Reproductive biology, embryonic development and matrotrophy in the phylactolaemate bryozoan Plumatella casmiana

Bryozoa is a phylum of aquatic, colonial suspension-feeders within the Lophotrochozoa. In the Phylactolaemata embryonic development occurs in an internal brood sac on the body wall accompanied by extraembryonic nutrition. Owing to previous contradictive descriptions, many aspects of their sexual reproduction require restudy. Consequently, this study analyses embryogenesis of the freshwater bryozoan Plumatella casmiana by serial sections, 3D reconstruction and transmission electron microscopy. Early embryos cleave and soon develop into blastulae with a small central cavity. The mesoderm forms by delamination starting from the distal side towards the proximal end. In later embryos two polypides form on the posterior side that ultimately will be covered by a ciliated mantle in the larva. Embryos increase in size during development and form temporary cell contacts to the embryo sac. Mesodermal cells of the embryo sac show signs of transcellular transport indicating that embryos are nourished by transferring nutrients from the maternal coelom towards the brood cavity. This study clarifies several details such as mesoderm formation and the onset of bud development. Embryos are connected to their respective embryo sacs by a variety of temporary cytoplasmic processes formed by both tissues during embryogenesis, including a ‘placental’ ring zone. Although ultrastructural data of these cell contacts are not entirely conclusive about their function, we suggest that embryos absorb nutrients via the entire surface. The close opposition of embryos to the embryo sac implies placentation as matrotrophic mode in phylactolaemate bryozoans, with embryo sacs acting as placental analogues.


Introduction
Bryozoans are a group of mostly colonial, suspension-feeding lophotrochozoans occurring in both marine and freshwater habitats (Ryland 1970(Ryland , 2005McKinney and Jackson 1989). Individual animals of a colony are termed zooids and are traditionally, but artificially, divided into a polypide and a cystid (Ryland 2005;Schwaha 2020a). The former comprises a ciliated tentacle crown (lophophore), the digestive tract, and associated neural and muscular tissue. The cystid is the body wall with the epidermis and a peritoneal layer termed endocyst and its cuticle, the ectocyst. Besides coloniality, the retraction process of the polypide into the cystid is one of the major characteristics of bryozoans (Mukai et al. 1997;Schwaha et al. 2020). There are two major clades of bryozoans, the Phylactolaemata, a group of sole freshwater bryozoans, and the Myolaemata (Schwaha et al. 2020). The latter comprises the exclusively marine Stenolaemata, with the Cyclostomata as the only extant clade, and the predominantly marine Gymnolaemata, which includes the most speciose and marine group of Cheilostomata that evolved from ctenostome-like ancestors (Waeschenbach et al. 2012;Taylor and Waeschenbach 2015). In contrast to cheilostome and cyclostome bryozoans, phylactolaemates and ctenostomes create a non-calcified ectocyst consisting of gelatinous or chitinous (sometimes sand-encrusted) material. Phylactolaemates, in general, make larger zooids and have a horseshoe-shaped lophophore, an epistome and a regular grid of body wall musculature (Wood 2014;Schwaha 2020b;Schwaha et al. 2020). Six or seven family-level clades belong to Phylactolaemata (Schwaha 2020b). Plumatellidae is the most speciose taxon with a branching colony morphology. Ectocysts of its members are gelatinous, particle-encrusted or chitinous. Colonies of most species are repent, but some are erect and branching (Wood 2014). Plumatellids are the most studied family and most investigations focus on Plumatella repens and P. fungosa.
Starting from pioneering research on phylactolaemate bryozoans of van Beneden (1839), Dumortier and van Beneden (1850) and Allman (1856), various aspects of their sexual reproduction and embryonic development were studied in the past (summarized in Table 1; reviewed in Mukai 1982;Reed 1991;D'Hondt 2005, Ostrovsky 2020), but data on the development from the zygote to the larva date back to the late nineteenth and early twentieth century and many aspects of phylactolaemate sexual reproduction, including fertilization, oviposition and embryonic development including mesoderm formation, remain unknown or require reinvestigation (see Hyman 1959;Mukai 1982).
Similar to many gymnolaemates (Ostrovsky 2013a, b, Ostrovsky et al. 2006, 2007, 2009a, phylactolaemates incubate their embryos in brood sacs. The embryonic development is accompanied by a temporary placenta-like structure that was previously considered as putative site of extraembryonic nutrition (Davenport 1891;Mukai 1982;Reed 1991;Hyman 1959;Ostrovsky 2013a, b;Ostrovsky et al. 2016). The contact cell zone referred to as 'placenta' forms a ring around the embryo in the genera Plumatella (Korotneff 1890, Kraepelin 1892, Braem 1897 and Hyalinella (Mukai 1982), a terminal 'placentulum'/'placenta-like neck' in Cristatella mucedo (Jullien 1890;Davenport 1891) or 'placental disc' in Fredericella sultana (Braem 1908b); its origin remains unclear, however. Also, while the term 'placenta' implies a nutritive function, these structures might simply anchor embryos inside the brood sac (Davenport 1891). One way to demonstrate that energy is being provided to the developing embryo from these tissues is through TEM showing exocytosis and endocytosis.
The present study analyses embryogenesis in the phylactolaemate bryozoan Plumatella casmiana by histological sectioning, 3D reconstruction and TEM. The main aims are to address open questions on the development of phylactolaemate bryozoans such as mesoderm formation or cell contacts during embryogenesis and its relevance for matrotrophy and extraembryonic nutrition.

Material and methods
Colonies of Plumatella casmiana (Fig. 1) were collected from the pond of the Faculty of Fisheries at the Kasetsart University, Bangkok, Thailand. Colony pieces were fixed in 2% glutaraldehyde (GA) in 0.01 M phosphate buffer (PB), pH 7.2, for 1 h and rinsed several times in PB. Samples were postfixed in 1% aqueous osmium tetroxide (OsO 4 ) for 1 h and rinsed in double-distilled water. Samples were afterwards dehydrated with acidified 2-2-dimethoxypropane (DMP) prior to embedding in Agar Low Viscosity resin (Agar Scientific Ltd, Essex, UK) using acetone as intermediate. Additional mounted zooids of sexually active Plumatella sp. from the teaching material of the University of Vienna was examined for overview images.

Sectioning and 3D reconstruction
Ribbons of serial semithin sections of 1.5-μm thickness were produced using a Histo-Jumbo diamond knife (Diatome, Biel, Switzerland) on a Leica UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany) (for details see Ruthensteiner 2008). Sections were stained with toluidine blue for 10-15 s at 60°C. Before sealing slides with a cover slip, single sections were chosen for resectioning to allow for TEM analysis. For this, excessive adhesion of sections was reduced by keeping slides on the heat plate for just a short duration until the water had evaporated.
Embryos of different developmental stages were photographed with a Nikon NiU compound microscope with a Nikon Ds-Ri2 camera (Nikon, Tokyo, Japan). Image stacks were then converted to grayscale with Photoshop CS6 (Adobe, San Jose, CA) and imported into the reconstruction software Amira 6.3 (Mercury Computer Systems, Chelmsford, USA). Images were automatically aligned using the alignment tool of Amira with some manual corrections. Structures were segmented manually with interpolation between sections. Surfaces were created with the SurfaceGen module of Amira. Optimisation of surfaces was conducted by iterative triangle reduction and smoothing steps. Snapshots of reconstructed embryos were taken with the Amira software.

Transmission electron microscopy
Utrathin sections of 60 nm were prepared by resectioning semithin sections (for details see Handschuh et al. 2013). Unsealed sections were first screened for embryos, which were afterwards isolated by cutting the borders of the section with a razor blade. Afterwards a drop of Milli-Q water or double-distilled water was placed next to the section. Sections were lifted to allow for waterflow underneath the section on one side first, followed by full detachment by subsequent mechanical lifting. The detached section was mounted on a trimmed resin block slightly larger than the section of interest. Prior to mounting of the section, a droplet of double-distilled water was placed on the block surface. Then, the detached section was transferred to the block surface with a needle. Adherence of the sections was achieved by placing the block in the oven for at least 30 min (and up to several hours) at 60°C. Afterwards, it was trimmed to a small area of interest. Subsequent ultrathin sections were placed on copper grids, contrasted with 1% uranyl-III-acetate (UA) for 8 minutes, followed by 8 min in 2.5% lead citrate (Laurylab, Brindas, France) with at least three rinses in double-distilled water after each medium. Transmission electron microscopy was performed on a Zeiss Libra 120 (Zeiss, Oberkochen, Germany). Images were taken with a post-column CCD camera (Sharp:eye TRS (2 × 2 k); Tröndle, Moorenwies, Germany) and the image SP software (Sysprogs OÜ, Kaiserslautern, Germany).

Position and formation of reproductive organs
Fertile zooids show a regular arrangement of polypide buds on the oral side of the cystid, followed by an ovary and the embryo sac towards the distal side (Fig. 2b). The ovary lies proximal of the embryo sac, which is closest to the orifice (Figs. 2, 3). The first developing female germ cells are detectable in the peritoneal layer of the endocyst. They gradually enlarge and differentiate into oocytes with a thin layer of peritoneal cells (Figs. 2c,3). Fully formed ovaries are grape-shaped, contain 8-12 mature oocytes and protrude into the coelomic cavity with the oldest (i.e. largest) oocytes situated in its proximal part (Figs. 3c,4a). The embryo sac forms slightly later than the ovary and is always closest to the orifice (Fig. 2b). The epidermal and the peritoneal layers of the endocyst are both involved in its formation (Fig. 3). Based on previous studies and for better comparability, these two maternal layers are herein designated as ectodermal layer and mesodermal layer of the embryo sac, corresponding to the epidermis and peritoneum, respectively. At the beginning of the formation of the embryo sac, the epidermal cells become highly prismatic and protrude towards the coelomic cavity to form a small internal bulge. Internally, the sac is lined by a thin peritoneum (Fig. 3). Similar to the polypide buds, the regular body wall musculature vanishes at the site of the proliferating embryo sac (Fig. 5a-d). Cell proliferation leads to an elongated embryo sac suspended in the coelomic cavity. The outer surface remains as a thin mesodermal layer of the embryo sac, the ectodermal, and internal layer remains as a thin layer in the proximal, suspended part of the sac, whereas it forms a dense plug of maternal ectodermal cells in the distal attachment area (Fig. 3d). These ectodermal cells stain less intense on the sections than surrounding cells (Fig. 4c).

Gonads
In young and more advanced ovaries, the younger/smaller oocytes are located closer to the body wall, whereas the older/larger ones are situated on the side protruding into the body cavity (Fig. 3c) each being surrounded by flattened follicle cells (Fig. 4a). Mature oocytes (see Table 2 for size) are oligolecithal with few yolk granules of various shape and size in the cytoplasm and contain a large nucleus usually with two nucleoli (Figs. 4a,d,6a,7d). Fully developed ovaries persist during embryonic development and can reach sizes similar to embryo sacs with advanced larvae. Although the ovary is often situated close to the embryo sac (Figs. 3, 4D), a direct contact was not found. Neither fertilisation nor oocyte transfer were observed in this study. Occasionally, however, single Spermatogenic tissue is usually formed simultaneously with female germ cells. It is differentiated as grape-like clusters along the funiculus (Fig. 4b). Individual sperm cells were sporadically found in the coelom.

Early stages
The earliest encountered stage was an embryo that consists of several blastomeres but lacks a blastocoel, i.e., a pre-blastula/ morula stage (Fig. 4c). The next stage was a blastula of approximately 20 cells with a small central blastocoel (Figs. 4d,5c,d,8a,9b). The embryonic cells show large nuclei with intensely stained nucleoli. In these early embryos, a thin unicellular ectodermal lining of the embryo sac still entirely surrounds the embryos in the proximal, suspended part of the sac (Fig. 4c). Starting from early blastula stages, the ectodermal cells vanish in this area (Fig. 5c, d). Consequently, the maternal ectodermal cells remain only in the upper/distal part of the brood sac, above the upper/posterior pole of the embryo as a dense cluster of large, often elongated, cells (Figs. 8,9,10,11,12). A central lumen is sometimes present in this cell mass (Fig. 10c).
The peritoneal cells that internally line the ectoderm cells of the embryo sac are cubic in shape in early stages. Nuclei of these cells slightly vary in size and amount of nucleoli.  Embryo sac formation occurs next to the ovary with its initial stage detectable by proliferation of the epidermal layer. b More progressed stage than in 'a' with the ovary and embryo sac protruding more into the body cavity of the zooid. c The oocyte number increases, and the largest and most mature oocytes are situated at the free end of the ovary towards the coelomic cavity. d Embryo sac with enclosed early embryo in the proximal part. The distal part towards the body wall is plugged by maternal cells from the epidermis. Abbreviations: b bud, e embryo, ebs embryo sac, ese embryo sac ectoderm, esm embryo sac mesoderm, mec maternal ectodermal cells, ooc oocyte, ov ovary Occasional dividing nuclei were encountered (Figs. 4c,d,5,10,11,12).
During mesoderm formation, the ectodermal lining of the embryo sac has disappeared at the proximal end. Cytoplasmic extensions of the blastomeres towards the mesoderm and the remaining ectoderm of the embryo sac are present in this developmental stage (Figs. 5d,10b,c). Some of these blastomeres are tightly adjoined to the maternal cells, although a direct cell-cell contact was not always found. The cytoplasmatic extensions appear more abundant in the proximal part of the embryo sac. In its distal part, less intensely stained cells of the maternal ectoderm show projections towards the embryo (Fig. 10d). In early stages, densely stained cytoplasmatic inclusions are present in cells of both layers of the embryo sac (Fig. 5c, d). Occasional coelomocytes are located adjacent to the embryo sac (Fig. 11a).

Mesoderm formation
The upper area of the embryo facing the maternal ectodermal cells at the attachment site of the embryo sac is referred to as posterior pole owing to the swimming direction of the fully developed larva. The opposite pole, at the proximal end of the embryo sac, is referred to as anterior pole. Mesoderm formation starts at the posterior pole of the embryo with dividing blastomeres of the blastula to form an internal layer via delamination (Figs. 4d,8b,9c,10d). This process gradually proceeds from the posterior pole of the embryo towards the anterior one (Fig. 8c). After mesoderm formation, the embryo is distinctly larger, elongated and bilayered (Table 2). At the posterior pole, the mesoderm is composed of cubic cells, whereas they are flattened in the anterior pole (Figs. 9d,11b,12). b Testes at the funiculus. Spermatids contain mostly the nucleus (insert). c Early cleavage stage with an embryo consisting of several blastomeres located at the free end of the embryo sac. The maternal ectodermal lining of the embryo covers the entire embryo (dashed line). d Oblique section of a blastula stage with commencing mesoderm formation (arrowheads indicate first mesodermal cells delaminating from the ectoderm). The maternal ectodermal lining persists (dashed line). Abbreviations: bwm body wall musculature, e embryo, ebs embryo sac, ed epidermis (ectodermal origin), ese embryo sac ectoderm, esm embryo sac mesoderm, fu funiculus; mec maternal ectoderm cells, nu nucleus, nuc nucleolus, ooc oocytes, ov ovary, ovc follicle cell, p peritoneum (mesodermal origin), sp sperm, yd yolk droplets At this stage, the ectodermal lining of the embryo sac is degenerating but remains as thin area at the posterior pole of the embryo (Figs. 8c,12a,b). The mesodermal layer of the embryo sac is unaltered compared with the previous stage. Some cells remain in close association with the embryo at the anterior pole (Figs. 8c, 11d-f).

Formation of polypide buds
At the end of mesoderm formation, both layers of the bilayered embryo start to proliferate and ingress on the posterior pole, indicating the first signs of bud formation (Figs. 6a,c,d,8d,13). The ectodermal cells of the embryo show cell divisions and together with the proliferating mesoderm (arrowhead) are present in the embryo sac ectoderm and mesoderm. d Cross-section close to the proximal end of the embryo sac. The embryo adjoins the embryo sac (arrowhead). The ectodermal lining of the embryo sac is being reduced (arrow) from its suspended end. Abbreviations: bwm body wall musculature, e embryo, ed epidermis (ectodermal origin), ese embryo sac ectoderm, esm embryo sac mesoderm, mec maternal ectoderm cells, pt peritoneum (mesodermal origin) . The ectodermal lining of the embryo sac remains at the posterior pole of the embryo up to the level of bud formation (Fig. 6b, c). At the anterior pole of the embryo, cytoplasmic extensions of its ectodermal cells form contact with the mesodermal cells of the embryo sac ( Fig. 11d-f).

Late developmental stages
Later stages show an increase in size of the embryo and the embryo sac. A comparison of the volume of the early reconstructed embryo (few cells) vs. the latest (almost fully formed larva) showed an increase of 37x in volume (Table 2). Both buds grow and differentiate within the forming larva (Figs. 8,14,15,16). The cells show no mitotic activity in later stages, and the embryo sac enlarges by flattening and elongation of its cells (Figs. 15,16). However, some mesodermal embryo sac cells in the proximal area remain voluminous (Figs.15,16a,b). The ectoderm of the embryo sac persists as few cells at its distal end (Fig. 8e). The epithelia of the anterior hemisphere of late embryos, which were not involved in polypide formation, thicken (Figs. 8e,15,  Slightly below the ring-shaped contact zone of the embryo, the body wall of the developing larva folds inwards and forms an annular groove (Figs. 8c,14c,15a,16a,b). The proximal lining of this groove represents the mantle fold, which subsequently will enlarge and grow in posterior direction, ultimately growing over and disrupting the ring-shaped contact zone. In fully formed larvae, the mantle extends over the entire length and only leaves a small larval aperture at the posterior pole (Fig. 15b). The fully formed larva has doubled in size compared with the stage of initial bud formation (see Table 2) and consists of a ciliated mantle and two enclosed ancestrular zooids.

Contacts between embryo and embryo sac
There are several contact zones between embryo and embryo sac during development. A close apposition of the embryo to the embryo sac is already present in the earliest observed stages (Figs. 4c; 5c, d) and persists until later bilayered Overview of the large thin-walled embryo sac next to the ovary with mature oocytes. The embryo occupies almost the entire cavity of the brood sac and has started to form buds in its distal region. b Ectodermal cells (asterisks) of the posterior pole of the embryo are in direct contact to the maternal ectodermal cells of the embryo sac. c Early bud formation. Ectodermal cells proliferate (arrows) and ingress, while the adjoining mesoderm thickens and surrounds the ectodermal ingression. d Establishment of contact between the embryonic ectoderm (asterisk) and the mesodermal layer of the embryo sac. The border between the ectodermal and mesodermal layer of the embryo sac is shown by a dashed line in b and c. Abbreviations: ebs embryo sac, eme embryonic ectoderm, emm embryonic mesoderm, mec maternal ectoderm cells embryonic stages (Figs. 4d,. Direct contact between the ectoderm of the embryo sac and the embryo via individual cytoplasmic extensions of the embryonic cells starts to form from blastula stages onwards (Figs. 5d,7a,c,10b,11a,b). Also, cytoplasmic extensions are formed by ectodermal cells of the embryo sac towards the embryo (Figs. 7b, 10d). Both types of connections are predominantly found in the distal part of the embryo sac. Since the ectodermal lining disappears from the proximal side of the embryo sac, the contacts between the two ectodermal layers ultimately get restricted to the posterior pole of the embryo where cytoplasmic extensions to the maternal ectodermal cells of the embryo sac form during later embryogenesis (Figs. 6b,7d,8d). Irrespective of the developmental stage of the embryo, maternal ectodermal cells in contact with the embryo possess a smaller nucleus and nucleolus when compared with embryonic cells. Regardless of the contact type, the cytoplasm of individual contact cells of the embryo sac commonly stains less intense than surrounding cells (Figs. 7a, b, 10d). Besides such ectodermal contacts, similar connections are established between early embryos (and also later stages) and the mesoderm of the embryo sac (Figs. 8c-e, 10c, 11d-f, 12D). Both, the ectoderm of the embryo and the mesoderm of the embryo sac, form cytoplasmic extensions (Fig. 11d-f).
Initially, contacts are not restricted to certain regions of the embryo but appear randomly distributed almost over the entire embryo. The mantle epithelium never shows contacts to the embryo sac. Once it has started to form, the contact areas are restricted to the posterior half of the embryo, most notably in the ring-shaped contact zone of later stages ('placenta'). There are either individual embryonic cells (Figs. 16c, e, f), or clusters of such that form the contact area by cytoplasmatic Fig. 7 Contact areas of embryos of Plumatella casmiana and the maternal ectoderm. Semithin sections. The border between the ectodermal and mesodermal layer of the embryo sac are shown by the dashed line in a-c. a Blastula stage in a bilayered embryo sac. The latter consists of a thick (outer) mesoderm and a thin inner lining of maternal ectoderm. One blastomere of the embryo is in contact (arrow) with the adjacent ectodermal cell of the embryo sac. The cytoplasm of the connected maternal ectoderm cell stains less intense than adjoining cells. b Detail of the embryo sac with a maternal ectodermal cell (arrow) in contact with the blastomere. c Detail of a blastula showing contact of a blastomere with the maternal ectoderm in the proximal area of the embryo sac. d Late embryo showing several ectodermal cells at the posterior pole of the embryo forming contact with the remaining maternal ectodermal cells (arrow). Abbreviations: e embryo, ebs embryo sac, eme embryonic ectoderm, emm embryonic mesoderm, mec maternal ectoderm cells, ov ovary extensions (Figs. 16b,c,17a,b). Similar contacts are occasionally formed with the mesodermal cells of the embryo sac. In these areas, the cells of the embryo sac are noticeably larger compared with the remaining thin lining (Fig. 16e, h). These larger cells sometimes stain less intense and can include dark inclusions (Fig. 16f).
The ring-shaped contact zone ('placenta') between embryo and embryo sac develops in the posterior half of the embryo in the proximal area of the ancestrular cystid (Fig. 6a, d). As mentioned above, the mantle fold forming proximally of this contact zone (Fig. 16a, b) will later break through this zone, disrupting direct contact between the cells of the embryo and the ES (Fig. 16c, d).

Ultrastructure of contact areas
The youngest investigated stage by TEM was a bilayered embryo (Figs. 18, 19). The ectoderm cells of the embryo are prismatic or cubic with a large basal nucleus. They contain mostly mitochondria and ribosomes, with few elements of endoplasmatic reticulum and occasional multivesicular bodies (Figs. 18a,b;19a,c). The apical side of prismatic cells have filiform cytoplasmatic processes between adjacent cells (Fig.  18a), or show regular short processes over the entire surface (Fig. 19a, c). The mesodermal cells of the embryo are smaller and contain mitochondria and ribosomes as well. Some The maternal ectoderm is in contact with some embryonic ectodermal cells (arrows). The mesoderm of the embryo sac is connected to the embryonic ectoderm via contact cells. e Late embryo with mantle fold formation. A distinct ring-shaped contact zone has formed at this stage. Abbreviations: b polypide bud, bwm body wall musculature of the maternal cystid, coc coelomic cavity, e embryo, eme embryonic ectoderm, emm embryonic mesoderm, ebs embryo sac, ese ectoderm of embryo sac, esm mesoderm of embryo sac, ed epidermis (ectodermal origin), mec maternal ectoderm cells, mf mantle fold of larval mantle, pac placenta cells, pt peritoneum (mesodermal origin). Colors: peritoneum and mesodermal layer of embryo sac, yellow; epidermis and ectodermal layer layer of embryo sac, green; ectoderm of embryo, red; mesoderm of embryo, blue; contact cells, violet Abbreviations: cnc contact cells, e embryo, eme embryonic ectoderm, emm embryonic mesoderm, esm mesoderm of the embryo sac, mec maternal ectoderm cells. Colors: Mesodermal part of embryo sac, yellow; ectodermal part of embryo sac, green; embryonic ectoderm, red; embryonic mesoderm, blue; contact cells, violet endoplasmatic reticulum is also present (Figs. 18a,c;19a). In addition, vacuoles of unknown content are found in their apical parts (Fig. 18a, d). Few irregular folds are sometimes present on the apical and basal side of these cells (Fig. 18a, d). The mesodermal cells of the embryo sac contain mitochondria, numerous free ribosomes and large swollen cisterns of endoplasmatic reticulum filled with flocculent material, the same which is present in large vesicles situated close to the apical cell membrane. Some of the vacuoles secrete their contents into the cleft between embryo and embryo sac (Fig.  19a, b). Apart from the vacuoles, the apical side of the cells form thick protrusions, sometimes with vesicles ( Fig. 19a) that in some cases were found to interdigitate with processes of the embryo (Fig. 19c, e).
In a more advanced stage, at the onset of bud formation, a close association of the embryonic and maternal tissues remains (Fig. 20). The ectodermal cells of the embryo have numerous cytoplasmic extensions of various shapes and sizes from their apical surface, some approaching the cells of the embryo sac. Cells of the latter also show numerous outgrowths towards the embryo that were thicker than the embryonic ones. The cells show prevalent euchromatin in the nuclei, high amount of endoplasmatic reticul um , some autophagosomes and numerous vesicles of various sizes (Fig. 20). Some of the latter are in open contact with the lumen of the embryo sac to secrete flocculent content (Fig. 20a, c, d). The side facing the maternal coelomic cavity shows a high amount of numerous small, pit-like invaginations on the membrane, potentially indicating endocytosis (Fig. 20b, e). The cytoplasm close to these vesicle formations contains numerous vesicles of various sizes, mitochondria and large multivesicular bodies (Fig. 20c, e).

Gametes, fertilization and zygote transfer
In the studied phylactolaemates, early female germ cells differentiate from the peritoneal layer (Metschnikoff 1871;Korotneff 1890;Davenport 1891;Kraepelin 1892;Braem 1897Braem , 1908b. At first, they are not distinguishable from other peritoneal cells until their nucleus enlarges and nucleoli become visible when differentiating into oogonia (Brien 1953). These grow to a cluster of oocytes with the oldest being closest to the proximal, suspended part of the developing embryo sac. The oocytes are surrounded by flattened follicle cells that together form the ovary (Reinhard 1881;Kraepelin 1892;Braem 1897;Mukai 1982). Whether a complete follicular envelope is present around each oocyte (as indicated from our sections) requires additional TEM analysis but was at least reported absent in Asajirella gelatinosa (Tazima et al. 1984). In addition, nurse-cells were reported being associated with growing oocytes in Lophopus crystallinus (Marcus 1934).
Sperm in phylactolaemates was detected as early as in 1839 by van Beneden, and several studies focused on spermatogenesis and sperm structure (see Table 1). The spermatogenic tissue is associated with the funiculus in Plumatella casmiana as in most phylactolaemates (Wood 2014). In most bryozoans, mature sperm leaves the coelom through pores at the tips of the tentacles (e.g. Okamura and Hatton-Ellis 1995;Ostrovsky 2008Ostrovsky , 2013bOstrovsky and Porter 2011). These pores are known from phylactolaemates (e.g. Marcus 1934Marcus , 1941, but whether they are used for sperm release has not been confirmed. Release of mature sperm was detected via a vestibular pore (Oda 1958;Wiebach 1953), which could also act as route for the sperm from other colonies (Ostrovsky and Porter 2011). Genetic fingerprinting of Cristatella mucedo has shown that outcrossing occurs (Jones et al. 1994;Freeland et al. 2003) although experimental work showed that selffertilization is possible too (Oka and Oda 1948).
Fertilization is internal in Phylactolaemata similar to other bryozoans (Reed 1991;Ostrovsky 2008). We did not find any signs of fertilization in the current study, but sperm had been previously recorded in the ovary of Plumatella (Korotneff 1890;Kraepelin 1892;Braem 1897) and male pronuclei have been found in Lophopus crystallinus oocytes (Marcus 1934). Fertilization has been described to occur in the embryo sac In some areas it has tight contact to the dissolving embryo sac ectoderm (arrowhead). c Embryo sac showing voluminous cells of the embryo sac mesoderm (asterisks) and an almost completely reduced ectodermal layer. The ovary is in tight association with the embryo sac with apparent contacts (arrows). d Ectodermal cell of the embryo showing extension towards the mesoderm of the embryo sac (arrow). Abbreviations: b polypide bud, eme embryonic ectoderm, emm embryonic mesoderm, ese ectoderm of embryo sac, esm mesoderm of embryo sac, mec maternal ectoderm cells, ov ovary (Marcus 1934) or in the ovary (Korotneff 1890), the latter b e i n g p r e s e n t i n m o s t a n d a l s o a l l o t h e r incubating bryozoans (Ostrovsky 2008).
Transfer of the zygote from the ovary into the embryo sac was hypothesized in two ways: (1) Amoeboid movement of the fertilized oocyte from the ovary into the embryo sac (Korotneff 1890;Brien 1953). This may involve zygotes to be circulated within the body cavity prior to implantation into the embryo sac (Metschnikoff 1871;Marcus 1934). Support for this mode of transfer by the present study is the presence of occasional free oocytes encountered in the body cavity.
(2) Direct transfer by the close apposition of the embryo sac with the ovary. By direct contact of these two tissues, the fertilized oocyte would be subsequently moved into the embryo sac (Braem 1897(Braem , 1908b. Although a close association of both structures has been described by other authors and has also been encountered in the present study, their direct apposition has not been confirmed. In some ctenostome bryozoans, ovulated oocytes are released via a supraneural coelomopore to be attached to the outer body wall and subsequently immersed into a brood sac similar to phylactolaemates (Braem 1951;Ström 1977). This mode was never observed in phylactolaemates, and internal oviposition was considered as ancestral (Silén 1944). There are many issues still requiring additional analyses for clarifying fertilization and the original mode of brooding.

Embryo sac
The phylactolaemate embryo is incubated within an embryo sac at the cystid wall, contrary to earlier observations stating that a ciliated embryo develops inside the ovary (Allman 1856; Reinhard 1881) because of its monolayered wall (see also Kraepelin 1892;Marcus 1934). Zygotes are incorporated in a double-layered structure, the embryo sac, which is reminiscent of polypide buds. The embryo sac (sometimes termed 'ooecium' in older literature, analogous to a functionally similar structure in marine bryozoans) was accordingly considered as modified polypide bud by some authors (Nitsche Alternatively, the embryo sac was considered as independent structure that develops via local thickening of the body wall involving cell enlargement and seemingly proliferation (Korotneff 1887a) which corresponds to the data of Brien (1936Brien ( , 1953 and our data. In contrast, embryo sac formation was also described as invagination of both layers of the cystid wall in P. fungosa and P. casmiana (Mukai 1982). The latter could not be confirmed by our current study. In general, development and formation of the embryo sac after oviposition is similar as previously described (Kraepelin 1892;Braem 1897;Marcus 1934;Brien 1953).
Internal brood sacs formed by non-calcified body walls are widespread among gymnolaemate bryozoans and evolved independently several times in cteno-and cheilostomes (Ostrovsky 2013a;Ostrovsky et al. 2006Ostrovsky et al. , 2009a. In some species of both clades, these sacs provide extraembryonic nourishment (Ostrovsky et al. 2009c(Ostrovsky et al. , 2016Schwaha et al. 2019), similar as found in phylactolaemates. Incubation chambers were considered homologous among the different bryozoan clades, with the phylactolaemate one (referred as 'embryonary') considered ancestral (Silén 1944). The distribution of reproductive patterns (including modes of fertilization, oogenesis and larval types), however, indicates that embryonic incubation, and also internal brood sacs, evolved independently in all large clades (Ostrovsky 2013a). In several ctenostomes, brood sacs are formed as invagination of the body wall that includes a zygote that is attached to the cystid after release by the polypide (Braem 1951). In contrast, brood chamber formation in cheilostomes is supposedly triggered by the development of the ovary (Silén 1945;Ostrovsky and Schäfer 2003). This is similar to phylactolaemates where an embryo sac only forms after the ovary has started to form.

Embryonic development
C o m p a r e d w i t h o t h e r b r y o z o a n s , c l e a v a g e i n phylactolaemates is generally regarded as derived and little studied. It is holoblastic and irregular and results in a morula based on serial semithin sections. a Bilayered embryo within thin embryo sac. The first bud forms from both embryonic layers in the posterior hemisphere. Mesoderm invagination and ectoderm ingression take place simultaneously. b Bilayered embryo with two buds developing next to each other. The first (older) bud is cup-shaped (to the right); the mesoderm of the second bud starts to invaginate and is accompanied by proliferation of the ectoderm. c Late embryo with both buds protruding from the posterior pole. The first bud is slightly larger than the second one. The mantle fold has formed halfway along the longitudinal axis. Abbreviations: es embryo sac, ecd ectoderm, eme embryonic ectoderm, emm embryonic mesoderm, mf mantle fold, msd mesoderm. Colors: blue, mesoderm of embryo; red, ectoderm of embryo; yellow, embryo sac followed by a hollow blastula (Allman 1856, Metschnikoff 1871Korotneff 1890;Kraepelin 1886Kraepelin , 1892Braem 1897Braem , 1908bMarcus 1934, reviewed in Reed 1991D'Hondt 2005;Santagata 2015). Development of Plumatella casmiana is similar to previous reports on phylactolaemates, but we found differences concerning the formation of various tissues.
Mesoderm formation in Plumatella fungosa starts approximately between the 20-30 (Brien 1953) or the 30-36 cell stage (Braem 1897), the former number being supported by our data on P. casmiana with approximately 30 cells. Its differentiation from the ectoderm starts from the posterior pole of the embryo as previously described (Kraepelin 1892;Braem 1897;Mukai 1982) but occurs via delamination instead of unipolar immigration/ingression of the prospective mesodermal cells into the blastocoel with subsequent alignment to the ectoderm (Korotneff 1887a, b;Davenport 1891;Kraepelin 1892;Braem 1897). Delamination of mesodermal cells occurs along the longitudinal axis of the embryo, from posterior to anterior. An endoderm is absent throughout development, although temporary endodermal cells were described that immigrate into the blastocoel at the posterior pole in P. fungosa (Braem 1897). These short-lived cells were neither found in subsequent studies (Mukai 1982) including the present one. Similar blastocoel-occupying cells were also described in mesoderm formation that either fill most of the blastocoel (Kraepelin 1892) or divide it into two parts (Braem 1897;Mukai 1982). Similar to the supposed endodermal cells, these could not be verified (Brien 1953, this study).
Most phylactolaeamates, including Plumatella casmiana, possess two polypide buds in the larva. Only Cristatella and Pectinatella have four, whereas Fredericella has only one (Schwaha et al. 2015). In accordance with previous descriptions (e.g. Braem 1897), bud formation starts when mesoderm formation is almost completed. During bud formation in P. casmiana, the embryonic ectoderm initially proliferates and ingresses towards the mesoderm, which then invaginates and proliferates as well. This differs from previous descriptions according to which both embryonic tissues simply thicken and fold inwards as in asexually produced buds (Braem 1897).

Embryo-maternal contact
A contact area between embryo and embryo sac, in form of the ring-shaped contact zone, was recognized by numerous authors and termed 'placenta' (Kraepelin 1886;Korotneff 1890;Braem 1897). This term suggests its nourishing function (Davenport 1891;Brien 1953;Hyman 1959;Mukai Fig. 15 Longitudinal sections of brooded larvae of Plumatella casmiana. a. The embryo sac is conspicuous. The larval mantle is ciliated (arrowhead) and in earlier stages restricted to the anterior pole of the larva. Halfway along the longitudinal axis the mantle forms a deep groove (arrows). Two almost differentiated buds are present in the larva. b Fully developed larva entirely surrounded by the mantle . The embryo sac is even less pronounced but still contains some larger cells. Abbreviations: b bud, cc coelomic cavity, dg digestive tract, ebs embryo sac, la larval aperture, l lophophore, lm larval mantle 1982), but its function as anchoring structure that holds the embryo in a certain position in the embryo sac was proposed as an alternative scenario (Kraepelin 1892;Braem 1897).
Elongate cylindrical cells, supposedly of embryonic and embryo sac origin, were described to form the placental ring in P. fungosa (Korotneff 1890). Initially, cell contacts to ectodermal cells of the embryo sac are present at the posterior pole that subsequently enlarge and elongate and make contact with the mesodermal cells of the embryo sac (Braem 1897). During further development, the number of embryonic contact cells increases and the placental ring-shaped contact zone forms in the equatorial region of the embryo (see also Kraepelin 1892). Previous studies on P. casmiana reported a cap of embryonic ectodermal cells on the posterior pole that participates in the formation of the placental ring (Mukai 1982), for which no evidence was found in the current study.
In Fredericella sultana individual cell contacts were described in posterior and anterior areas of the embryo (Braem 1908b).
The current results on P. casmiana show a more complex situation: before the formation of the placental ring, the embryo and the embryo sac establish temporary individual contacts. First indications of such contacts start at the blastula stage when blastomeres form cytoplasmic extensions towards mesodermal and ectodermal cells of the embryo sac. In addition, some cells of the maternal ectoderm form projections towards the embryo in the distal part of the embryo sac (see also Braem 1897). The function of these temporary individual contacts remains unclear. These randomly distributed contact cells are much later replaced by the ring-shaped contact zone (placenta). During early bud formation, embryonic cells at the anterior pole form individual cytoplasmic extensions towards the mesodermal cells of the embryo sac. The latter form similar processes towards the embryo. When the embryo forms the second bud, several neighbouring cells on the posterior pole of the embryo form cytoplasmic extensions towards the maternal ectodermal cells. Such a prominent contact of the is not yet ciliated and a mantle fold is present in the equatorial region. Posterior of the mantle fold, the ring-shaped contact zone ('placenta') of elongated cells of the larval ectoderm is in contact with the mesoderm of the embryo sac (partly seen in a). The embryo sac is rather thin but still includes voluminous cells (arrowheads), some with cytoplasmatic inclusions (arrows). c, d Oblique cross section of an advanced larva showing contact areas of the larva to the cells of the embryo sac. The ringshaped 'placenta' is disrupted where the mantle fold overgrows the larva in posterior direction. e-i Contact cells (asterisks) in early larvae. These cells are either formed by cytoplasmatic extensions of embryonic/larval cells (e, f), or their origin is unclear (g, h). In some cases they appear to belong to the embryo sac (i). Abbreviations: b bud, e embryo/ larva, ecd ectoderm of the larva, ebs embryo sac, ma larval mantle, msd mesoderm of the larva, mf mantle fold, pac placenta cells maternal and embryonic ectoderm was not previously described in such late developmental stages. In general, contacts of the maternal and embryonic ectoderm establish earlier and persist longer than previously described. Cell extensions are not formed over wides areas, but rather individual cell-cell contacts, thus termed contact cells here.
Groups or individual embryonic cells form the large ringshaped contact zone, termed 'placenta' by previous authors, in P. casmiana, but it is not entirely clear whether contacts are solely formed from the embryonic side or whether cytoplasmic extensions of the mesodermal cells of the embryo sac contribute as well. In other phylactolaemates, the 'placenta' or contact zone was described to consist of both 'fetal' and 'uterine' cells in Fredericella sultana (Braem 1908b). In Cristatella mucedo, the so-called 'placentulum' was also mentioned as fusion of maternal and embryonic cells (Jullien 1890). A nutritive function of the latter was suggested (Davenport 1891, as 'placenta-like neck of the ooecium'). Lophopus crystallinus appears to lack any large contact zone between embryo and embryo sac altogether (Marcus 1934). Although generally similar, reports of these particular contact structures and zones are commonly based on single, and old, observations. It would be necessary to obtain material from several, sexually active colonies of different, families and to apply more recent methods such as used in this study in order to get a better insight into variation, structure and function of these contact zones. Establishment of the ring-shaped contact zone of the bilayered embryo by ectodermal cells of the embryo (arrows) that connect to the mesoderm of the embryo sac. Both layers of the embryo are thickened on the posterior side towards the body wall, indicating early bud formation. c, Contact cells forming at random locations in the anterior hemisphere of an early embryo (a, b). Abbreviations: e embryo, ebs embryo sac, mec maternal ectoderm cells

Ultrastructural evidence of matrotrophy
The term matrotrophy defines parental, extra-vitelline nutrition during offspring gestation. Different variants of matrotrophy evolved independently in more than 20 animal phyla including bryozoans (Ostrovsky et al. 2016). Its most elaborated form is placentotrophy with a placenta referred to as an intimate apposition or fusion of embryonic and maternal tissues providing substance exchange (Mossmann 1937). During incubation, a prominent increase in embryonic size and the formation of the temporary structures by the parent and/or embryo are indicative of matrotrophy (Ostrovsky et al. 2016). Embryonic enlargement during development accompanied by embryo sac growth were previously described in phylactolaemates (e.g. Davenport 1891), but not recognized as indication of matrotrophy. If contact cells or zones lack any specific nourishing function, embryos probably absorb nutrients via the entire ectodermal surface. In contrast, if intracellular transport does occur in the cells providing contacts between the embryo and the embryo sac, they could be considered as some sort of placental analogue.
Cytological details show that the mesodermal cells of the embryo sac have largely convoluted surfaces and include numerous vesicles that empty their flocculent content into the space between the embryo sac and the embryo in Plumatella casmiana. Similar mechanisms have been shown for the Fig. 18 Ultrastructure of the anterior hemisphere of a bilayered embryo and embryo sac shortly before bud formation in Plumatella casmiana (TEM). a Overview of embryonic epithelia and embryo sac. The embryo shows high prismatic ectoderm and flattened mesodermal cells. Ectodermal cells possess filiform extensions on their lateral sides. b Detail of an embryonic ectodermal cell showing filiform processes, mitochondria, numerous free ribosomes and a multivesicular body in the apical part of the cell. c Detail of a mesodermal cell of the embryo sac. In contrast to the embryonic cells, it shows a lower density of ribosomes, but much more vacuoles and endoplasmatic reticulum. Presumed site of exocytosis towards the lumen of the embryo sac is marked by an arrow. d Detail of the mesoderm of the embryo including large vacuoles with unknown content (asterisks). Arrow shows an extension of the cell membrane towards the basal lamina. Abbreviations: bl basal lamina, eme embryonic ectoderm, emm embryonic mesoderm, er endoplasmatic reticulum, esm mesoderm of the embryo sac, mi mitochondria, mvb multivesicular body, nu nucleus, nuc nucleolus, ri ribosomes placental analogue of a matrotrophic cheilostome (Moosbrugger et al. 2012). The internal side of the embryo sac facing the body cavity also shows surface increasements in the form of infoldings in P. casmiana and is probably a sign of  b Mesodermal cell of the embryo sac facing the maternal coelom and showing several pits (arrows) that presumably indicate endocytosis. c Large autophagosomes, one in the cell of the embryo sac and another located in the brood cavity. d Vesicle of a cell of the embryo sac (to the left) emptying its contents into the space between the embryo sac and the embryo. The embryonic cell shows a presumed endocytotic vesicle (wide arrow). e Embryo sac cell showing highly increased surfaces facing the maternal coelom and embryo sac lumen. Abbreviations: as autophagosomes, er rough endoplasmatic reticulum, mi mitochondria, mvb multi-vesicular body, nu nucleus, ve vesicles endocytosis. In addition, the abundance of mitochondria, endoplasmatic reticulum and multivesicular bodies indicates that these cells are synthetically active and probably transport nutrients from the body cavity to the embryo sac lumen. A similar condition was recently shown in the placental analogue of cyclostome bryozoans (Nekliudova et al. 2021). Autophagosomes in the cells of the embryo sac are indications of membrane recycling as found in other matrotrophic bryozoans (Moosbrugger et al. 2012;Nekliudova et al. 2019). In matrotrophic bryozoans, the embryo receives nutrients from the brood sac lumen and shows an increase of its surface and signs of endocytosis in several bryozoans (Woollacott and Zimmer 1975;Hughes 1987;Moosbrugger et al. 2012;Schwaha et al. 2019;Nekliudova et al. 2019). Indications for endocytosis were rarely encountered in the current study from embryos of P. casmiana, but their outer surface is increased by abundant filiform cytoplasmic processes that face the embryo sac lumen and are probably involved in nutrient uptake.
In contrast to most matrotrophic gymnolaemates ( Woollacott and Zimmer 1975;Hughes 1987;Moosbrugger et al. 2012;Ostrovsky 2013a, b;Nekliudova et al. 2019), developing phylactolaemate embryos are completely surrounded by cells of the embryo sac once incubated. In most areas, the surface of the embryo is situated very close to the nutrient-providing cells of the embryo sac, which is characteristic of placental analogues of various animal clades (Ostrovsky et al. 2016). Uptake of nutrients in phylactolaemate embryos occurs via absorbotrophy (i.e. histotrophy) from the embryo sac cavity, with the embryonic external epithelium being in close contact with the embryo sac. This merits the terms placentation with the entire embryo sac wall acting as placental analogue. Histotrophy is thus an equivalent to placentotrophy in this case.
No implantation of the embryonic cells in the embryo sac was detected during our study (see also Ostrovsky 2013b). In Plumatella casmiana, cytoplasmic processes of embryonic cells in the contact zone are often surrounded by mesodermal cells of the embryo sac that are larger and thicker. These cells show different staining properties and have dark inclusions. So far there are no direct indications of intercellular transport between these cytoplasmic processes. Ultrastructural details of the ring-shaped contact zone are required to recognize its function since it is also possible that its cells have a nourishing function in later development.

Evolution of sexual reproduction in Phylactolaemata
Most phylactolaemates produce numerous small oligolecithal eggs (although just one becomes an embryo) which are reminiscent to the ancestral condition characteristic of zygotespawning of (mostly marine) gymnolaemates with planktotrophic larvae (Ostrovsky 2013a). The origin of embryonic incubation could be a prerequisite for the evolution of matrotrophy. The combination of numerous small oocytes and (presumably) matrotrophic incubation in an embryo sac associated with the cystid wall is known, for example, in the enigmatic bryozoan Labiostomella gisleni (Silén 1944;Ostrovsky 2013a, b). The transition to nourished embryos and non-feeding, short-lived larvae appears most likely. Among phylactolaemates, there are additional modifications from this reproductive mode with multiple oligolecithal oocytes and matrotrophic incubation: in Fredericella the amount of oocytes is reduced to very few. Plumatella evelinae possibly has mesolecithal oocytes instead of oligolecithal ones, and Lophopus crystallinus has putative nurse cells.

Conclusions
This study provides the first detailed analysis of the embryonic development of a phylactolaemate bryozoan, Plumatella casmiana, using 3D-reconstruction of several developmental stages. The mesoderm forms via delamination, not by unipolar immigration as previously described. Temporary contacts between the embryonic and maternal cells are abundant. These are largely formed by extensions of the embryonic ectodermal cells towards cells of the embryo sac, but few mesodermal cells of the brood sac also form such extensions. A ringshaped contact zone forms in late embryos, but its definitive function remains to be revealed. Ultrastructural details of the mesodermal cells of the embryo sac indicate transcellular transport presumably delivering nutrients from the coelom to the embryo sac cavity. Embryonic cells with filiform extensions are closely opposed to the wall of the embryo sac, which indicates placentation.