Evolutionary traces of miniaturization in a giant—Comparative anatomy of brain and brain nerves in Bathochordaeus stygius (Tunicata, Appendicularia)

Appendicularia comprises 70 marine, invertebrate, chordate species. Appendicularians play important ecological and evolutionary roles, yet their morphological disparity remains understudied. Most appendicularians are small, develop rapidly, and with a stereotyped cell lineage, leading to the hypothesis that Appendicularia derived progenetically from an ascidian‐like ancestor. Here, we describe the detailed anatomy of the central nervous system of Bathochordaeus stygius, a giant appendicularian from the mesopelagic. We show that the brain consists of a forebrain with on average smaller and more uniform cells and a hindbrain, in which cell shapes and sizes vary to a greater extent. Cell count for the brain was 102. We demonstrate the presence of three paired brain nerves. Brain nerve 1 traces into the epidermis of the upper lip region and consists of several fibers with some supportive bulb cells in its course. Brain nerve 2 innervates oral sensory organs and brain nerve 3 innervates the ciliary ring of the gill slits and lateral epidermis. Brain nerve 3 is asymmetric, with the right nerve consisting of two neurites originating posterior to the left one that contains three neurites. Similarities and differences to the anatomy of the brain of the model species Oikopleura dioica are discussed. We interpret the small number of cells in the brain of B. stygius as an evolutionary trace of miniaturization and conclude that giant appendicularians evolved from a small, progenetic ancestor that secondarily increased in size within Appendicularia.

Consequently, appendicularians efficiently remove particles from the seawater as small as 160 nm, including bacteria and viruses (Lawrence et al., 2018). Preyed upon by numerous larger animals including cnidarians, ctenophores, chaetognaths, and fishes (Miller et al., 2011;Purcell, 2003;Spriet, 1997), appendicularians form a crucial link that channels organic material from the submicron range into the oceanic food web, bypassing the microbial loop. Since appendicularian houses are regularly discarded and rapidly sink to the bottom, they remove carbon directly from the photic zone and indirectly, yet effectively, from the atmosphere (Berline et al., 2011;Hansen et al., 1996;Katija et al., 2017). Indeed, appendicularian houses can in certain areas contribute up to 83% of the total carbon deposited to the sea floor (Alldredge et al., 2005). Since the houses of giant appendicularians like Bathochordaeus spp. are underrepresented by conventional sampling methods, their role in carbon flux estimates has not been accounted for historically (Robison et al., 2005).
The peculiar feeding mechanism of appendicularians requires a finely tuned regulation of tail beats and arrests (Conley et al., 2018;Selander & Tiselius, 2003). Most animals can also reject unwanted particles by temporarily detaching the mouth from the buccal tube of the house and reversing the direction of the ciliary beat in the gill slits (Deibel, 1986;Fenaux, 1986). In addition, discarding a house, swimming, and inflating a new house requires completely different movements (Glover, 2020;Lohmann, 1933;Spriet, 1997). These complex behaviors are coordinated by a nervous system, consisting of a central brain and peripheral nerves, that innervate musculature, cilia, and epidermis or receive input from sensory cells.
Taxonomically, Appendicularia belongs to Tunicata, the possible sister taxon of Craniota and is therefore important for understanding the early evolution of vertebrates (Braun et al., 2020;Delsuc et al., 2006). Unlike other tunicates, appendicularians retain the body division into trunk and postanal tail throughout their lives. In this, they resemble larval ascidians and appendicularians are accordingly also called larvaceans. As most appendicularians are small, develop rapidly, and with a stereotyped cell lineage, it has been suggested that they evolved through heterochrony from an ancestor with a free-swimming tadpole-like larva and a sessile, ascidian-like adult (Garstang, 1928;Stach et al., 2008;Stach & Turbeville, 2005).
According to this hypothesis, the heterochronic, more specifically progenetic, miniaturization of appendicularians constitutes an apomorphic feature. Alternatively, it had been suggested that swimming freely represented a plesiomorphic trait inherited unaltered from the last common ancestor of Chordata (Swalla et al., 2000;Wada, 1998).
The latter hypothesis is based on the hypothesis that Appendicularia was the sister taxon to all remaining Tunicata, a phylogenetic position that has since been recovered consistently in molecular phylogenetic studies (Delsuc et al., 2018;Kocot et al., 2018) and also in a cladistic analysis of morphological characters (Braun et al., 2020).
Morphologically, little is known of these giant appendicularians beyond their original species descriptions (Garstang, 1937;Lohmann, 1933). In this study, we investigated Bathochordaeus stygius Garstang, 1937, a giant appendicularian from the Eastern Pacific, to expand the knowledge of the morphological disparity of appendicularians. Moreover, this study allowed us to identify anatomical traces of miniaturization in the nervous system of B.
stygius from which we conclude that giant appendicularians evolved from a small, progenetic ancestor that secondarily increased in size within Appendicularia.

| MATERIALS AND METHODS
Two specimens of B. stygius were collected from MBARI (Monterey Bay Aquarium Research Institute, California, USA) using a remotely operated vehicle, Ventana, and gentle suction (see Table 1; Robison, 1993). Animals showed normal regular tail beat and were healthy looking, undamaged, corresponding to the description and depictions rendered in the species description by Garstang (1937;Figure 1a,b and Supporting Information: Figures S1 and S2). Animals were maintained alive until they could be fixed onshore using a solution of 1% paraformaldehyde, 2.5% glutaraldehyde in 0.2 mol l −1 sodium cacodylate buffer (pH 7.2), and adjusted to an osmolarity of approximately 800 mOsm with the addition of sodium chloride.
Primary fixation was stopped after 1 h with three buffer rinses.  Figure  Thus, the resulting model is on the one hand smoother and more live-like in outer appearance and some of the major organ systems and at the same time contains less information on their substructure or even no information (e.g., the pericardium; see Supporting Information: Figure 2).

| RESULTS
Upon capture, animals showed normal regular tail beats and appeared healthy looking, undamaged, corresponding to the description and depictions rendered in the species description by Garstang (1937; Figure 1a (f) Brain, brain nerves, and nuclei in lateral view from the left side. (g) Brain, brain nerves, and nuclei in oblique dorsal view. br, brain; cf, ciliated funnel; es, esophagus; fb, forebrain; fo, Fol's oikoplast; hb, hindbrain; ht, heart pericard; mo, mouth opening; mu, musculature; N1 l/r , left/right first brain nerve; N2 l/r , left/right second brain nerve; N3 l , left third brain nerve; oso, oral sensory organ; nc, notochord; np, neuropil; nt, nerve cord; stl, left stomach lobe; sv, sensory vesicle; tf, tail fin; red asterisks, nuclei of second brain nerves.
Morphologically, the brain can be divided into an anterior part  Figure S3). Fiber tracts, that is, a neuropil area, can be traced through the posterior part of the forebrain, the hindbrain, and into the nerve cord (Figures 1g, 2, and 4).
The forebrain makes up the anterior two-thirds of the brain in length. It contains 67 cells identified by their nuclei (Figure 3 and In the posterior part of the brain, we unambiguously identified 35 cells (see Table 2). Again, some cell membranes could not be Several nerves and neurites leave (respectively enter) the brain, and posteriorly, the brain extends into the nerve cord or nerve cord ( Figures 1-3 and 5). The most prominent and most voluminous nerve leaving the brain is the most anterior first brain nerve. It is a paired nerve that bifurcates immediately after leaving the brain anteriorly.
The first brain nerve is composed of numerous neurites ( could be identified in the first brain nerve on the right side, and two somata in the first brain nerve on the left side. Some 40 µm posterior to the first brain nerve, the second brain nerve that is also paired leaves the brain ventrally in the anterior direction (Figures 1f,g, 2a,d, 5, and 6e). In fact, the second brain nerve on the right side leaves the brain slightly anterior to its left counterpart. The nerves consist of a single neurite that runs underneath the epidermis around the mouth opening. The right second brain nerve, therefore, passes between the ciliary funnel and the epidermis. The somata giving rise to the second brain nerves are situated in the anterior part of the forebrain (Figure 1f,g). The neurites of the second brain nerves eventually branch with a short F I G U R E 3 Three-dimensional (3D) reconstruction of the brain of Bathochordaeus stygius. (a and b) In gray: Cells with a volume less than 12, 600 µm 3 . In red: Cells with a volume of 15,600 µm 3 and more. (a) Brain and brain nerves in lateral view from the left side. (b) Brain and brain nerves in oblique dorsal view. (c and d) In gray: Nuclei with a volume less than 1480 µm 3 . In red: Nuclei with a volume of 1630 µm 3 and more. (c) Brain and brain nerves in lateral view from the left side. (d) Brain and brain nerves in oblique dorsal view. fb, forebrain; hb, hindbrain; N1 l/r , left/ right first brain nerve; N2 l/r , left/right second brain nerve; N3 l , left third brain nerve; np, neuropil; nt, nerve cord; sv, sensory vesicle.
branch running dorsally to the epidermis of the posterior margin of the mouth opening. The other branch continues anteriorly, branches some more times, and contacts three complex oral sensory organs situated in the epithelium of the lateral and anterior margin of the mouth and the anterior part of the mouth cavity ( Figures 5 and 7) on each side.
The left third brain nerve and the nerve cord leave the brain together, the brain nerve just left of the nerve cord. Different from the other brain nerves, the third brain nerve is asymmetrical (Figures 1e-g and 5). Its counterpart on the right side is part of the nerve cord and branches off from the rest of the nerve cord about 500 µm posterior to the brain. Nevertheless, we will call this right-sided nerve "right third brain nerve." The third brain nerve consists of three neurites on the left side (Figure 8a,b) and two neurites on the right side. Both third brain nerves branch into a ramus branchialis that runs toward the elongated ciliated rings of the gill slits on each side and another branch (ramus epidermalis) that projects toward the adjacent lateral epidermis ( Figures 5 and 8).

| DISCUSSION
Most of our knowledge about appendicularians is generalized from detailed studies of Oikopleura dioica (Ferrández-Roldán et al., 2019). This is true for molecular, developmental, and ecological studies, but even for morphological or evolutionary studies. In O. dioica the brain consists of 78-150 cells (Braun & Stach, 2019;Glover, 2020;Nishida et al., 2021;Søviknes et al., 2005; see also Supporting Information: Table 1), which-like the small size and the high developmental speed among other things-have been considered as indications of neotenic acceleration during evolution (e.g., Garstang, 1928;Stach et al., 2008;Stach & Turbeville, 2005).
The anatomy of the nervous system in several Oikopleura species has been investigated by light microscopy (Lohmann, 1933;Martini, 1909aMartini, , 1909b and details of the anatomy of the brain have been investigated by electron microscopy in O. dioica (Nishida et al., 2021;Olsson et al., 1990). We showed that details of the anatomy of the nervous system in the mesopelagic giant B. stygius correspond to morphological structures described for Oikopleura spp.,  | 7 of 14 but at the same time revealed notable differences. Like in Oikopleura spp., the first pair of brain nerves consists of several fibers that leave the brain anteriorly. Also, each of the first brain nerves features nuclei outside the brain, belonging to a small group of cells that were called bulb cells by Bollner et al. (1986) and supportive cells by Nishida et al. (2021). Different from Oikopleura spp., however, the first brain nerves in B. stygius project into the epidermis of the posterior rim of the mouth. This corresponds to the upper lip region in Oikopleura spp. In contrast, in Oikopleura spp., the first pair of brain nerves project into the conspicuous ventral sense organs that in turn consist of 30 cells, each with a single conspicuous sensory cilium (Bollner et al., 1986) and are situated laterally on the ventral side of the animal. No such organs, or indeed ciliated sensory cells, are found in B. stygius in the region of the first brain nerves. Instead, the epidermis cells in that region are covered by the house rudiment and therefore seem to be part of the oikoplastic epithelium. The ventral sense organ in O. dioica has been shown to derive from a placode-like thickening during development . This anterior placode expresses some of the genes that are also expressed in the olfactory placode of vertebrates, such as eyeA, six1/2, or pitx Mazet, 2006). Interestingly, the expression domain of these genes is ring-like around the future  (Olsson et al., 1990). Given the structural similarities in B. stygius, it is plausible to assume that the second brain nerves serve a corresponding function in this species.
In O. dioica, the third brain nerve of the different sides of the body differs; it contains two neurites on the right side and three on the left (Olsson et al., 1990). While we could show that the left third brain nerve in B. stygius consists of three neurites as well, we were unable to show the number of neurites in the right counterpart. Like in O. dioica, however, we could trace the neurites of the third brain nerves to the region of the ciliary rings of the gill slits and the lateral epidermis. Light microscopy, however, does not F I G U R E 5 Semischematic drawing of the brain, brain nerves, and their targets in Bathochordaeus stygius. Anterior is to the left and dorsal is to the top. cr, ciliary ring; en, endostyle; mo, mouth opening; N1 l/r , left/right first brain nerve; N2 l , left second brain nerve; N3 l/r , left/right third brain nerve; oso, oral sensory organ; rb, ramus branchialis (of the left third brain nerve); re, ramus epidermalis (of the left third brain nerve).
F I G U R E 6 Light micrographic details of the pair of first brain nerves. Sections are from anterior (a) to posterior (e). Dorsal is to the top of the images. (a) Left and right first brain nerve in the dorsal epidermis; note the nucleus of one of the nerve cells in the left first brain nerve. (b) Two nuclei are seen in the left first brain nerve. (c) Note the nucleus in the right anterior brain nerve. (d) Neurites of the left anterior brain nerve entering the brain anteriorly. (e) Neurites of the right anterior brain nerve entering the brain anteriorly. c, ciliated funnel; ci, cilia; hr, house rudiment; ne, neurites; nu, nucleus; nu*, nucleus in the first brain nerve; N1 l/r , left/right first brain nerve.
suffice to demonstrate synaptic contacts; transmission electron microscopy or specific labeling would be necessary for validation.
The pair of third brain nerves differs from O. dioica in another aspect as well; the third brain nerve in B. stygius does not leave the brain directly (and therefore by definition does not constitute a brain nerve). Instead, it accompanies the nerve cord for about 500 µm before branching off toward the right gill slit and epidermis, demonstrating at the same time evolutionary plasticity and constancy.
Different from O dioica, we did not find a pair of dorsal neurites projecting into the dorsal oikoplastic epidermis (Cañestro et al., 2005;Olsson et al., 1990). These neurites might be lacking in B. stygius, they might be incorporated into the first brain nerve, or we may have missed these neurites due to the technique used in the present study because these neurites might have been on sections, we were unable to analyze.
Earlier molecular phylogenetic results positioned Appendicularia as the sister taxon to the remaining tunicates and concluded that the last common ancestor of Tunicata was a free-living, swimming organism (e.g., Wada, 1998). This hypothesis contrasted with the more traditional hypotheses that regarded Appendicularia as an evolutionarily progenetic form derived from a sessile tunicate ancestor with a tadpole larva (Garstang, 1928;review in Stach & Turbeville, 2005). This "progenesis hypothesis" (also neoteny hypothesis or pedomorphosis) was based on several observations: (i) features that are larval in ascidians, such as the tail or the statocyst, are present in adult appendicularians (Cañestro et al., 2005;Garstang, 1928;Lohmann, 1933); (ii) ontogenetic events, for example, cell fate determination or gastrulation, occur comparatively early in appendicularians (Delsman, 1910(Delsman, , 1912Nishida & Stach, 2014;Stach et al., 2008); (iii) most appendicularians are miniaturized, that is, they show a body size comparable to ascidian larvae at sexual maturity; (iv) appendicularians show eutely (constancy in cell numbers) in several (but not all) tissues (Kishi et al., 2017;Nishida et al., 2021;Søviknes et al., 2007), a trait that is thought to be correlated with miniaturization.
With a trunk length larger than 1 cm, and an overall body size of 3-4 cm, species in the genus Bathochordaeus have been called giant appendicularians and in size are more similar to many solitary ascidians or cephalochordates (e.g., Kott, 1985;Poss & Boschung, 1996;Shenkar & Swalla, 2011). Could this be a plesiomorphic trait for Appendicularia? A brain length of approximately 220 µm in B. stygius is twice the brain length observed in O.
F I G U R E 8 Light micrographic details of the third brain nerve of Bathochordaeus stygius. Dorsal is to the top of images. (a) Third brain nerve on the left side next to the nerve cord. (b) Left third brain nerve immediately after the branching into the ramus branchialis (two neurites) and the ramus epidermalis (one neurite). (c) Ramus epidermalis of the left third brain nerve running underneath the lateral epidermis. (d) Ramus branchialis of the left third brain nerve close to the ciliary ring cells of the left gill slit. (e) Enlarged light micrograph of the ramus branchialis of the left third brain nerve close to the ciliary ring cells of the left gill slit. ci, cilia; cj, cell junction; crc, ciliary ring cell; ep, epidermis; gs, gill slit; hr, house rudiment; icm, intracellular matrix; N3 l rb , ramus branchialis of the left third brain nerve; N3 l re , ramus epidermalis of the left third brain nerve; nu, nucleus; nt, nerve cord; arrowheads point to individual neurites.
F I G U R E 9 Phylogenetic relationship of selected chordate representatives combined after several authors. Apomorphies that evolved in the stem lineages marked by colored and numbered rectangles include: ① notochord, nerve cord/neural tube, endostyle; ② molecular characters; ③ tunic with cellulose, heartbeat reversal; ④ external filtering house, rotation, and translocation of tail, miniaturization, progenesis; ⑤ dorsal translocation of mouth, increase in body size (but not brain), invasion of mesopelagial.