Comparative localization of serotonin-like
immunoreactive cells in Thaliacea informs tunicate
phylogeny

Alberto Valero-Gracia, Rita Marino, Fabio Crocetta, Valeria Nittoli, Stefano

Tiozzo, Paolo Sordino

► To cite this version:

Alberto Valero-Gracia, Rita Marino, Fabio Crocetta, Valeria Nittoli, Stefano Tiozzo, et al. Comparative localization of serotonin-like immunoreactive cells in Thaliacea informs tunicate phylogeny. Frontiers in Zoology, 2016, 13, pp.45. 10.1186/s12983-016-0177-6. hal-01388004

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Comparative localization of serotonin-like
immunoreactive cells in Thaliacea informs
tunicate phylogeny

Valero-Gracia *et al.*
RESEARCH

Open Access

<figure><img src="image_3.png" /></figure>

CrossMark

# Comparative localization of serotonin-like immunoreactive cells in Thaliacea informs tunicate phylogeny

Alberto Valero-Gracia<fnref n="1" />†, Rita Marino<fnref n="1" />†, Fabio Crocetta<fnref n="1" />,², Valeria Nittoli<fnref n="1" />, Stefano Tiozzo³ and Paolo Sordino<fnref n="1" />*

## Abstract

**Background:** Thaliaceans is one of the understudied classes of the phylum Tunicata. In particular, their phylogenetic relationships remain an issue of debate. The overall pattern of serotonin (5-HT) distribution is an excellent biochemical trait to interpret internal relationships at order level. In the experiments reported here we compared serotonin-like immunoreactivity at different life cycle stages of two salpid, one doliolid, and one pyrosomatid species. This multi-species comparison provides new neuroanatomical data for better resolving the phylogeny of the class Thaliacea.

**Results:** Adults of all four examined thaliacean species exhibited serotonin-like immunoreactivity in neuronal and non-neuronal cell types, whose anatomical position with respect to the nervous system is consistently identifiable due to α-tubulin immunoreactivity. The results indicate an extensive pattern that is consistent with the presence of serotonin in cell bodies of variable morphology and position, with some variation within and among orders. Serotonin-like immunoreactivity was not found in immature forms such as blastozooids (Salpida), tadpole larvae (Doliolida) and young zooids (Pyrosomatida).

**Conclusions:** Comparative anatomy of serotonin-like immunoreactivity in all three thaliacean clades has not been reported previously. These results are discussed with regard to studies of serotonin-like immunoreactivity in adult ascidians. Lack of serotonin-like immunoreactivity in the endostyle of Salpida and Doliolida compared to *Pyrosomella verticillata* might be the result of secondary loss of serotonin control over ciliary beating and mucus secretion. These data, when combined with other plesiomorphic characters, support the hypothesis that Pyrosomatida is basal to these clades within Phlebobranchiata and that Salpida and Doliolida constitute sister-groups.

**Keywords:** Comparative neuroanatomy, Evolution, Immunohistochemistry, Thaliaceans, Tunicata, Zooplankton

## Background

Thaliacea is a class of pelagic tunicates that undergo alternation of generations between the sexual blastozooid stage and the asexual oozooid stage (reviewed in [1]). This clade comprises three orders: Pyrosomatida, Salpida, and Doliolida [2]. Despite a rich literature describing the anatomical characters of thaliaceans, the phylogenetic position within orders is still disputed. Most authors proposed a nested position of Thaliacea within the class 'Ascidiacea', thus recognized

as a paraphyletic group formed by the Stolidobranchiata, Aplousobranchiata and Phlebobranchiata clades [3–6]. This view suggests that the thaliaceans, with their planktonic life style, diverged from a benthic ancestor. However, there is no consensus on the relationships among Thaliacean orders. Some authors proposed that Pyrosomatida and Salpida group independently from Doliolida [7–9], while more recent works suggested that Pyrosomatida branched off first, and that Salpida and Doliolida are sister groups [10–12]. To date, molecular phylogenetic analyses based on ribosomal markers have been hindered by long-branch attraction [6]. Analyses based on morphological characters have not overcome such error, mainly because of the lack of a more

<footnote>* Correspondence: paolo.sordino@szn.it</footnote>

<footnote>†Equal contributors</footnote>

<footnote n="1">¹Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohm, Villa Comunale, 80121 Naples, Italy</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
comprehensive taxon sampling, particularly covering all three thaliacean orders [11, 12].

When molecular and morphological phylogenies con-
flict, neuroarchitectural traits offer a wealth of hitherto
largely-unexploited characters which can make valuable
contributions to phylogenetic inference even among dis-
tantly related groups (e.g., tardigrades, onychophorans,
kinorhynchs and priapulids) [13–17]. However, when
adopting neural characters, extensive sampling of crown-
group representatives is required to assess the origin of
evolutionary traits. In thaliaceans, comparative anatomy is
particularly problematic due to the complexity of their life
cycles and the difficulty of comparing homologous struc-
tures. As a consequence, it is essential to sample taxa
across all orders.

To better understand thaliacean phylogenetic relation-
ships, we analysed the distribution of serotonin-like
immunoreactivity in specimens from the three orders
and at different stages of their life cycle. Monoamine
serotonin is an ancient and conserved neurotransmitter
found throughout Opisthokonta [18]. Serotonin can trig-
ger several physiological functions that range from regu-
lation of ciliary band activity [19], to feeding circadian
patterns [20], and influencing emotional state [21]. In
addition to neurotransmitter functions, serotonin has
also non-neurogenic roles. For instance, it affects cardiac
morphogenesis and neural crest cell migration during
early mammalian and chicken embryonic development
[22–24], modulates gastrulation in echinoderms and in-
sects [25–27], and plays a role in the determination of
left-right asymmetry in amphibians and birds [28, 29].
Cellular distribution of serotonin is a reliable biochem-
ical trait to infer phylogenetic hypotheses due to the
ancestral nature of this amine, its diffuse role in nervous
transmission, and its metabolic and developmental
functions [13, 17, 30, 31]. Moreover, the precise classifi-
cation and description of serotonin-like immunoreactive
cells is needed to improve taxonomic comparability [31].
Serotonin-like immunoreactivity in thaliaceans has been
described in oozooids of *Doliolum nationalis* (Borgert,
1893) (Doliolida) and *Thalia democratica* (Forsskål in
Niebuhr, 1775) (Salpida) [32–34]. Immunoreactivity to
serotonin was observed in both species in different or-
gans such as cerebral ganglion, intestine, pericoralon
bands, and in a structure termed the ‘placenta’, a single
layer of flattened follicle cells that covers the embryo
during development [9, 35]. Recently, Braun and Stach
classified serotonin-like immunoreactive cells of Ascidia-
cea, Appendicularia and Thaliacea in three types: one
neuronal and two non-neuronal, spherical and elongated
respectively. Each of these cell types has a conserved tis-
sue type-specific distribution [34]. However, cell lineage
studies are needed to elucidate the origin of serotonin-
like immunoreactive cells.

To understand the evolution of the serotonergic sys-
tem in Thaliacea, three additional species were examined
at different successive life cycle stages, including a mem-
ber of the order Pyrosomatida. Immunohistochemistry
against acetylated and tyrosinated α-tubulins was com-
bined with nuclear staining in order to provide overall
anatomical landmarks of the nervous system and an
antibody against 5HT serotonin was used to describe the
distribution of serotonin-like immunoreactive cells. Our
study provides a more complete description of thalia-
cean serotonergic nervous system, with the aim of better
understanding the course of neurotransmitter system
evolution in this group of invertebrate chordates.

Results

Organization of the serotonergic nervous system in the pyrosomatid *Pyrosomella verticillata* (Péron, 1804)

Pyrosomes form tubular colonies consisting of barrel-
shaped individual animals (oozooid) that bud off near
the posterior closed end of the colony [36]. The nervous
system of the pyrosomatid oozooid is an ovoid mass
which comprises two regions with contrasting develop-
ment and function, the neural gland connected to the
ciliated funnel, and a voluminous cerebral ganglion [37].
Mature zooids of the tetrazooid colony showed serotonin-
like immunoreactivity in neuronal cells of the cerebral
ganglion and in the visceral nerve (medial posterior nerve,
mpn) running antero-posteriorly and encircling the
cerebral ganglion (Fig. 1a–e). The peribranchial tube ex-
hibits two lateral tufts of α-tubulin-positive cilia crossed
by the serotonin-like immunoreactive mpn fibres (Fig. 1f).
Serotonin-like immunoreactivity was also detected in
spherical cell bodies on the pericoralon bands around the
oral siphon (Fig. 1c), in two bilaterally symmetrical
antero-posterior rows within the endostyle (Fig. 1g), and
in a single row in a structure identified as the pyloric
gland (Fig. 1h). Early forming and young primary blasto-
zoooids growing in the *P. verticillata* tetrazooid colony
exhibited axons labelled with the anti-α-tubulin antibody,
but no serotonin-like immunosignals were observed (data
not shown).

Organization of serotonergic nervous system in the
salpids *Thalia democratica* and *Ihlea punctata* (Forsskål in
Niebuhr, 1775)

*Thalia democratica*

A thorough description of the structure of *T. democrata*
cerebral ganglion has been provided by Lacalli and
Holland [38]. Serotonin-like immunoreactive neurons
were found in the posterior half of the cerebral ganglion
(Fig. 2a, b, c). A central cluster of serotonin-like immu-
nopositive perikarya was localized near the posterior
margin of the neuropil (Fig. 2b). In addition, the cere-
bral ganglion of *T. democrata* exhibited two paired
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Localization of serotonin-like immunoreactivity, acetylated α-tubulin, and DAPI in *Pyrosomella verticillata* tetrazooid colony. **a** Adult blastozooids (b1 and b2), overview. Oral siphons (os) and cerebral ganglia (cg) highlighted. **b** Mature blastozooid highlighting oral siphon (os), pericoronal bands (pb), ciliated funnel (cf), gills (g), peribranchial tube (pt) and with motor nerves (anterior (an), lateral (ln), posterior (pn) and medial posterior (mpn) nerves) extending from the cerebral ganglion (cg). **c** Detail of the ciliated funnel (cf) and cerebral ganglion (cg) in dorsal view. **d, e** Light (d) and confocal (e) magnification of the cerebral ganglion (cg) (lateral view) in connection with the ciliated funnel (cf). **f** Detail of mpn crossing a peribranchial tube (pt). **g** Detail of the endostyle (serotonin-like immunopositive cells marked with arrowheads), with grayscale invert editing to highlight serotonin-like immunoreactive cell shape (inset). **h** Detail of the posterior part of one adult zooid, highlighting the pyloric gland (pg), with grayscale invert editing to highlight serotonin-like immunoreactive cell shape (inset)</figcaption></figure>

clusters of serotonin-like immunoreactive neurons lat-
erally (Fig. 2b). Depth color-code analysis of serotonin-like
immunoreactivity suggests that a loose bundle of nervous
fibres extends ventrally through the neuropil from the
central core (Additional file 1). Nervous fibres projecting
from the ventral margin of the cerebral ganglion were
found to adjoin anteriorly to the optic bundles of the eye
(Fig. 2c). Double labelling for serotonin and acetylated α-
tubulin suggested that some of the lateral serotonin-
like immunoreactive neurons extend fibres as would
be expected in case of motor neurons (Fig. 2c). As
reported by Pennati et al. [33], serotonin-like immu-
noreactivity was detected on the pericoronal bands
(Fig. 3a), in the digestive system (oesophagus and in-
testine) (Fig. 2d, e) and on the posterior end of the
branchial septum (Fig. 2d). In the first organ, immu-
noreactive cell bodies have an elongated morphology

and are organized in a single row (Fig. 2a), in the
second one they are both spherical and elongated and
are organized in single and multiple rows (Fig. 2d, e),
while in the third one serotonin-like immunoreactive
cells are both spherical and elongated, and form two
bilateral rows (Fig. 2d). Serotonin-like immunopositive
cells were not seen in ciliated funnel (data not
shown) and endostyle (Fig. 2f).

Although the anatomy of oozooids and blasto-
zooids of *T. democratica* is similar in many respects,
serotonin-like immunoreactivity was not detected in
the early aggregate blastozooids derived by stro-
bilation from a posterior stolon of the oozooid
(Fig. 2g-i). In blastozooids at developmental stage II
sensu Brien [39], labelling of α-tubulins highlighted
neural fibres running along the pericoronal bands
and in a visceral longitudinal nerve extending to the
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Localization of serotonin-like immunoreactivity, acetylated α-tubulin, and DAPI in *Thalia democratica*. **a–f** Adult oozooids. **g–i** Aggregate blastozooids. **a** General view of the anterior region that contains the ciliated funnel (cf), endostyle (en), cerebral ganglion (cg), and pericoronal bands (pb), with grayscale invert editing to highlight serotonin-like immunoreactive cell shape in the pericoronal bands (inset). **b** Detail of the cerebral ganglion highlighting peripheral (arrowheads) and central (encircled) serotonin-like immunoreactive cells, and fibres projecting ventrally through the neuropil (arrowhead in the inset). **c** Detail of the cerebral ganglion highlighting eye (e), neuropil (np) (arrow indicates α-tubulin and serotonin co-labelled neuron), and motor nerves (mn) extending from peripheral serotonergic neurons (arrowhead indicates α-tubulin immunoreactive nerve). **d** Detail of mouth (mo), oesophagus (oe) and branchial septum (bs). **e** Magnification of intestine (in) and branchial barrier (bb), with grayscale invert editing to highlight serotonin-like immunoreactive cell shape (inset). **f** Detail of the endostyle. **g** General view of early aggregate blastozooids at developmental stage I sensu Brien [39]. **h, i** Details of aggregate blastozooids at developmental stage II sensu Brien [39] highlighting ciliated funnel (cf), cerebral ganglion (cg), pericoronal bands (pb), visceral nerve (vn), and eleoblast (el)</figcaption></figure>

eleoblast (i.e., a specialized epithelial organ of some thaliaceans) [38] (Fig. 2g-i).

### Ihlea punctata

In the *I. punctata* oozooids, serotonin-like immunopositive neurons were scattered at the ventral posterior margin of the cerebral ganglion, near the exit of axonal projections extending from it (Fig. 3a). Serotonin-like immunoreactivity was also encountered in spherical cell bodies along the pericoronal bands (Fig. 3b) and in regularly arranged rows in the oesophagus (Fig. 3c). No endostyle was observed in any of the *I. punctata* oozooids examined.

### Organization of the serotonergic nervous system in the doliolid *Doliolina muelleri* (Krohn, 1852)

In comparison with salps and pyrosomes, doliolids have a long generation time and their life cycle encompasses different zooids [40]. Their typical body plan is barrel-shaped with two wide siphons and 8-9 circular muscle bands. The neural complex of Doliolida groups the cerebral ganglion (the central nervous system, composed by neurons and the neuropil), the neural gland (an ectodermal structure of unclear function), and the ciliated funnel, sometimes called "vibratile organ" [36]. The cerebral ganglion of *D. muelleri* phorozooids is localized dorsally in the middle of the body, and long nerves
<figure><img src="image_6.png" /><figcaption>**Fig. 3** Localization of serotonin-like immunoreactivity and tyrosinated α-tubulin in *Ihlea punctata* oozooids. **a** Cerebral ganglion (cg) with eye (e) and motor nerves (mn) extending from it. **b** Pericoronal bands (pb), serotonin-like immunopositive cells marked with arrowheads. **c** Oesophagus (oe), serotonin-like immunopositive cells marked with arrowheads</figcaption></figure>

emerge from it elongating anteriorly and posteriorly (Fig. 4a). Two clusters of 3–4 serotonin-like immunoreactive neurons are seen laterally in the cerebral ganglion (Fig. 4b), in close proximity to neurons projecting motor nerves (Fig. 4c). A continuous row of serotonin-like immunoreactive spherical cells was seen at the junction of the pericoronal bands (Fig. 4d, e). This region has been previously described as the ciliated funnel in Doliolida [32, 41] but it is probably not homologous to the funnel that links the neural complex (neural gland) to the branchial chamber [42]. Few and sparse spherical and elongated serotonin-like immunoreactive cells were found in the initial tract of the digestive system (mouth and oesophagus) (Fig. 4f). Serotonin-like immunoreactivity was not detected in pericoronal bands and endostyle (Fig. 4g, h).

The barrel-shaped zooid growing in one side of the head of *D. muelleri* tadpole larvae gradually takes on the adult form while the larval tail degenerates (Fig. 5a–c). No serotonin-like immunoreactivity was overall detected in cell bodies or nerves from larvae and young zooids. In young zooids, α-tubulin marked major nerves that appeared to connect a fibre plexus within the neural ganglion to the entire body (Fig. 5a', b) and a bundle of fibres running through the ciliated funnel (Fig. 5c). No α-tubulin immunoreactivity was observed in tadpole larvae attached to the young zooids (Fig. 5d, d').

## Discussion

### Serotonin-like immunoreactivity in the nervous system, implications for brain evolution

Based on the localization of serotonin-like immunoreactive neuronal cells with descending projections through the neuropil, Hay-Schmidt [13, 43–47] suggested that an orthogonal organisation of the nervous system was likely present in the last common ancestor of chordates, an idea previously proposed by Garstang [48]. This ancestral condition should be observed also in thaliaceans due

to the phylogenetic placement of this clade within ‘ascidians’. We found that serotonin-like immunopositive neurons are symmetrically distributed in the cerebral ganglion of the examined Doliolida and Salpida species and that, at least in *T. democratica*, serotonin-like immunoreactive tracts project transversally through the neuropil (Additional file 1) [34]. Recent works based on the gene expression study of orthologous transcription factors during development suggests that the ascidian CNS holds molecular evidence of brain compartment homology with vertebrate fore-, mid-and hindbrain [3, 49]. In thaliaceans, it will be of great interest to compare gene expression pattern of transcription factors involved in the differentiation of the three organizing centres in the vertebrate brain: the anterior neural ridge, the zona limitans intrathalamica and the isthmic organizer (e.g. *Fgf8*, *Fgf17*, *Fgf18*, *Sfrp1/5*, *Hh*, *Wnt1*) [50]. This would help in understanding to which degree the homologous neuroectodermal signalling centers that pattern deuterostome bodies were conserved or diverged in Thaliacea. However, evidence of chordate features in ascidians does appear before metamorphosis, while thaliaceans examined in the present study are all post-metamorphic stages. This suggests that caution is needed when interpreting gene or protein expression patterns in mature forms of thaliaceans.

Based on the expression of several pituitary markers (e.g., *Pitx*, *Pax2/5/8*, *Six1/2*), the ciliated funnel of ascidians has been suggested to be homologous to the adenohypophysis, a major organ of the vertebrate endocrine system that regulates various physiological processes such as stress, growth, and reproduction (reviewed in [51]). In thaliaceans, the ciliated funnel could be responsive to the detection of olfactory information from the environment thus eliciting specific behavioural responses [33]. The evidence presented here concerning the absence of serotonin-like immunoreactive cells in the ciliated funnel of the examined specimens is in agreement with similar reports in appendicularians, ‘ascidians’ and
<figure><img src="image_7.png" /><figcaption>**Fig. 4** Localization of serotonin-like immunoreactivity, acetylated α-tubulin, and DAPI in *Doliolina muelleri* phorozooid. **a** Dorsal view of the whole mount phorozooid, highlighting oral siphon (os), pericoronal bands (pb), endostyle (en), motor nerves (mn), cerebral ganglion (cg), gills (g), oesophagus (oe), and atrial siphon (as). **b, c** Cerebral ganglion with lateral clusters of serotonergic neurons (arrowheads), and motor nerves protruding from it (mn 1–7) at different magnifications. **d, e** Pericoronal bands (pb), with grayscale invert editing to highlight serotonin-like immunoreactive cell shape (inset). **f** Initial tract of the digestive system highlighting stomach (st) and serotonergic cells in the mouth (mo). **g** Anterior part of the specimen highlighting pericoronal bands (pb), endostyle (en), and gills (g). **h** Lateral view of the endostyle highlighting the long cilia protruding from it (encircled)</figcaption></figure>

salpids [32, 34, 52–55]. While discounting the use of the serotonergic system in the ciliated funnel of tunicates, this finding suggests that the prominent role played by the local production of serotonin in the pituitary gland is an acquired feature of vertebrates.

## Serotonin-like immunoreactivity in non-neural tissues

The tunicate endostyle, a structure homologous of the vertebrate thyroid, is a ventral U-shaped organ made by folds of the pharyngeal epithelium that secretes mucus for filter feeding [56]. Each mirror-image side of the tunicate endostyle displays between five and nine zones of distinctive cells, including supporting and glandular zones as well as zones with iodinating capacity [57, 58]. In stolidobranch, aplousobranch and phlebobranch ascidians, serotonergic cells were exclusively found in the

lateral portion of the endostyle, between zone seven (known to have iodinating capacity), and eight (which consists of ciliated cells) [34, 54, 55, 59–61]. Based on our analysis, serotonin-like immunoreactivity in the endostyle of thaliaceans was detected only in *P. verticillata*, in a lateral zone near a band of ciliated cells, just as in ascidians. The observation that salpids and doliolids lack serotonin-like immunoreactivity in the endostyle provides support for an evolutionary scenario in which Pyrosomatida is the first group branching from the class Thaliacea [11]. However, the absence of serotonin-like immunoreactivity in the endostyle of salpids and doliolids could be a character associated with independent changes in the control of thyroid hormone production rather than an ancestral state; however, this has not yet been verified.
<figure><img src="image_8.png" /><figcaption>**Fig. 5** Localization of acetylated α-tubulin and DAPI in tadpole larvae and young zooids of *Doliolina muelleri*. **a–d'** Light (**a**, **d**) and confocal (**a'**, **b**, **c**, **d**) images of a single tadpole larva (**d**, **d'**) connected with a young zooid (**a**, **a'**, **b**, **c**) highlighting cerebral ganglion (**cg**), ciliated funnel (**cf**), dorsal appendix (**da**), endostyle (**en**), mesoblast (**me**), major nerves (**mn**) and notochord (**nt**); area of contact between zooid and tadpole larvae marked with asterisk (*). **b**, **c** Dorsal view of the young zooid. **d**, **d'** Detail of the tadpole larva</figcaption></figure>

The peripharyngeal (pericoronal) bands of pyrosoma-
tids, salpids and doliolids are rich in ciliated cells and
could have a role in mechanoreception [62]. The pres-
ence of serotonin-like immunoreactive spherical cells in
the pericoronal bands, of ascidians as are in thaliaceans
suggests a phylogenetic link between these two tunicate
classes [34, 54, 55, 59–61].

The post-pharyngeal digestive tract of tunicates con-
sists of mouth, oesophagus, stomach, intestine, and
anus [63, 64]. Digestive functions are also ascribed to
the pyloric gland, an organ that begins at the globular
gland that encrusts and opens to the intestine. The
tunicate pyloric gland is composed of tubules and
ampullae that grow from the outer wall of the stomach
and is considered to be one of the major synapo-
morphies of the group [65, 66]. In ascidians, the
occurrence of spherical and elongated cell bodies that
are serotonin-like immunoreactive is reported in
distinct tracts of the digestive system, including
oesophagus, stomach and intestine [32, 34, 52, 54, 55].
In our work, serotonin-like immunoreactive cell bodies
in the oesophagus of Salpida and Doliolida species, as
seen in ascidians, likely reflect a plesiomorphic condi-
tion. Conversely, serotonin-like immunoreactivity in
the pyloric gland of *P. verticillata* seems to be an inde-
pendently derived character.

The serotonergic system is not required in immature
forms

We did not detect serotonin-like immunoreactivity in *Thalia democratica* juveniles (blastozooids), in larvae and young asexual zooids of doliolid or in sexual hermaphrodite blastozooid stages of the pyrosomatid examined. The lack of expression of 5HT suggests that serotonin acquires its functionality only in mature thaliancean zooids, thus not having a role in early development. However, serotonin expression may be still present but not detectable with our methods due to low levels or poor permeabilization, prompting for transcriptional activity studies of genes belonging to the serotonin biosynthetic pathway.

Phylogenetic relationships within Thaliacea

Due to their classification as chordates, the subphylum
Tunicata has been central to discussions on the evolu-
tion of deuterostomes and craniates [66–76]. None-
theless, the internal phylogenetic relationships of the
tunicate class Thaliacea remain uncertain. Thaliacea is
recovered as monophyletic regardless of the number of
taxa analysed, the molecular data type used, or the
phylogenetic method applied, with the exception of one
study which used partial 28S rDNA sequences [10].
Almost all studies agree in grouping Thaliacea as sister
group of Phlebobranchiata, one of the classical 'Ascidiacea' groups (where 'Ascidiacea' = Phlebobranchiata + Stolidobranchiata + Aplousobranchiata). We assume Phlebobranchiata as out-group for our phylogenetic comparison, due to the placement of this clade as adelphotaxon of Thaliacea in many studies [4, 7, 66, 77].

A scheme summarizing the differential spatial distribu-
tion of serotonin-like immunoreactivity among organs in
Thaliacea is shown in Fig. 6.

Character comparisons suggesting that pyrosomatids
originated early in the evolutionary history of thaliaceans
include the presence of serotonergic cells in the endo-
style and pyloric gland as in the phlebobranch *Phallusia
mammillata* [54]. This condition is not present in the
Salpida or Doliolida species examined, as discussed
above. Since the presence of serotonin-like immunoreac-
tivity alone cannot be considered as an uncontroverted
character of phylogenetic value, we supplemented our
molecular data with ten morphological and life cycle
characters extracted from the literature [32]. Apomor-
phies such as the existence of inner longitudinal vessels
in branchial basket and the presence of ontogenetic
rudiment of atrial opening are common features shared
just between Pyrosomatida and Phlebobranchiata [66].
The ciliated funnel is a very variable organ both with
respect to its anatomy and its topology. It is associated
with the cerebral ganglion in appendicularians, pyroso-
matids and ‘ascidians’, but not in salpids nor in doliolids
[37, 78–80]. Otherwise, the topology of the ciliated

funnel in salpids is distinct from that of pyrosomatids
and ‘ascidians’ in that it is not continuous to the pericor-
onal bands [37, 42]. Further, the presence of dorsal
lamina, branchial tentacles and distinct muscle bands
used in jet propulsion in Salpida and Doliolida [66]
supports a sister group relationship between these two
thaliacean orders. By applying a principle of parsimony,
these characters (Fig. 7) seem to favour the phylogenetic
hypothesis in which Pyrosomatida, an order often classi-
fied within the ‘ascidians’ [11, 80, 81], and not Doliolida
[6], is the first branching group from Thaliacea.

**Conclusion**

Here we present a study of serotonergic immunoreactivity
in the three thaliacean orders, and provide a first descrip-
tion of the pyrosomatid serotonergic system. The analysis
of the distribution of serotonin-like immunopositive cells
in adult thaliacean oozooids appear to depict shared char-
acters with ascidians. Remarkably, serotonin-like immuno-
reactivity is not present in immature thaliacean zooids,
suggesting that this amine is not crucial for the morpho-
genesis of the species examined. Differences in serotonin-
like immunoreactive arrangement in endostyle, initial tract
of the digestive system and pyloric gland, plus a review of
life cycle and morphological data, prompt us to support
the phylogenetic hypothesis in which Pyrosomatida is the
first Thaliacean order that diverged from the Ascidiacea
clade, thus positioning Salpida and Doliolida as sister
groups. Data from more species and the support of

<figure><img src="image_9.png" /><figcaption>**Fig. 6** The serotonin-like immunoreactive nervous system in Thaliacea. serotonin-like immunopositive cells in adult *Pyrosomella verticillata*, *Thalia democratica* and *Doliolina muelleri*</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 7** Phylogenetic characters. Comparison of serotonin-like immunoreactivity distribution and selected plesiomorphies in thaliacean orders and in the phlebobranch species, *Phallusia mammillata*. Non-neural data from [35, 52, 61, 76, 77]</figcaption></figure>

molecular based phylogenetic and/or phylogenomic analyses will be crucial to make more robust the relationships among different clade of Thaliaceans.

# Methods

## Animal collection and identification

Samples were collected in the Western Mediterranean using vertical plankton tows-200 µm mesh size-in the localities of Rade de Villefranche-sur-Mer (France) (43° 42'18"N 7°18'45"E) (*Pyrosomella verticillata* and *Ihlea punctata*) and Gulf of Naples (Italy) (40°48'5"N 14°15' E) (*Thalia democratica* and *Doliolina muelleri*). Specimens were identified under stereomicroscope following taxonomic keys in [82] and [83].

## Whole mount immunocytochemistry and imaging

Specimens were fixed in 4 % paraformaldehyde/0.1 M MOPS pH 7.4 containing 0.5 M NaCl, overnight at 4 °C. After several washes in phosphate buffered saline (PBS),

samples were treated with 0.5 mg/ml cellulase (Sigma C1184) in PBS pH 5.5 for 10 min at 37 °C in order to partially digest the tunic and facilitate antibody penetration. Following this, incubations were carried out on a rotating shaker. Specimens were permeabilized for 20 min in PBS/0.25 % Triton X-100 (PBTr). A blocking step was performed with 30 % heat-inactivated Normal Goat Serum (NGS) in PBTr for 2 h, prior to incubating specimens with primary antibodies-anti-5HT (serotonin) (Immunostar 20080), anti-tyrosinated α-tubulin (Sigma T9028), and anti-acetylated α-tubulin (Sigma T6793)-diluted 1:300 in PBS containing 0.1 % Tween-20 (PBST) and 30 % NGS, for 60 h at 4 °C. After extensive washes in PBST, samples were incubated at 4 °C overnight with secondary antibodies-goat anti-rabbit IgG-Alexa 488 and goat anti-mouse IgG-Alexa 647-diluted 1:400 in blocking buffer (1 % BSA in PBST). All samples were washed thoroughly in PBS. All specimens except *Ihlea punctata* were counterstained with DAPI (1 µg/ml in PBS) for
nuclear labelling. Control experiments were run in parallel by omitting primary antibodies.

Image acquisition was performed on Zeiss LSM 510 Meta and Leica SP5 confocal microscopes. Z-stack images were analyzed and processed with Fiji and Photoshop CS6 (Adobe). Figure plates were made with Illustrator CS6 (Adobe). Brightness/contrast, inversion and colour balance adjustments where applied, were applied to the entirety of the image and not to parts thereof.

## Additional file

**Additional file 1:** Visual assessment of serotonin-like immunoreactivity in *Thalia democratica* cerebral ganglion. (A–E) Five consecutive frontal sections ranging dorsal to ventral from Z = 14 to Z = 18 every 4.54 µm, showing elongating serotonin-like immunoreactive bundle (squared line). (F) Color-coded 2D image from hyperstacks Z = 14–18, showing different depth distribution of lateral clusters of serotonin-like immunoreactive neurons (arrows), central cluster of serotonin-like immunoreactive neurons (dashed line) and serotonin-like immunoreactive nervous fibre bundle (squared line). (JPG 1000 kb)

## Acknowledgments

We are grateful to Alessandro Minelli (University of Padova, Italy) and John Kirwan (Lund University, Sweden) for critical reading and commenting the manuscript. The authors would also thank Santiago Valero-Medranda (Spain) for illustrating Figs. 6 and 7, and the fishery services at MEDA-Stazione Zoologica Anton Dohrn (Italy). Sampling at the Observatoire Océanologique de Villefranche-sur-Mer (France) have been done with the support of EMBRC-France.

## Funding

AVG is supported by the Marie Curie ITN 'Neptune' program (grant no. 317172), and was supported by an Erasmus-Leonardo fellow (Universidad Autónoma de Madrid and Spanish National Government), and a Réseau d'Activité Scientifique André Picard mobility grant (Pierre et Marie Curie University and French Italian Government) during the early stage of this study. FC and VN have been supported by a SZN PhD fellowship and a MIUR PON Grant (PONa3_00239), respectively.

## Authors' contributions

AVG, FC, ST, VN and PS participated in the collection of specimens. AVG, RM, and ST carried out immunocytochemical experiments and confocal laser-scanning microscopy. AVG, ST and PS conceived the paper and drafted a first version. All other authors assisted in drafting the manuscript. All authors read and approved the final manuscript.

## Competing interests

The authors declare that they have no competing interests.

## Declarations

Experiments were performed in accordance with the European Union animal welfare guidelines [European Communities Council Directive of September 22, 2010 (2010/63/UE)]. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

## Author details

¹Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy. ²Institute of Marine Biological Resources and Inland Waters, Hellenic Centre for Marine Research, GR-19013 Anavyssos, Greece. ³Observatoire Océanographique, CNRS, Sorbonne Universités, UPMC Univ Paris 06, Laboratoire de Biologie du Développement de Villefranche-sur-Mer, 06230 Villefranche-sur-Mer, France.

Received: 3 May 2016 Accepted: 16 September 2016

Published online: 29 September 2016

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