teleosts: in fact teleost genomes show a dramatically wide repertoire of genes involved in pigmentation, produced both by tsgd and small-scale gene duplications. many pigmentation genes in teleosts gained other roles, distributing ancestral functions to duplicated genes or acquiring new one after the split. in this paper, we studied all the rab and rab genes present in the genomes of the amphioxus b. lanceolatum and the zebrafish d. rerio to shed light on the evolution of this subfamily of deuterostomes and gain insight into their role during chordate development.  evolutionary history of rab and rab in deuterostomes to study the evolutionary history of the rab and rab we performed a phylogenetic reconstruction starting from a database of manually curated supergroup rab iii sequences. we included sequences from vertebrates, urochordates, cephalochordates, hemichordates and echinoderms. since rabs are very similar to other small gtpase proteins, we only included sequences giving the higher blast scores with known rabs. to obtain the best phylogenetic resolution we included representative members of rab supergroup iii: rab rab rab rab7l rab rab. regarding rab and rab our analysis allowed to identify two genes in amphioxus and five in zebrafish genomes. phylogenetic analysis revealed four strongly supported clades: rab rab rab and a clade formed by rab7l rab32lo, rab and rab among these, rab rab and rab represent phylogenetically robust monophyletic groups, while the rab7l results as sister group of rab32lo and rab32/rab.fig. evolution of the rab and rab subfamily in deuterostomes. numbers at the branches indicate replicates obtained using the maximum likelihood estimation method. sites were used for tree inference. colored boxes highlight three classes of proteins: rab7l present in invertebrates and vertebrates, rab32lo present in protostomes, echinoderms and cephalochordates, the rab and rab cluster present in all the deuterostomes. black arrow indicates the acquisition of falk amino acid stretch at the stem of rab7l rab32lo, rab32 and rab evolution. triangles indicate monophyletic clades belonging to rab supergroup iii rab32los, previously named rab32b, were described as already present in choanoflagellates, sponges and cnidarians, and therefore considered as leca gene. moreover, we found rab32lo in protostomes and in deuterostomes. strikingly, our study highlighted its loss in olfactores prompting us to name it rab32lo. unfortunately, our phylogenetic survey did not clarify the relationships among the vertebrate rab and rab and invertebrate rab32/ genes due to the high degree of sequence similarity. the phylogenetic tree shows that amphioxus possesses a rab32lo and a unique rab32/ member. zebrafish presents three rab, and two rab genes. strikingly, the position of rab32b in the tree is inconsistent with species phylogeny, probably due to its fast evolutionary rate. a vertebrate-specific phylogenetic analysis was performed to elucidate the relationship existing between rab and rab proteins in this group. with the aim to distinguish between rab and rab proteins, we compared the three conserved rab domains across several deuterostome species, using the human rab6a as reference: the p-loop located at amino acids 20– the switch i at amino acid positions 38– and the switch ii at amino acids 69–. to better understand the conservation of protein domains, we also added all human proteins of rab supergroup iii and one representative of other five rab supergroups. the alignment shows a high degree of conservation for three known domains in deuterostomes. the general core sequence of the p-loop is gxxxxgkt in all subfamilies. we found that the 2nd residue is diagnostic for rab classification in vertebrates. switch i and switch ii are highly conserved across deuterostomes, showing sequence divergence only in zebrafish rab32b. moreover, we identified for the first time the existence of an ultra-conserved stretch of four amino acids located at the end of switch i that is present only in rab rab rab32lo and rab7l subfamily members among rab supergroup iii. the presence of two conserved histidine residues exclusively in rab32lo switch ii confirms the fact that rab32lo is a distinct class within supergroup iii, supporting our phylogenetic data. in order to clarify the poorly resolved relationship among invertebrate rab32/ and vertebrate rab and rab we searched for an evolutionary signature of conserved introns, analysing the gene structure of human rab and rab in comparison with branchiostoma, ciona and lottia rab32/ genes. strikingly, we found two introns that show conserved position and phase, suggesting a common ancestral origin.fig. alignment of rab supergroup iii domains. the alignment shows the amino acid conservation of three rab domains: the p-loop, involved in trafficking, the switch i and the switch ii important for supporting the binding with other molecules. downstream of switch i, four ultra-conserved amino acids are highlighted, named falk. the white background indicates changes in amino acid composition during evolution. at the bottom, human representatives of other rab supergroups are reported to show the absence of falk rab and rab synteny conservation to further improve knowledge on the evolutionary history of the rab and rab subfamilies, we analysed their chromosomal neighbourhood genes in available deuterostome genomes. we searched in genomes of invertebrates and, among vertebrates, we selected the lamprey p. marinus for the agnathans, the elephant shark c. milii and spotted gar l. oculatus as representatives of non-teleost fish, d. rerio for teleosts, the lizard a. carolinensis in the sauropsida clade, and h. sapiens and m. musculus among mammals. we did not find chromosomal conservation in lamprey. on the other hand, we discovered a high degree of synteny conservation within both gnathostome rab32s loci and rab38s loci. nevertheless, the genes flanking rab are different from those surrounding rab to demonstrate the orthology of syntenic genes we performed ad hoc phylogenies of tab, nox, fzd and stxbp genes.fig. rab and rab synteny conservation in gnathostomes. the rab and rab loci harbour several genes conserved across evolution; in blue boxes rab genes we studied, in red boxes rab genes that are physically linked to tyrosinase. during the gnathostome evolution, the rab is always linked to grm while rab is linked to grm the scheme 3b ’ shows the functional relationship between rab and tyr during mammalian melanosome biogenesis. the position of the genes above or below the chromosome indicates their transcriptional orientation on positive or negative strand, respectively the existence of a chromosomal region conserved between h. sapiens and some teleosts, containing rab grm and tyr genes has been already described in a comparative study. our syntenic survey across the vertebrates extended the conservation to the holocephalan elephant shark, the non-teleost actinopterygian spotted gar, the anole lizard and mice. moreover, we also showed that rab genes are always linked to a grm family member. collectively, our data suggest an ancestral chromosomal linkage between a rab32/ and grm1/ gene, already present in the vertebrate’s common ancestor. we carried out the phylogeny of the grm family to demonstrate the orthology among vertebrate grm genes and grm genes, respectively, further supporting our hypothesis of an ancient chromosomal linkage. in more detail, we found one rab always coupled with the melanoma oncogene grm, while one rab is always close to grm a gene playing a fundamental role in many human disorders such as schizophrenia and autism. among zebrafish rab paralogues we found a higher degree of syntenic conservation in the rab38a locus than in the rab38b and rab38c loci. for instance, we found that only the zebrafish rab38a maintained the linkage with the tyr gene, as is the case in the unique tetrapod rab paralogue. similarly, in elephant shark and spotted gar this linkage is retained in only one of the two rab paralogons. additionally, in tetrapod fohl1a and fohl1b, and the coiled-coil domain containing proteins, belonging to a big family whose role is completely unknown, are duplicated on the same chromosomal stretch. the mammalian rab loci show an interesting peculiarity, they present several insertions of receptors belonging to the same family derived from tandem gene duplication events. on mouse chromosome beside the two fohl1s, there are fifteen vomeronasal receptors, known for their crucial role in mouse ultrasensitive chemodetection and fourteen olfactory receptors, belonging to a very large family in mice, explaining their extraordinary odour sensitivity and ability to discriminate scents. interestingly, in the orthologous position, the human chromosome harbours seven tripartite motif proteins, which are involved in a plethora of biological processes, in particular the immune response. on the other hand, we did not find any synteny conservation in rab loci of invertebrate deuterostomes. the unique trace of microsynteny was found in the sea urchin and amphioxus gene scaffolds, between rab32lo and tim an evolutionarily conserved transporter involved in metabolite import by mammalian mitochondria. rab and rab gene expression patterns in amphioxus and zebrafish to investigate the possible role of rab genes in the amphioxus b. lanceolatum, we cloned the rab32lo and rab32/ genes and studied their expression pattern by whole mount in situ hybridization in embryos at different developmental stages. unfortunately, rab32lo was not detectable by in situ hybridization at any analysed stage, confirmed by the low levels of expression detected by real-time rt-pcr experiments. rab32/ was first observed at the gastrula stage in presumptive notochord territories, and later during development, at the neurula stage, it appears clearly in the notochord, from the most rostral part along the length of the embryo without reaching the caudal part. the rab32/ expression in the notochord has been confirmed by the transverse section. at the pre-mouth larval stage of development, the rab32/ expression in the notochord turns off and a novel territory of expression is visible in the pharynx region.fig. rab32/ expression pattern during amphioxus embryogenesis. rab32/ has been observed in notochord presumptive territories at the gastrula stage, while later in development at the neurula stage it is expressed in notochord, mainly in the rostral part. d is a vibratome section of the neurula specimen showed in b-c, at the level of the vertical dashed line. at the pre-mouth larval stage, rab32/ expression in the notochord turns off while a positive signal appears in the pharynx region the same approach was used to investigate the expression pattern of rab and rab during zebrafish embryogenesis. it was impossible to amplify rab32b from embryonic and adult tissues, suggesting that this gene is either expressed at very low levels or only under specific environmental conditions. rab32a expression has been detected in the presumptive posterior axial mesoderm starting from 6 h post-fertlization, as it was already described by thisse and collaborators. during embryo elongation, the rab32a positive cells were localized near the animal pole and form a longitudinal band in the dorsal midline, the developing notochord that includes the tail bud region. moreover, at this stage, a signal is detectable in the kupffer’s vesicle, a small but distinctive epithelial sac, situated posteriorly near the yolk. during segmentation, the expression of rab32a becomes weaker in the notochord, while it starts to be expressed in the eyes, as well as in cells that appear to be migrating melanoblasts according to their position. starting from the long-pec stage, the expression in migrating melanoblasts disappears and is retained only in the retinal pigmented epithelium of the eye and in the notochord. at this stage, a strong signal becomes visible in the swim bladder, the organ that adult teleosts use for buoyancy and breathing, and persists up to the protruding-mouth larval stage.fig. rab32a expression pattern during zebrafish embryogenesis. rab32a has been observed in the presumptive posterior axial mesoderm at hpf and in the developing notochord and kupffer’s vesicle at hpf. at hpf, it is present in rpe, notochord and migrating neural crest cells. at hpf the signal disappears in neural crest cells, but persists in rpe and notochord and appears in the swim bladder. the expression in the swim bladder persists at hpf larvae regarding rab paralogues, rab38a is expressed across the pharyngula embryonic period. embryos at hpf show a faint signal in rpe and a strong one in migrating neural crest cells, presumably migrating melanoblasts. at this stage, a low level of expression is detectable in the mid-ventral region of brain as well. later in development expression is only observed in the rpe. rab38b is expressed only at late developmental stages; at hpf it is expressed in a small region of the pharyngeal arch and in the developing swim bladder, while at hpf only the signal in the swim bladder persists. the gene rab38c is expressed from to hpf with a strong signal in the head.fig. rab38s expression pattern during zebrafish embryogenesis. rab38a is expressed across the pharyngula embryonic period: at hpf there is a faint signal in rpe and a strong one in migrating melanoblasts and a low level of expression is detectable in the mid-ventral region of brain; at hpf the expression is visible only in the rpe. rab38b is expressed only at late developmental stages: at hpf in a small region of the pharyngeal arches and in the developing swim bladder, while at hpf only in swim bladder. the gene rab38c gene is strongly expressed in the head region. lateral view in all images except d and f that are ventral discussion rab and rab toolkit in animal evolution our phylogeny evidences three distinct branches: rab7l rab32lo and rab32/rab which share the highly conserved falk stretch downstream of the switch i domain and emerged after splitting from rab rab and rab groups. although the falk function is still unknown, its high degree of sequence conservation in all analysed proteins suggests that it could be responsible for important functional properties. it has been suggested that the rab32/ already described as rab32a and rab32lo, previously named rab32b, present in the last eukaryotic common ancestor were already implicated in late endosomal or lysosomal trafficking. rab32lo is present in unicellular eukaryotes to chordates, and here we showed its loss in the lineage of olfactores. this event could be related to the loss of ancestral functions during chordate evolution, although the biological functions of these proteins should be further investigated in the future. detailed analysis of the conserved rab functional domains allowed to identify few diagnostic residues of vertebrate rab and rab. similarly, two histidines are distinctive residues of the rab32lo switch ii region. our analyses suggest that the phylogenetic signal is weak for the heterogeneous evolutionary rate of this group of proteins, leaving the issue of vertebrate rab and rab origin unresolved. a possible hypothesis is that vertebrate rab and rab arose from a prevertebrate rab32/ through a vertebrate genome duplication. the study of intron conservation, considered a diagnostic tool for tracing back the evolutionary history of genes, showed the existence of a conserved intron code supporting a common origin for all the subfamily members. furthermore, the parallel evolutionary history of rab32/ and grm1/ genes is consistent with our hypothesis of a duplication event in vertebrates. in fact, in invertebrates, similarly to the rab32/ a single pre-duplicative grm1/ exists as demonstrated by the dedicated phylogeny. interestingly, the chromosomal proximity of rab to grm and rab to grm indicates a duplication of an ancestral chromosomal region comprising rab32/ and grm1/. the retained microsynteny that we found from elephant shark to humans hints at a functional correlation between these two gene duplets. it would be challenging to find out whether the integrity of these linkages is necessary for their functioning. collectively our phylogeny, intron and synteny conservation analyses suggest that rab and rab derived from a vertebrate genome duplication, therefore they are both ohnologs of the pre-vertebrate rab32/ gene. we found a very high degree of chromosomal conservation among vertebrates. in particular the high similarity between c. milii rab38a and tetrapod rab loci confirms that cartilaginous fishes mimic the tetrapod genomic organization better than teleosts. surprisingly, a sharp lack of synteny involving only one side of the rab38a of spotted gar and zebrafish loci suggests a rearrangement event in the actinopterygii. vertebrates have been affected by several rounds of genome duplication that have increased the repertoire of pigmentation genes mainly in teleosts. the present survey highlights the expansion of the rab and rab driven by a mixture of gene and genome duplications that allowed the emergence of novel functions. in zebrafish the rab and rab reached a total of five members through the tsgd, plus single gene duplication in the rab lineage. the role of tsgd in the evolution of teleost pigmentation diversity is well-known resulting in 30 % more pigmentation genes than in tetrapods. in fact, other genes involved in pigmentation are also duplicated such as mitf, kitl, tyr and tyrp explaining the extremely diversified body colour patterning of teleosts. intriguingly, we noticed in gnathostomes the existence of a specific linkage among two well-known players of the pigmentation process; the tyr and rab retained in all analysed tetrapods and in the elephant shark, spotted gar and zebrafish rab38a paralogons. it has been demonstrated that the delivery of tyr, tyrp and tyrp to the maturing melanosomes is required to initiate pigmentation. rab and rab have been shown to mediate the transport of tyr and tyrp by interacting with the ubiquitous trafficking machinery. taking into consideration that rab38a is the sole rab member to be expressed in zebrafish pigment-producing cells and its chromosomal vicinity to tyr, we hypothesize a bystander gene regulation modality during vertebrate melanosomes biogenesis by means of a locus control region, as previously demonstrated for developmental genes. expression profile in zebrafish and amphioxus in mammals, melanocytes, platelets and mast cells, rich in lros, exhibit a high level of rab, whereas rab is restricted to melanocytes and lung epithelial cells. in zebrafish, we have identified two rab and three rab genes. rab32a is expressed in the embryonic pigmentary lineage, including both melanocytes and rpe, as described in other vertebrates. moreover, it is expressed during notochord development, a feature that seems to be specific of zebrafish. recently it has been proposed that rab32a function is required for correct formation of the notochord vacuoles in zebrafish. finally, rab32a is expressed in the natatory vesicle of zebrafish, also known as the swim bladder, an organ whose relationship with the tetrapod lung is still debated. considering that invertebrate rab32/ has an important role in melanogenesis, as demonstrated by a functional study in c. intestinalis, and that this role is maintained in vertebrate rab we can assume rab function in melanogenesis as ancestral. in zebrafish this gene has acquired new roles in the formation of other lysosome related organelles. zebrafish shows a varied scenario of rab32s and rab38s expressions with a high spatio-temporal diversification due to genomic events that occurred during the evolution of vertebrates. the three zebrafish rab38s are expressed in distinct embryonic territories, including pigmented cells, swim bladder and nervous system, unravelling a functional paralogous diversification. distinct types of duplications have permitted the functional specialization in pigmentation of one of the paralogues. the tsgd provided further raw genetic material for the evolution of teleost pigmentation patterns. the expression data of the three zebrafish rab38s are an example of ancestral function distribution among duplicates. finally, the amphioxus rab32/ expression territories in the notochord and pharynx are consistent with transient zebrafish rab32a expression in the notochord and rab38b in pharyngeal arches. this findings suggest possible ancestral roles of the prechordate rab32/ which seem to be lost in tetrapods.  our study focused on genes belonging to the rab family known to be involved in melanosome formation in mammals: rab and rab phylogenetic, intron conservation and synteny analyses of these genes in deuterostomes point towards an evolutionary scenario based on a duplication of a single invertebrate rab32/ gene giving rise to vertebrate ohnologs rab and rab additional duplicates arose in bony fishes by teleost-specific genome duplication and expression pattern of rab paralogues in zebrafish evokes sub-functionalization event. finally, the discovery of a chromosomal linkage between the rab or rab gene with a grm or grm opens new perspectives on possible conserved bystander gene regulation across the vertebrate evolution. however, future studies are necessary to investigate the presence of shared enhancer and to test functionally its regulative properties. 
among all membrane-enclosed organelles, melanosomes are the cellular site for synthesis, storage and transport of melanin granules, making them an excellent model for studies on organelle biogenesis and motility. we studied the molecular evolution of rab supergroup iii in deuterostomes by phylogenetic reconstruction, intron and synteny conservation. finally, the discovery of a chromosomal linkage between the rab or rab gene with a grm opens new perspectives on possible conserved bystander gene regulation across the vertebrate evolution. the regulation of cellular membrane trafficking in all eukaryotes is a very complex mechanism, mostly regulated by the rab family proteins. we analysed the rab and rab genes in the teleost zebrafish and in the cephalochordate amphioxus, gaining insight on their evolutionary history following gene and genome duplications. specific rab proteins, as rab and rab have been shown to play a key role in melanosome biogenesis. we discovered a novel amino acid stretch, named falk, shared by three related classes belonging to rab supergroup iii: rab7l rab32lo and rab32/rab among these, we demonstrated that the rab32lo class, already present in the last common eukaryotic ancestor, was lost in urochordates and vertebrates. phylogenetic, intron conservation and synteny analyses point towards an evolutionary scenario based on a duplication of a single invertebrate rab32/ gene giving rise to vertebrate rab and rab the expression patterns of rab paralogues highlight sub-functionalization event. pigmentationamphioxuszebrafishsyntenyphylogenyintron conservationgene duplication, genome duplicationmarie curie career integration grant pcig09-ga-2011-293871pcig09-ga-2011-293871d’aniello salvatore issue-copyright-statement© the author 2016 moreover, the chromosomal linkage of rab or rab with a member of the glutamate receptor metabotropic family has been retained in all analysed gnathostomes, suggesting a conserved microsynteny in the vertebrate ancestor.
 the regulation of cellular membrane trafficking in all eukaryotes is a very complex mechanism, mostly regulated by the rab family proteins. among all membrane-enclosed organelles, melanosomes are the cellular site for synthesis, storage and transport of melanin granules, making them an excellent model for studies on organelle biogenesis and motility. specific rab proteins, as rab and rab have been shown to play a key role in melanosome biogenesis. we analysed the rab and rab genes in the teleost zebrafish and in the cephalochordate amphioxus, gaining insight on their evolutionary history following gene and genome duplications.  we studied the molecular evolution of rab supergroup iii in deuterostomes by phylogenetic reconstruction, intron and synteny conservation. we discovered a novel amino acid stretch, named falk, shared by three related classes belonging to rab supergroup iii: rab7l rab32lo and rab32/rab among these, we demonstrated that the rab32lo class, already present in the last common eukaryotic ancestor, was lost in urochordates and vertebrates. synteny shows that one zebrafish gene, rab38a, which is expressed in pigmented cells, retained the linkage with tyrosinase, a protein essential for pigmentation. moreover, the chromosomal linkage of rab or rab with a member of the glutamate receptor metabotropic family has been retained in all analysed gnathostomes, suggesting a conserved microsynteny in the vertebrate ancestor. expression patterns of rab and rab genes in zebrafish, and rab32/ in amphioxus, indicate their involvement in development of pigmented cells and notochord.  phylogenetic, intron conservation and synteny analyses point towards an evolutionary scenario based on a duplication of a single invertebrate rab32/ gene giving rise to vertebrate rab and rab the expression patterns of rab paralogues highlight sub-functionalization event. finally, the discovery of a chromosomal linkage between the rab or rab gene with a grm opens new perspectives on possible conserved bystander gene regulation across the vertebrate evolution. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords pigmentationamphioxuszebrafishsyntenyphylogenyintron conservationgene duplication, genome duplicationmarie curie career integration grant pcig09-ga-2011-293871pcig09-ga-2011-293871d’aniello salvatore issue-copyright-statement© the author 2016 