Deep-sea ctenostome bryozoans: revision of the family Pachyzoidae, with description of a new genus and three new species from Zealandia

Pachyzoidae is a little-known family of deep-sea ctenostome Bryozoa that until now was monospecific for Pachyzoon atlanticum. Originally described from the Atlantic Ocean, the genus was also found off southeastern New Caledonia in deep waters of the geological continent of Zealandia. Pachyzoon atlanticum forms globular to flat round colonies, living on soft, muddy to sandy bottoms with a few rhizoidal cystid appendages extending from the basal, substrate-oriented side. In this study, we investigate additional pachyzoids, collected between 1965 and 2015 from over 40 sites around New Zealand, by means of detailed morphological and histological investigations. In total, several hundred colonies were encountered in the NIWA Invertebrate Collection, comprising two new species of the genus Pachyzoon, P. grischenkoi sp. nov. and P. pulvinaris sp. nov., and the new genus and species Jeanloupia zealandica gen. et sp. nov.. The genus Jeanloupia is characterized by small disc-shaped colonies with highly elongated peristomes and a quadrangular aperture, distinct from the round apertures of the genus Pachyzoon. Pachyzoid species differ in colony structure and shape, apertural papillae and polypide features such as tentacle number or digestive-tract details. Cystid appendages are non-kenozooidal, but may originate from laterally flanking kenozooids. Based on published images, alleged P. atlanticum from New Caledonia is re-interpreted as P. pulvinaris n. sp.. Morphological characters support alcyonidioidean relationships, as previously suggested. First observations on pachyzoid reproduction show macrolecithal oocytes and brooding of embryos, which seems to be the general pattern for this family. The occurrence of three new Zealandian species in a comparatively small geographical area far from the Atlantic indicates a high possibility of more species to discovered.


Introduction
Bryozoa is a phylum of colonial suspension feeders.Colonies are composed of iterated modules, zooids, consisting of an exterior body-wall (cystid) and internal soft tissues.Most of the latter comprises a gut and an eversible and retractable tentacle crown with associated neural and muscular tissue [1][2][3].Depending on the taxonomic clade, the cystid may be cuticularized in various ways or mineralized via calcium carbonate incorporation.
Two clades can be distinguished among bryozoans: Phylactolaemata, a small group of freshwater bryozoans, and Myolaemata, which is predominantly marine [3].Myolaemata is divided into the sister-taxa Stenolaemata and Gymnolaemata.The latter comprises the paraphyletic Ctenostomata and monophyletic Cheilostomata [4].
The deep sea includes the most widespread and least known habitats on earth, harboring many bizarre forms adapted to the challenging conditions of hundreds to thousands of meters of depth, with variable substrata and food availability.Numerous bryozoans, most prominently gymnolaemates, have been recorded from a variety of deep-sea habitats.Three ctenostome families occur almost exclusively in the deep sea and are adapted to live on soft bottoms: Aethozoidae d 'Hondt, 1983(emend. Reverter-Gil et al. 2016), Clavoporidae Soule in Osburn, 1953, and Pachyzoidae d 'Hondt, 1983, the latter two from the same superfamily [see [5].Aethozoids are bizarre single-zooid ctenostomes with appendages; they comprise four genera [6].Clavoporids are club-shaped with a kenozooidal stalk of various morphologies and a capitulum carrying the feeding autozooids [e.g.[7,8]; five genera have been described.Pachyzoidae is represented by the sole genus and species Pachyzoon atlanticum d' Hondt, 1983, which was recorded from various deep habitats of the North Atlantic Ocean [see [9] and nominally from New Caledonia [10].Morphologically it has not been studied in detail.
Between 1965 and 2015, additional pachyzoid samples were collected by seven cruises conducted by NIWA (and its predecessor, the New Zealand Oceanographic Institute) in New Zealand waters.These yielded 366 pachyzoid colonies, comprising three new species and one new genus.Most were found off eastern South Island, with a few samples also collected in the Tasman Sea.Detailed histological analyses were carried out, allowing characterization of the new taxa and amplification of the family diagnosis with soft-body morphological features.For that purpose, material from the type species Pachyzoon atlanticum was analysed.

Materials and Methods
Samples were collected by trawls, epibenthic sleds or box corers from 42 stations over a depth range of 750-3480 m (Table 1, Fig. 1).Shipboard primary fixation was either in seawater-formalin or unknown, followed by storage in ethanol.
Specimen documentation and imaging was conducted using a Nikon SMZ25 (Nikon, Tokyo, Japan) stereomicroscope equipped with a Ds-Ri2 camera or a Hirox RH2000 microscope (Hirox, Tokyo, Japan).For histology, colonies or pieces of colonies were dehydrated in acidified dimethoxypropane followed by several rinses in pure acetone before infiltration in Agar low-viscosity resin (Agar, Stansted, UK).Cured resin blocks were serially sectioned with a Diatome HistoJumbo diamond knife (Diatome, Switzerland) at 1 µm thickness on a Leica UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany).Sections were stained with toluidine blue and sealed in resin.Analysis and documentation were conducted using a Nikon NiU microscope equipped with a Ds-Ri2 camera.Section series were converted to greyscale and enhanced in contrast with FIJI [11] before being imported into the reconstruction software Amira 2021.1 (ThermoFisher).Structures of interest were manually segmented and afterwards displayed as surface models.Surrounding tissues were displayed as volume renderings.Snapshots were taken using Amira software.

Family Pachyzoidae d'Hondt, 1983 Description
Colonies free-living, discoidal or globular, usually one to several mm in size.Colonies with c. 10-100 autozooids.Autozooids polygonal, normally tightly arranged, with orifices in close proximity on frontal side of colony.Orifices radially symmetrical or quadrangular, apertural papilla or long peristome may be present.Vestibular wall long.Autozooids with non-kenozooidal rhizoids attaching colony to substrate on basal side.Lateral kenozooids at colony margin with rhizoid processes common.Rhizoids usually non-muscular, muscles rare.Cystid cuticle wrinkled and often arborescent, commonly with attached shell or test material from foreign particles, often foraminiferans.Lophophore with 24-32 tentacles.Digestive tract short with elongated cardia, caecum usually vestigial, rarely pronounced; anus vestibular.Funicular muscle from caecum to body wall present or absent.Retractor muscles attaching at lophophoral base, foregut and cardiac portion of midgut.Parietal and apertural muscles thin and diffusely dispersed in zooids, not concentrated into regular bundles.Duplicature bands numerous on oral polypide side or totally lacking.Orificial and diaphragmatic sphincter not detected.Collar elongated, highly wrinkled, radial or quadrangular.Interzooidal pore plates simple with few special cells.Spermatogenic tissue at lateral zooidal walls, more basally.Ovaries basally associated, oocytes generally macrolecithal, internal brooding probably in tentacle sheath.

Remarks
We define true peristomes as elongated structures of the frontal side at the orifice.These are rigid and not retractable, whereas apertural papillae are simple, papillar rims around the orifice that show more variation in size and shape as different degrees of polypide retraction affect it.In many samples, part of the retracted vestibular wall is often protruded from zooidal orifices, which gives the impression of peristomes.The term peristome has been previously used for such tubular structures [8,10] Description Colony dome-shaped, flattened in frontobasal axis, 2.3-4.4 mm in diameter (Fig. 2).Zooids occurring as slender tubes in frontobasal axis (Figs. 3, 4d, 5a), about 1 mm long and 350 µm in diameter; cuticle complex, multilayered and sculptured with numerous surface elaborations on frontal and lateral side (Fig. 4a, e).Basal side of colony with thinner cuticle (Fig. 4d), thick and thin rhizoid cystid appendages attaching to substratum particles (Fig. 4e, f ).Laterally bordered by kenozooids (Fig. 4e).Orifice on frontal side, externally inconspicuous and little pronounced (Fig. 2c,  3a).Vestibular wall extending into zooidal tube about 1 3 of entire zooidal height in frontobasal axis (Figs.4a, 5a), vestibulum filled with irregular flocculent material similar to ectocyst covering (Fig. 4a, b, e).Polypide with 24 tentacles; gut short with very small caecal pouch; anus highly vestibular terminating almost at diaphragm (Figs. 3, 5a).Retractor muscles originate from vertical cystid walls and insert at lophophoral base and on esophagus-cardia transition (Fig. 3).Collar epithelium large, conical with radial spikes from where large collar emerges (Fig. 4c).Spermatogenic tissue located on anal vertical zooidal wall.Multiple duplicature bands present, 5-6 on oral and two on anal side of polypide (Fig. 3).Vestibular muscles diffuse in distofrontal area of zooid.
Distribution P. atlanticum was first described from muddy to sandy bottoms at 800-1600 m depth in the North Atlantic [8].Additional records are from off Iberian coasts at similar depths [12,13], summarized in 9].

Remarks
The species identified as P. atlanticum by d'Hondt & Gordon [10] is here considered to be P. pulvinaris (see below), in which case P. atlanticum is   1).
Etymology Honorific for bryozoologist Andrei V.
Distribution Southwest Pacific Ocean; most samples collected southeast of South Island, New Zealand, one sample from the Tasman Sea off northeastern North Island; 760-1586 m.
Remarks Pachyzoon grischenkoi sp, nov.occurs principally in or on the surface layer of terrigenous-foraminiferal ooze.Its flattened disc-shaped colony bears some resemblance to the free-living Arctic ctenostome Alcyonidium disciforme [14].Whereas colonies of the latter always develop a central hole once reaching a certain colony size, such a hole is generally missing in P. grischenkoi.Despite the superficial similarities of both species, clear differences are found in the cuticle, which is branching/ arborescent in P. grischenkoi and smooth in A. disciforme.Also, multiple duplicature bands on the oral polypide side are only found in this species and P. atlanticum whereas in Alcyonidium four regular bands are usual [15,16].The more flattened colony shape, differences in tentacle numbers (24 in P. atlanticum and 32 in P. grischenkoi) and different gut structure, particularly the very small intestine of P. grischenkoi, clearly distinguishes P. grischenkoi sp.nov.from P. atlanticum.Encrusting particles common on basal and lateral sides, and frontally between apertural papillae (Fig. 11).Vestibular wall elongated, with deeply immerged retracted polypide along frontobasal axis of colony (Figs.5c, 12).Lophophore with 32 tentacles, digestive tract with short foregut and cardia, caecum vestigial, slender elongated intestine (Fig. 12c, d).

Pachyzoon pulvinaris
Retractor muscles inserting at foregut and cardiac portion of gut (Fig. 12c, d).Duplicature bands not present.
Distribution Southwest Pacific Ocean; most samples collected off southeastern South Island, New Zealand, some samples from the Tasman Sea west of North Island.Also, off southwestern New Caledonia [10]; the depth Remarks Pachyzoon pulvinaris sp.nov.can occur in high numbers in each sample; 113 colonies were collected from one station in the Bounty Trough.Colonies often show deformations owing to fixation can make measurements difficult; reliable metric data depend on the leastdistorted specimens.Size measurements are thus difficult to compare and individual zooid sizes could not be determined.However, the typical cushion-shape of colonies and polypide features clearly distinguish this species from other congeners.Nominal Pachyzoon atlanticum described from New Caledonia by Gordon & d'Hondt [10] is here considered to be P. pulvinaris, as overall colony size and shape conform more to this species than to P. atlanticum.The little information provided shows rather globular colonies more similar to P. pulvinaris sp.nov.rather than the more flattened ones of P. atlanticum.Additional polypide features such as the general gut structure and different tentacle number (24 in P. atlanticum, 32 in P. pulvinaris sp.nov.) could support this, but data for the New Caledonian species is missing.Partially protruded vestibular walls, interpreted as peristomes as found in the New Caledonian samples, were also detected in some colonies in the current study.However, the frequency of such characters is relatively rare based on our analysis of over 100 colonies.
Etymology Alluding to its occurrence in the geological continent of Zealandia, which includes New Caledonia and New Zealand and the adjacent seafloor.

General structure and diagnostic characters of pachyzoid ctenostomes
Sequence data are so far lacking for any pachyzoid, and indeed for any deep-sea ctenostome (e.g.clavoporids, aethozoids).Consequently, achieving a phylogenetic understanding of deep-sea ctenostomes is challenging.The present analysis clearly shows numerous obvious differences between the four pachyzoid species, but their relatedness to each other and to other ctenostomes remains enigmatic.In agreement with previous assessments [8,17], we can conclude from our analysis that there are several morphological characters that support a closer relationship of pachyzoids to alcyonidioidean ctenostomes.This particularly relates to: 1) a dense zooidal arrangement of polygonal zooids; 2) diffuse and spread-out parietal and apertural musculature; 3) high tentacle number as in other alcyonidioideans [5]; 4) gut morphology with a particularly short or vestigial caecum, lack of cardiac prominence and vestibular anus [18]; and 5) at least for the genus Pachyzoon, a circular orifice.Individual zooid sizes are usually difficult to observe externally as their boundaries are not clear unless the obscuring covering is partially removed or colonies are damaged to show internal structures.For P. pulvinaris even, no zooid sizes were determined as zooidal wall were often broken und in dissected colonies also untraceable.We therefore consider the general colony shape and details in polypide features more reliable characters for species distinction in pachyzoids.
The quadrangular apertural shape in Jeanloupia zealandica begs the question of its affinity to pachyzoids, since apertural shapes are usually diagnostic at family level [5].Apertures in Alcyonidioidea are circular (Alcyonidiidae, Clavoporidae), quadrangular (e.g.Pherusellidae) or bilateral (Flustrellidridae).Hence it remains ambiguous whether pachyzoid colony morphology and shape evolved independently in the genera Pachyzoon and Jeanloupia.
Duplicature bands are a common feature of all bryozoans with some victorellid and many vesicularioidean ctenostomes showing reductions [15].Usually there are four bands, two on the anal side and two on the oral side of the polypide.Some ctenostomes have a few additional bands [19,20], and some cheilostomes also show four on each side [3], but multiple oral-sided duplicature bands as are found in P. grischenkoi sp.nov.and P: atlanticum have not been described.Their function and significance remain unknown.
The cuticle in pachyzoids is composed of multiple layers similar to other alcyonidioideans [19,21], but also shows a reticulate or dendritic pattern on its outermost layer, such as is found in Haywardozoon [22], Pherusella [21] and Sundanella [20].All of these genera are clearly closely related, but the lack of sequence data for Haywardozoon and Pachyzoidae hampers knowing whether Rhizoids are, as previously indicated, non-kenozooidal [8] and are hence cystid appendages.They are found in all pachyzoid species on the basal colony side and are essential for anchoring colonies in soft sediment.Based on histological analysis of the current study, specific musculature is present only in rhizoids of P. grischenkoi sp.nov.. Its thick cuticle also indicates that movement is likely to be restricted.Possibly hydrostatic pressure increase by polypide retraction could act in movement of the rhizoids.Pachyzoid biology remains virtually unknown, but it is highly unlikely that active colonial movement is possible.Whereas lunuliform cheilostomes have polymorphs with movable bristles [e.g.[23] and conescharellinids have a righting behavior conferred by non-muscular kenozooidal props [24], such structures and behavioral repertoires are lacking in ctenostomes.

Reproductive aspects of pachyzoids
The current study discovered the presence of some large macrolecithal oocytes in pachyzoids as well as apparent embryo brooded within the zooid, probably the tentacle sheath, in P. grischenkoi sp.nov..These characters indicate that lecithotrophy and brooding probably are a general feature of the family, similar to other ctenostomes [8].This differs from other deep-sea ctenostomes such as aethozoids [6], the genus Haywardozoon [22] and probably the genus Pierrella [25], which are zygotespawners with numerous, smaller oligolecithal oocytes.Deep-sea species of Clavoporidae, however, are also brooders (Schwaha, pers.observation).
Lecithotrophic development and associated coronate larvae are short-lived and hence are generally considered to be correlated with a reduced dispersal rate [see [26][27][28].Determination of the numbers of zooids per mature colony revealed that Jeanloupia zealandica remains rather small in size, with not more than 11 zooids.In Pachyzoon, the number of zooids per colony ranges from c. 20 in P. atlanticum, c. 50 in P. pulvinaris and almost 100 in P. grischenkoi.This implies that species with fewer zooids reach sexual maturity earlier.Since oocytes and embryos appear voluminous, it is possible that larvae result in ancestrulae with multiple zooids, which would confer greater stability on the soft sediments where they live.Although smaller fragmented parts of colonies have been observed in the current study, we have no indication whether fragmentation, as a dispersal form, is present in pachyzoids.

Distribution of pachyzoids
With the current study, we extend the distribution of Pachyzoidae over a wider geographic range to include New Zealand.The previous report of P. atlanticum from New Caledonia pertains to P. pulvinaris (see above), which gives it a wider Zealandian distribution in the Southwest Pacific.It seems that pachyzoids have endemic ranges, with P. atlanticum occurring exclusively in the North Atlantic and the other three species pertaining to the geological continent of Zealandia, which is mostly submerged [29].However, likely sampling bias clouds our understanding of the true distributional ranges of pachyzoids, since most samples from the current analyses were from similar areas, mostly southeast of South Island, where all three species may co-occur at the same station (e.g.NIWA Stn S154-see Table 1).There is a high likelihood that pachyzoids may be easily overlooked in other sample analyses or that sampling techniques may be inadequate for capturing them.
So far, all pachyzoid bryozoans are restricted to deeper water ranging from c. 600 m to over 3000 m depth, with most samples occurring around 1000 m depth.As with other deep-sea bryozoans, there is little information on their ecology, including their diet.Given the abundance encountered in the current study, there is a high possibility of finding more samples to discover other aspects of their general biology.The often camouflaged and nondescript appearance of pachyzoids renders them difficult to recognize, especially for non-bryozoologists.

Conclusions
The analysis of deep-sea samples revealed abundant colonies of pachyzoids belonging to three new species, including one new genus.This shows that the diversity of these bryozoans is much higher than previously known and that they probably constitute an important part of deep-sea ecosystems.

Fig. 1
Fig. 1 Map showing the distribution of Pachyzoidae in the Zealandian region.Latitude and longitude markers are shown at 1° intervals.The white line shows the boundary of the geological continent of Zealandia

Fig. 10
Fig. 10 Pachyzoon grischenkoi sp.nov.Reproductive details.Semithin sections.a Basal and lateral body wall showing spermatogenic tissue on the lateral wall.b Basal colony area showing ovaries with oocytes of different sizes.c Embryo inside of maternal, degenerated zooid.d Early embryo.Note also cup-shaped cuticular structures.e Detail of embryo showing high amounts of yolk.Abbreviations: bbw basal body wall, fbw frontal body wall, int intestine, l lophophore, ooc oocytes, pm parietal muscles, rm retractor muscle, te testis

Fig. 11
Fig. 11 Pachyzoon pulvinaris sp.nov.General overview.a Multiple colonies.b Two different colonies, viewed from basal side (upper) and frontal side (lower).c Detail of frontal side of a colony showing multiple apertural papillae.d Close-up of basal side showing cystid appendages.e, f Lateral view of two colonies with basal side with more cystid appendages and less-prominent apertural papillae (e) or opposite (f).g Broken colony showing frontal area with functional polypides and basal area without.h Detail of apertural papillae on frontal side.Abbreviations: app apertural papilla, cy cystid appendage, oz old zooid, po polypide

Fig. 12
Fig. 12 Pachyzoon pulvinaris sp.nov., 3D reconstruction based on histological sections.a Volume rendering of frontal colony area.b Same as (a) but with reconstructed polypide features displayed.c Close-up of polypide.d Same as (c) but from opposite side.Abbreviations: a anus, app apertural papilla, ca cardia, cae caecum, es esophagus, fbw frontal body wall, int intestine, l lophophore, ph pharynx, rm retractor muscles, v vestibulum

Fig. 13
Fig. 13 Pachyzoon pulvinaris sp.nov.Histological details. a Basal colony side with non-kenozooidal cystid appendages, unrestricted in contact with the main body cavity (arrow).Note the thick cuticle of the appendages following the remaining cuticle.b Basal colony area showing multiple layers of zooidal generations from basal to frontal, with degenerated polypides.c Apertural papilla with cross-section of vestibular wall.d Zooid with several large oocytes.Abbreviations: bb brown body, bbw basal body wall, cy cystid appendage, fbw frontal body wall, l lophophore, oz old zooid, v vestibulum, vw vestibular wall

Fig. 14
Fig. 14 Jeanloupia zealandica gen.et sp.nov.General overview.a Colony from frontal view.b Lateral view of colony.c Colony with seven zooids, frontal view.d Lateral view of two small colonies of three zooids each.e Basally broken colony showing zooidal borders and brown bodies.f Lateral view of peristome showing wrinkled surface.g Detail of frontal apertures showing rectangular orifices.Abbreviations: bb brown bodies, bbw basal body wall, o orifice, ps peristome, v vestibulum, vw vestibular wall

Fig. 15
Fig. 15 Jeanloupia zealandica gen.et sp.nov.3D reconstruction based on histological sections.Volume rendering showing elongated wrinkled peristomes on the frontal side of colony.b Same view as (a), but with volume displayed transparently and polypide details shows as surface models.c Opposite view of (a) and (b) with more transparent volume rendering.d Frontal view of the polypide.e Basal view of polypide.Abbreviations: a anus, bbw basal body wall, ca cardia, cae caecum, es esophagus, int intestine, l lophophore, ph pharynx, ps peristome, rm retractor muscles

Fig. 16
Fig. 16 Jeanloupia zealandica gen.et sp.nov.Histological details. a Longitudinal section of peristome.b Cross-section of basal area of peristome showing the collar.c Ovary with vitellogenic oocyte surrounded by follicle cells.d Oocytes in basal zooidal area.Abbreviations: bbw basal body wall, c collar, l lophophore, o orifice, ooc oocyte, ps peristome, psw peristomial wall, v vestibulum, vw vestibular wall

Table 1
Station data for Pachyzoidae from Zealandia

Table 1 (
continued) a Data from Muséum National d'Histoire Naturelle, Paris , but in pachyzoids peristomes are present only in Jeanloupia gen.nov.(see below).