Luigi Caputi Science Reviews - Biology, 2024, 3(2), 22-32

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Evolutionary Genomics of Tunicates

Luigi Caputi, PhD

Indipendent Researchers, Via Arrigo Davila 37, 00179, Rome, Italy

https://orcid.org/0000-0002-5724-1943

https://doi.org/10.57098/SciRevs.Biology.3.2.3

Received June 21, 2024. Revised July 01, 2024. Accepted July 03, 2024.

Abstract: Tunicates are the closest living relatives of vertebrates and are a group comprised exclusively of

marine animals. In the current review, I aim to present some of the most interesting aspects of tunicate genomics

within an evolutionary context to a non-specialized scientific audience. Tunicates are of scientific interest for

many reasons. First, their phylogenetic position, as well as their internal evolutionary relationships, are heavily

debated. Second, multiple species have been studied as developmental biology and evolutionary models. Third,

some tunicate species play crucial ecological roles and functions. Lastly, tunicates have become an interesting

field of study in evolutionary genomics since the beginning of the new millennium. Tunicate genomes are

atypical within chordates, bearing many hints of divergence and differentiation. In addition, their genomes are

fast-evolving and highly plastic, with functional genetic units uncharacteristically often organized in operons.

It is likely that continued research efforts surrounding tunicates will continue to challenge current

understanding of the mechanisms driving molecular adaption as well as evolutionary genomic processes.

Keywords: Tunicates, Chordates, Genomics, Operons, Genome Plasticity.

Tunicates and the Evolution of Chordates.

The phylum Chordata includes three sub-

phyla, namely Craniata (or Vertebrata, which in-

cludes humans), and two non-vertebrate subphyla,

Cephalochordates and Urochordates (or tunicates)

(Maisey 1986; Cameron et al., 2000; Delsuc et al.,

2006; Delsuc et al., 2008; Stach, 2008; Zhong et al.,

2009). The primary feature shared by all chordates

is the presence of a notochord within a tadpole-like

body plan developed at the end of embryogenesis,

hence the source of the phylum’s name (Lemaire,

2011). Notwithstanding the long-known close evo-

lutionary relationships between chordates (Turbe-

ville et al., 1994; Cameron et al., 2000), as well as

their monophyly (Turbeville et al., 1994; Cameron

et al., 2000; Bourlat et al., 2006; Stach, 2008; Satoh et

al., 2014), the exact evolutionary relationships be-

tween the three subphyla have been long-debated,

and a consensus regarding this issue has yet to be

reached.

There are three main hypotheses that attempt

to explain the evolutionary relationships of chor-

dates (Figure 1).

Science Reviews - Biology, 2024, 3(2), 22-32 Luigi Caputi

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Figure 1: The three hypotheses on chordates evolutionary relationships: (a) the ‘Ofactores hypothesis’, (b) the Atriozoa hypothe-

sis’, and (c) Notochordata hypothesis’. Modified from Stach, 2008.

Notably in each, the relative location of tuni-

cates within the Chordata evolutionary tree varies.

In the first hypothesis, named the ‘Atriozoa hypothe-

sis’, tunicates are considered a sister taxon to the

Cephalochordata. In the second hypothesis, called

the ‘Olfactores hypothesis’, Tunicata is placed as a sis-

ter taxon to Craniata. Finally, the ‘Notochordata hy-

pothesis’ postulates that Craniata and Cephalochor-

data are sister taxa (Stach, 2008). The Notochordata

hypothesis has been widely accepted by the scien-

tific community for more than a century, while the

Atriozoa hypothesis is of mere historical interest,

having been rejected decades ago (Stach, 2008). The

Olfactores hypothesis is also an older hypothesis,

and was principally invoked only due to the contro-

versial interpretation of unusual fossil records (Jef-

feries, 1986).

Despite its surrounding controversy, the Ol-

factores hypothesis has become the main competi-

tor of the Notochordata hypothesis since the begin-

ning of the new millennium, mainly due to novel

genomics insights via technical advances in genome

sequencing. The most obvious reason for support-

ing the Notochordata hypothesis was the high re-

semblance of a Craniata body plan. However, large

datasets reflective of genomics and phylogenomic

analyses have since challenged this notion and

spurred the recent turn towards the long-neglected

Olfactores hypothesis (Delsuc et al., 2006; Delsuc et

al., 2008; Dunn et al., 2008; Swalla & Smith, 2008).

The Olfactores hypothesis has now become the

most widely accepted hypothesis.

Deuterostomia is a major evolutionary group

which includes three clades (Echinodermata, Hem-

ichordata, and Chordata). More recently (Satoh et

al., 2014), it has been suggested that a general revi-

sion of Deuterostomia is required to fully describe

the evolutionary relationships between higher ani-

mals. According to the proposed revision, Bilateria,

the Echinodermata and Hemichordata phyla will

form a new superphylum (Ambulacraria), while

Cephalacordata, Urochordata and Vertebrata (the

last two jointly forming the Ofactoria group) will be

recognized as proper phyla, with Chordata catego-

rized as the other superphylum within Deuterosto-

mia.

Phylogeny of tunicates.

Tunicates are clearly pivotal in higher animal

evolution despite the uncertainty surrounding their

phylogenetic position in the animal tree of life. The

understanding of phylogenetic affinities within tu-

nicates is also a matter of debate, but new molecular

data is working to provide further insights.

Tunicates are commonly divided into three

classes, namely Ascidiacea (or ascidians), Appen-

dicularia (or larvaceans) and Thaliacea (or thalia-

ceans) (Lemaire, 2011). Each of the three classes

bears distinctive life-history traits and developmen-

tal modes. The number of species included in all the

Luigi Caputi Science Reviews - Biology, 2024, 3(2), 22-32

24

three classes is highly uncertain, since molecular

techniques are revealing a higher-than-expected

number of cryptic, morphologically hard-to-distin-

guish, species (Lopez-Legentil and Turon, 2006; Ca-

puti et al., 2007; Pérez-Portela et al., 2013; Plessy et

al., 2024). The most biodiverse class are the ascidi-

ans, with more than 2,500 species known and com-

monly classified into three orders (Phlebobranchia,

Aplousobranchia and Stolidobranchia) based on

branchial sac structure and molecular data (Stach

and Turbeville, 2002; Lemaire, 2011). The most evi-

dent developmental and lifestyle characteristic of

the ascidians is that all species live as free-swim-

ming tadpole-like larvae that later undergo meta-

morphosis resulting in a sac-like sessile solitary or

colonial adult life form (Lemaire, 2011). From a his-

torical point of view, benthic species capable of col-

onizing ports and other areas in the proximity of

densely populated human settlements such as Ciona

intestinalis and C. robusta have become, thanks also

to the easiness of finding,, ideal model species for

evolutionary and developmental studies. Appen-

dicularia and Thaliacea (or thaliaceans) consist of

exclusively planktonic species. Like the ascidians,

thaliaceans also undergo metamorphosis to form an

adult organism (Lemaire, 2011). However, unlike

ascidians, the species belonging to this class retain

larval-like morphology for their entire lifespan

(Bone, 1998). Consequently, Appendicularia is of in-

terest from an evolutionary development point of

view (Delsuc et al., 2006).

The most recent studies on the evolutionary

relationships between tunicates have challenged

the canonical three-class partition. A molecular

phylogeny based on a large dataset found at least

four major clades (Tsagkogeorga et al., 2009; Kocot

et al., 2018; Delsuc et al., 2018). The first class was

determined to consist of only Appendicularia and

the Thaliacea and Ascidiacea classes were not con-

firmed. Rather, the second class included all Tha-

liacea, plus ascidians Phlebobranchia and Aplouso-

branchia, while Molgulide alone formed the third

class and Styelidae with Pyuridae the fourth (Figure

2).

Science Reviews - Biology, 2024, 3(2), 22-32 Luigi Caputi

25

Figure 2: Phylogenetic relationships between major clades of tunicates. Modified from Delsuc et al., 2018.

Genomic Features of tunicate genomes.

Tunicates show a remarkable number of

unique genomics features within chordates. From

the presence of operons to unusually fast evolution-

ary rates and highly packed genomes to a disrupted

Hox genes cluster, tunicates display divergent char-

acteristics not evident in other chordates.

Fast-evolving genomes.

Tunicate species are among the fastest evolv-

ing chordates in terms of molecular evolution. Spe-

cies belonging to the Ciona genus such as C. intesti-

nalis and C. robusta, show evolutionary rates ap-

proximately 50% faster than other chordates, while

O. dioica show evolutionary rates of up to three time

faster (Berna and Alvarez-Valin, 2014). Two pro-

cesses are thought to be involved. First, in some spe-

cies (for instance, O. dioica) the absence of many

genes related to DNA repair has been noted, albeit

not definitive (Berna and Alvarez-Valin, 2014). The

second suspected process involves high lineage-

specific selective pressure acting on tunicate ge-

nome coding regions (Berna and Alvarez-Valin,

2014). One study (Yokomori et al., 2014.) reported

that the C. intestinals proteome showed high intra-

specific diversity, efficient purifying selection, and

a substantial percentage of adaptive amino acid

substitutions, resulting in a two to six times higher

per-year mutation rate compared to Vertebrata and

Luigi Caputi Science Reviews - Biology, 2024, 3(2), 22-32

26

Cephalochordata. Research on the evolutionary dy-

namics of Wnt genes in chordates reinforced the no-

tion of tunicates diverging from cephalochordates

and vertebrates (Martí-Solans et al., 2021). The au-

thors interestingly opposed the “extraordinary ge-

nomic stasis in cephalochordates” to the “liberal

and dynamic evolutionary patterns of gene loss and

duplication in urochordate genome” (Martí-Solans

et al., 2021). Apart from the most well-known and

studied case, the Hox genes cluster (see below),

other examples of extraordinary gene gain, loss and

amplification are seen in the evolution of Metal-

lothioneins, a family of small proteins binding met-

als such as cadmium, zinc, copper, iron and mer-

cury (Calatayud et al., 2021), or by the evolution of

gene networks related to the development of olfac-

tory organs, eyes, hair cells and motoneurons

(Fritzsch & Glover, 2024).

Compact genomes.

Tunicate genomes are generally considered to

be compact, with patterns of extensive gene loss

(Berna and Alvarez-Valin, 2014). Rapid variations

in genome size occur mainly through a phenome-

non known as whole-genome duplications, or

bursts in the activity of transposable elements (TEs)

(Piegu et al., 2006). O. dioica, whose genome is par-

ticularly small for even a tunicate, notably lacks el-

ements found in the most ancient families of animal

retrotransposons (Denoeud et al., 2010). Recently, a

study examined the genome size of various Appen-

dicularea and found that genome size increased

with body length, and that, although no evidence

was found for whole-genome duplications, the

global amount of TEs strongly correlated with ge-

nome size (Neville et al., 2019). Non-autonomous

TEs, and particularly short interspersed nuclear el-

ements (SINEs), explained as much as 83% of inter-

specific genome size variation.

Genomic rearrangements.

Due to their fast evolutionary rates, tunicate

genomes have also undergone extensive genomic

rearrangements (Denoeud et al., 2010; Aase-Reme-

dios & Ferrier, 2021). A striking example of the level

and frequency of this is the comparison of the chro-

mosomal architecture of the two closely related,

conspecific, and morphological cryptic species C.

intestinalis and C. robusta. These two species live in

partial sympatry in the English Channel (Caputi et

al., 2007), where hybrids can be found. Chromoso-

mal alignments between the two Ciona revealed the

existence of numerous chromosomal inversions,

which likely contributed to genetic isolation and

speciation (Satou et al., 2021). Patterns of chromoso-

mal rearrangements are even more extreme in cryp-

tic species belonging to the genus Oikopleura (Plessy

et al., 2024).

Conserved non-coding elements in Tunicates.

Another interesting characteristic of the tuni-

cate genome is the so-called ‘Olfactores conserved

non-coding elements’ (Sanges et al., 2013; Ambrosino

et al., 2019). These elements, while significantly as-

sociated with transcription factors showing specific

functions fundamental to animal development,

such as multicellular organism development and

sequence-specific DNA binding, are highly syntenic

within vertebrates. However, synteny is not pre-

served between tunicates and vertebrates, again

showing that tunicate genomes are highly diver-

gent within chordates.

Disrupted Hox gene cluster and chromosomal rearrange-

ments in tunicates.

Hox genes are determinants of the body plan

along the antero-posterior axis (Lewis, 1978). In

many species along the Bilateria tree of life, they are

clustered such that the order of the genes along the

chromosome corresponds with the order of their ex-

pression along the body (collinearity - Lewis, 1978).

Collinearity is also referred to as ‘spatial collinear-

ity’, while ‘temporal collinearity’ refers to the tim-

ing of Hox gene expression and the subsequent for-

mation of structures in the developing body plan. In

Figure 3, Hox gene arrangements in the chromo-

somes of key species across the Deuterostomia tree

of life are shown.

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27

Figure 3: Hox gene arrangements in deuterostomes. Modified from Gaunt et al., 2018.

Of note among the tunicate species presented,

O. dioica represents a unicum within chordates, hav-

ing a disrupted cluster of Hox genes via the absence

of central Hox genes (Gaunt et al., 2018). Notwith-

standing the cluster disruption, Hox genes display

a mode of expression which is ‘spatially collinear’

(Seo et al., 2004). Duboule describes this as ‘trans-

collinearity’ (Duboule, 2007). In the C. intestinalis

genome, Hox genes are only partially dispersed

(Pascual-Anaya et al., 2013, Sasakura and Hozumi,

2018). Ciona shows residual spatial collinearity in

the developing larval nervous system and in the ju-

venile gut during metamorphosis (Ikuta et al., 2004,

Nakayama et al., 2016). Experimental knock-downs

of Ciona Hox genes have demonstrated that they

play minor roles in larval development but major

roles during metamorphosis (Ikuta et al., 2010, Sasa-

Luigi Caputi Science Reviews - Biology, 2024, 3(2), 22-32

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kura and Hozumi, 2018). Neither of the above uro-

chordate species displays obvious temporal colline-

arity in Hox gene expression (Seo et al., 2004).

The re-emergence of Operons in higher Metazoa.

Operons are defined as clusters of co-regu-

lated genes with related functions (Osbourn and

Field, 2019). Operons were classically considered as

a common feature of prokaryote genomes; however,

recent work has described functional gene cluster-

ing in many eukaryotes, from yeast to animals. At

the beginning of the 1990s, the first genomic struc-

tures similar to classical prokaryotic operons were

found in the genome of Caenorhabditis elegans, a

nematode worm (Zorio et al., 1994). Contrary to

prokaryotic operons, C. elegans operons produce

polycistronic mRNA which is then trans-spliced

into individually translated monocistronic mRNAs

(Osbourn and Field, 2019). Moreover, genes within

C. elegans operons are not typically related by se-

quence or function.

The origin of trans-splicing is debated and ap-

pears to be an unevenly distributed process across

the animal kingdom. Trans-spliced operons have

been found in insects such as the fruit fly (Drosophila

melanogaster), chaetognaths (Spadella cephaloptera)

and, among chordates, exclusively in tunicates. In C.

intestinalis, approximately 20% of its genes reside in

operons containing a high proportion of single-

exon genes (Berna and Alvarez-Valin, 2014). In the

extremely compact genome of the Appendicularia

O. dioica, about 27% of the entire genome is orga-

nized in operons, again primarily consisting of sin-

gle-exon genes (Ganot et al., 2004). Importantly, re-

cent studies on the O. dioica genome found that, in

this highly fragmented and scrambled genome (Seo

et al., 2001), operon structure is not preserved be-

tween cryptic, anatomically identical species. This

strongly suggests the absence of selective evolution-

ary pressure in maintaining their functionality and

very existence, further obscuring the evolution of

operons in tunicates. (Plessy et al., 2024). Ultimately,

there is a need for greater understanding of the evo-

lutionary forces that caused a reemergence of oper-

ons in tunicates.

Genomics and adaptation in tunicates.

Genomic diversity is a key component that

dictates the ability of tunicates to adapt to new en-

vironments, making some species invasive pests

(Micael et al., 2020; Santos et al., 2023). The high

evolutionary rates of tunicate genomes is reflected

by the high level of within-species diversity of ge-

nome proteomes, as well as the high percentage of

adaptive amino acid substitutions (Tsagkogeorga et

al., 2012). This has led to lineage-, genus- and spe-

cies-specific adaptive mechanisms that ensure the

success and persistence of tunicates in modern-day

oceans.

Pelagic tunicates, such as Oikopleuridae, are

known to quickly respond during phytoplankton

oceanic blooms, mainly due to their fast life cycle

(Sordino et al, 2020). A recent study on salps (sea

squirts, Thalia spp.) (Castellano et al., 2023) used ge-

nome comparative analyses to reveal an abundance

of repeats and G-quadruplex (G4) motifs, a feature

typical of tunicates capable of alternate sexual and

asexual reproduction. This may allow salps to be ca-

pable of asexual reproduction at birth, enabling

bloom formation in optimal conditions. The sessile,

invasive, and colonial species Botryllus schlosseri

shows clear genetic and epigenetic differentiation in

its global population (Gao et al., 2022) presumably

due to variations caused by their local environment.

Another salp species, Styela clava, has been found to

have an expanded genome (compared to Ciona

spp.), possibly due to an increased number of trans-

posons (Wei et al., 2020). Specifically in this species,

the heat-shock protein 70 family repertoire is ex-

panded, likely from horizontal gene transfer from

bacteria (Wei et al., 2020), possibly playing a role in

the high degree of adaptability of S. clava to new en-

vironments.

The Oikopleura genome: challenging basic

biological intuitions.

Appendicularian tunicates are one of the ma-

jor components of ocean zooplankton (Hopcroft

and Roff, 1998) and are among the fastest hetero-

trophic responders to phytoplanktonic ocean

blooms (Sordino et al., 2020). The best studied Ap-

pendiculararea genus is Oikopleura, characterized

by its very short life cycle of four days. As stated

above, many genomic features including trans-

poson diversity, developmental gene repertoire,

physical gene order, and intron-exon organization

are shattered in this genus and among cryptic, mor-

phologically similar species. Chromosome arms

and sex-specific regions appear to be the primary

Science Reviews - Biology, 2024, 3(2), 22-32 Luigi Caputi

29

unit of macrosynteny conservation. Regarding mi-

crosynteny, scrambling did not preserve operon

structures; however, this happens without any ap-

parent loss of functionality, suggesting the absence

of selective pressure to maintain operon structure

even between closely related species (Denoeud et al.,

2010). The Oikopleura genome seems to challenge

some basic biological assumptions. Indeed, the fact

that similar, almost indistinguishable morphologies

may be based on largely divergent genomes (and

vice versa) is not an intuitive notion. As a corollary,

this also means that genetic distances cannot be

used to predict morphological similarities between

species, and vice-versa (Plessy et al., 2024). Another

interesting consequence of the unique features of

the Oikopleura genome is that the basic chordate

body plan, morphology, and its development is es-

sentially preserved, strongly suggesting that global

similarities of genome architecture in Metazoa are

not crucial for the preservation of ancestral mor-

phologies (Denoeud et al., 2010).

Conclusion.

Tunicates are fascinating animals. The aim of

the present review was to present several of their

unique and interesting characteristics to an unfa-

miliar audience. First, tunicates are pivotal in un-

derstanding higher animal evolution and the tran-

sition from invertebrates to vertebrates. This is not

a simple task, partially because they are highly di-

vergent and derivative at the molecular and ge-

nomic level, notwithstanding the fact that they are

these closest living relative of vertebrates. Second,

tunicates exhibit astonishing features. The possess

an operon-like organization of many genes, despite

operons not being positively selected and being a

trait of genome organization that disappeared early

on in evolutionary history. Tunicates also have a

partial or total disruption of the Hox gene cluster

that does not seem to affect their development. Tu-

nicate genomes may showcase genome re-shuffling

between extremely closely related species, but ob-

served large differences in genomic architecture

does not appear alter their morphology. For the

above reasons, research on tunicates has challenged

our knowledge of organismal evolution and ge-

nome function and future studies are likely to con-

tinue to unveil or run contrary to current under-

standing of molecular evolution.

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