Comprehensive review of previously published data
As a prerequisite for attempting to identify the specimens originally presented by Boletzky (1978–1979, 1980, 1982, 1985) as well as those introduced here (Specimens A–F and G–R, respectively), previously published data on cirrate egg dimensions, structure, and color were assembled and taxonomically arranged (Table 2). These data on cirrate eggs were differentiated by their location within the female reproductive tract, i.e., inside the ovary (ovum without or with chorion), the proximal oviduct (ovum with chorion), or the distal oviduct (ovum with chorion and egg capsule), and additionally following spawning (ovum with chorion and egg capsule). Furthermore, information on geographic and bathymetric distribution were added. Particular care was taken to identify those taxa, where a considerable gap between chorion and egg capsule would be present following spawning, a feature potentially specific of certain cirrate taxa and first observed by Boletzky (1982). However, more recent observations show that egg capsule size may increase when the freshly secreted egg capsule material comes into contact with seawater and subsequently solidifies (Vecchione et al., 1998).
Table 2 Comprehensive list of ovum, chorion, and egg capsule dimensions as well as characteristics differentiated by location within the female reproductive tract—data on species distribution obtained from Hochberg et al., (1992, 2014) and Collins and Villanueva (2006), values given in millimeters No spawned egg capsules have yet been documented for any species from the bathy-to-abyssopelagic taxa Cirroteuthis, Cirrothauma, and Stauroteuthis (Cirroteuthidae). However, ova with chorion and surrounding egg capsule are reported from the distal oviduct of several female specimens and here the egg capsules are medium in size, measuring from about 11 × 6 to 14 × 9 mm (Table 2). Where known, these egg capsules show a rather smooth external aspect and a brown color. Taking chorion dimensions from ovary and proximal oviduct into account, the gap between chorion and egg capsule must be very small.
In the mesopelagic to hadal Grimpoteuthitidae (Cryptoteuthis, Grimpoteuthis, Luteuthis), data on eggs are available for two of the three genera. While egg characteristics are unknown for Cryptoteuthis, species in the other two genera produce large egg capsules, measuring from about 18 × 11 to 26 × 17 mm (Table 2). However, egg capsules in Luteuthis are reported to possess a smooth external structure with light color, while those of Grimpoteuthis are showing irregular structuring with a muddy brown to greenish color. Recently, an egg capsule measuring 19 × 14 mm attached to a large colony of the deep-sea octocoral (Cnidaria: Octocorallia) species Chrysogorgia artospira (Alcyonacea: Chrysogorgiidae) was unequivocally shown to be a representative of the genus Grimpoteuthis (Shea et al., 2018). As the particular behavior of using octocorals as substrate for egg capsule deposition has not been documented from other cirrate taxa yet, it must at present be assumed to be a specific feature of selected or potentially all species pertaining to that particular genus. Where available, the data suggest the presence of a large gap between chorion and egg capsule, at least in freshly spawned specimens (Table 2).
Species in the meso-to-bathypelagic Cirroctopodidae (Cirroctopus) produce large egg capsules ranging in size from about 16 × 10 to 22 × 14 mm. The external structure of the egg capsule has been reported as smooth to irregular with beige or tan color (Table 2). However, ova with chorion and surrounding egg capsule found free in the mantle cavity of a single female specimen of Cirroctopus hochbergi showed significant variation in size (ca. 9 × 5 to 16 × 10 mm), external structuring (smooth, irregular, circular or longitudinal ridges, circular lines), as well as color (tan or khaki). While such a degree of egg capsule variation has not yet been documented from any other cirrate species, this finding could indicate that caution has to be taken when using egg capsule characteristics as taxonomic proxies (O'Shea, 1999). Nonetheless, the available data suggest that there is a small or even significant gap between chorion and egg capsule (Table 2).
In the epi-to-bathypelagic Opisthoteuthidae (Opisthoteuthis) data on egg characteristics are available for several species (Table 2). In general, the egg capsules are small to medium in size, measuring from about 7 × 4 to 14 × 9 mm. Externally, the egg capsule is smooth with little or significant irregular ornamentation and white to brown color. Based on chorion dimensions from ovary and proximal oviduct, the gap between the chorion and egg capsule must be very small.
These findings for all four cirrate families result in a matrix using egg capsule dimensions, presence of a gap between chorion and egg capsule, as well as geographic and bathymetric distribution as key features for the identification of cirrate eggs collected without an associated adult specimen. In the following section, these key features as well as other data will be used to identify a broad range of early cirrate life stage specimens down to at least genus level.
Specimen A
Description
Collected in 1974 on the Meriadzec Terrace, northeastern Atlantic Ocean at 2860 m depth during cruise BIOGAS 5 (Table 1, Fig. 1). The size of the partially ruptured egg capsule is estimated to be around 13 × 10 mm, its external structure shows high and sharp ridges arranged in a hexagonal pattern, the color is brown (Fig. 2a). The chorion measures about 12.5 × 9.5 mm, leaving practically no gap towards the surrounding egg capsule. At about 10 mm length, the large external yolk sac almost entirely fills the internal volume of the chorion. A small embryo can be seen lying on top of the external yolk sac. Its dimensions are: 5 mm total length (TL), 4 mm mantle length (ML), 2.5 mm mantle width (MW), 3 mm fin length (FL), 2 mm fin width (FW), and 1 mm arm length (AL). The primary web has not yet formed, the arm tips are rounded, no cirri are visible, but sucker rudiments can be discerned.
Remarks
Based on egg capsule dimensions, absence of a gap, as well as locality and bathymetry, this specimen is identified as a representative of the Cirroteuthidae, most likely Cirrothauma murrayi.
Specimen B
Description
Egg capsule obtained in 1974 on the Biscay Abyssal Plain, northeastern Atlantic Ocean from 4715 m depth during cruise BIOGAS 6 (Table 1, Fig. 1). The egg-shaped egg capsule measures 12 × 9 mm and has a coarse surface with inconspicuous longitudinal striations and beige color (Fig. 2b). As in the previous specimen, the chorion with about 11.5 × 8.5 mm almost entirely fills the inner volume of the egg capsule, thus leaving no gap between the two structures. An embryo can be seen lying on top of the large external yolk sac that at about 11 mm length fills out almost the entire inner volume of the chorion (Fig. 2b). The extracted embryo is measured at 6.5 mm TL, 5 mm ML, 3 mm MW, 3 mm FL, 2 mm FW, 1.5 mm AL, and 0.8 mm funnel length (FuL). No primary web and cirri are present yet, but sucker rudiments can be seen lining the arms with their rounded arm tips (Fig. 2c). The funnel is prominent, but no funnel organ is visible. The translucent mantle and fin tissues permit discerning several internal structures, such as a simple (i.e., unilobular) internal yolk sac, elongated gills, branchial hearts, the distal part of the intestine, and elongated fin cartilages with pointed ends (Fig. 2c).
Remarks
The key features of the egg capsule (dimensions, absence of a gap, locality, and depth) and embryo (gill shape) lead us to assigning this specimen to the Cirroteuthidae, presumably Cirrothauma murrayi.
Specimen C
Description
This specimen was collected in the Laccadive Sea, Indian Ocean during cruise GALATHEA II (1950–1952) at 4425 m depth (Table 1, Fig. 1). The elongated, almost elliptical egg capsule measures 24 × 11 mm and has an irregularly structured and coarse surface with beige color as well as a slightly deformed apical area (Fig. 2d). The chorion is about 23 × 10.5 mm in size and contains a large external yolk sac measuring 14 × 9 mm as well as an attached embryo. The embryo measures 11 mm TL, 10 mm ML, 6 mm FL, 4 mm FW, and 1 mm AL. The eyes are small and covered by the short, reflected arms with their rounded arm tips (Fig. 2d). A primary web and cirri have not formed yet, while sucker rudiments are already present. The semi-transparent mantle tissues reveal the presence of a U-shaped shell as well as elongated fin cartilages with rounded ends.
Remarks
Based on egg capsule dimensions, presence of a U-shaped shell as well as locality and bathymetry, this specimen is identified as a representative of the genus Grimpoteuthis. While the presence of a large gap between chorion and egg capsule was shown to be a key feature of this taxon (Table 2), this particular character might only be present for a given time during ontogeny (see specimens H and M below).
Specimen D
Description
Egg capsule collected 1950–1952 on the Hikurangi Plateau, southwestern Pacific Ocean at 4520 m depth during cruise GALATHEA II (Table 1, Fig. 1). At 12.5 × 8 mm, this egg capsule has an egg-shaped form and shows a fairly smooth external structuring with faint longitudinal striations predominantly at both ends as well as a brown color (Fig. 2e). The embryo found inside has 5 mm TL, while the large external yolk sac is 7.5 mm in length (not figured). The embryo's fins are well developed, but no primary web has formed yet. The arms are short and have rounded tips, but they do not show any cirri.
Remarks
Taking bathymetry, locality, as well as egg capsule characteristics and dimensions into consideration, this specimen is identified as a member of the Cirroteuthidae, most likely Cirrothauma.
Specimen E
Description
Collected 1950–1952 during cruise GALATHEA II on the Hikurangi Plateau, southwestern Pacific Ocean at 4625 m depth (Table 1, Fig. 1). Oval egg capsule measuring 12.5 × 8 mm with a rough and sandy surface and beige color. The chorion is 12 × 7.5 mm large and there is practically no gap between chorion and egg capsule (Fig. 2f). The external yolk sac measures 7 × 5 mm with almost the remainder of the volume inside the chorion being occupied by the embryo. The embryo has the following dimensions: 11 mm TL, 9 mm ML, 5 mm FL, 3 mm FW, 2 mm AL, and 0.8 mm eye diameter (ED). The primary web has not formed yet and cirri are absent, too. The arms have rounded tips and show sucker rudiments (Fig. 2f).
Remarks
Based on egg capsule dimensions, absence of a gap, as well as locality and collection depth, this specimen is identified as a representative of the Cirroteuthidae, most likely Cirrothauma.
Specimen F
Description
Egg capsule obtained from 1218 m depth on the Kerguelen Plateau, southern Indian Ocean in 1975 during cruise MD04/BENTHOS (Table 1, Fig. 1). The egg capsule measures 16 × 9 mm and externally has numerous longitudinal striations and a brown color (Fig. 2g). The chorion is much smaller, measuring 11 × 6 mm—the gap between chorion and egg capsule is filled with a gelatinous matter (Boletzky, 1982). Inside the chorion, a small external yolk sac of ca. 5 × 4 mm size can be seen, attached to the yolk sac is an embryo (Fig. 2g). The embryo measures 5.5 mm TL, 4 mm ML, 3 mm MW, 2.5 mm FL, 2 mm FW, 1.5 mm AL, 0.9 mm ED, and 0.5 mm FuL. Its primary web has not yet formed, the arm tips are rounded, and there are no cirri yet, but sucker rudiments can be seen (Fig. 2h). Notable are the posterior edges of the fins, which are continuous with the posterior mantle end as well as the fact that the eyes are not yet covered by the corneal skin. In addition, the funnel organ is already visible at this stage.
Remarks
Using egg capsule dimensions, presence of a large gap, fin and mantle morphology as well as locality and bathymetry as relevant characters, this specimen is identified as a representative of the genus Cirroctopus.
Specimen G
Description
This single egg capsule was attached to the wall of an aquarium by a female cirrate (Opisthoteuthis sp.) collected alive from the Monterey Canyon, northeastern Pacific Ocean in 1992 at 200–400 m depth (Table 1, Fig. 1). The elongated egg capsule measures 10 × 4 mm and is predominantly smooth with occasional sediment particles embedded into its exterior (Fig. 3a). The egg capsule has a beige color and tightly encloses a chorion measuring 6 × 3 mm (Fig. 3b). There is no gap between chorion and egg capsule as well as no sign of an embryo.
Remarks
Based on egg capsule dimensions and absence of a gap as well as sampling locality and depth, this specimen is identified as pertaining to a representative of the genus Opisthoteuthis, potentially O. californiana. However, unpublished data as well as adult specimens collected by Frederick G. Hochberg indicate the presence of a yet undescribed species of Opisthoteuthis along the California coastline (Daniel Geiger, personal communication).
Specimen H
Description
Collected in 1972 during cruise Y7210A on the Tufts Abyssal Plain, northeastern Pacific Ocean at 3900 m depth (Table 1, Fig. 1), this egg capsule measures 19 × 12 mm. It has an elongated egg-shaped form, irregular external structuring, and a muddy greyish color (Fig. 3c). At 12 × 8 mm the chorion is much smaller than the egg capsule and contains a large external yolk sac that almost entirely occupies its inner volume. No sign of an embryo is found (Fig. 3c).
Remarks
Given egg capsule dimensions and structuring, the presence of a large gap, and locality as well as bathymetry, this specimen is identified as pertaining to a species within the genus Grimpoteuthis. More specifically, this egg capsule is very likely either from an adult of G. bathynectes or G. tuftsi, as these two species have been found to inhabit the exact area—in fact, during the same haul on cruise Y7210A, an adult male specimen of G. tuftsi (SBMNH 35146) was collected (Voss & Pearcy, 1990).
Specimen I
Description
Egg capsule collected in 2008 during cruise TAN0803 on the Macquarie Ridge Complex, southwestern Pacific Ocean at 1087–1160 m depth (Table 1, Fig. 1). Measuring 15 × 8 mm, the rounded egg capsule has numerous longitudinal striations and a light brown color—note the smooth, white-colored chorion that can be seen through an artificial rupture of the egg capsule (Fig. 3d). Using MRI, the internal composition of the egg capsule can be analyzed: the chorion is much smaller than the surrounding egg capsule and measures 11.5 × 7.5 mm, the gap being filled with some kind of organic (gelatinous?) matter (Fig. 3e). The external yolk sac is smaller than the chorion as well, measuring 10 × 6 mm, but no sign of an embryo is found. On average, the thickness of the egg capsule is ca. 150 µm and that of the chorion ca. 100 µm.
Remarks
Based on the presence of a large gap and egg capsule dimensions, form, and structuring as well as sampling locality and depth, this egg capsule is identified as a representative of the genus Cirroctopus.
Specimen J
Description
This single egg capsule was collected during cruise TAN0803 on the Macquarie Ridge Complex, southwestern Pacific Ocean at 1400–1615 m depth (Table 1, Fig. 1). In its overall aspect it is very similar to the previous specimen. However, it measures 16 × 9 mm and on one side is covered by a white cristalline matter (Fig. 3f). The opposite side shows conspicuous imprints that are here judged to be the area of attachment of the egg capsule to a hard substrate (Fig. 3f). MRI data reveal the presence of organic (gelatinous?) matter in-between the chorion and egg capsule. The chorion measures 12 × 7.5 mm. The large external yolk sac in turn measures 9.5 × 5.5 mm, but there is no sign of an embryo (Fig. 3g, h). Average thickness of chorion and egg capsule are the same as for the previous specimen.
Remarks
In consequence, this egg capsule is identified as pertaining to a species of Cirroctopus as well.
Specimen K
Description
Collected in 1998 during cruise NZOI SOP on the Hikurangi Plateau, southwestern Pacific Ocean at 1003–1108 m depth (Table 1, Fig. 1), this egg capsule measures 12 × 9 mm. It has a relatively smooth exterior with some irregular structuring and an olivine color (Fig. 3i). At one end, the egg capsule is very likely to have been attached to a hard substrate. Using MRI, the chorion can be readily discerned: it measures 11.6 × 8.5 mm, thus leaving practically no gap towards the egg capsule (Fig. 3j). The external yolk sac is relatively large and at 9.5 × 7 mm fills the inner volume of the chorion to some extent (Fig. 3k). In contrast to specimens I and J, the average thickness of the chorion is about 150 µm, while that of the egg capsule is 300 µm.
Remarks
According to the label found with this museum specimen, the egg capsule was assigned to Cirroctopus hochbergi. However, based on egg capsule dimensions and structuring, absence of a gap, as well as sampling locality, this specimen is here identified as pertaining to a member of the Cirroteuthidae, most likely Cirroteuthis.
Specimen L
Description
Severely ruptured egg capsule collected in 2008 during cruise TAN0803 on the Macquarie Ridge Complex, southwestern Pacific Ocean at 1087–1160 m depth (Table 1, Fig. 1). The original dimensions of the egg capsule are difficult to ascertain, but it closely resembles specimens I and J with regard to external structuring and color (Fig. 4a). The chorion is significantly smaller than the egg capsule and measures 11 × 4 mm. Using contrast-enhanced µCT it can be shown that the chorion contains an embryo attached to the largely amorphous mass of the external yolk sac (Fig. 4b). At about 1 mm ML, this embryo represents the smallest cirrate embryo on record. It has a large rounded mantle with proportionally very large eyes (Fig. 4c). The primary web has not formed yet and cirri are not discernible as well, although a few sucker rudiments are present. A reconstruction of the shell shows this organ to be open U-shaped (Fig. 4d).
Remarks
The embryonic features in combination with the egg capsule characteristics underline the previous assessment of this type of egg capsule pertaining to a species within the genus Cirroctopus.
Specimen M
Description
Single egg capsule collected in 2000 during cruise DIVA-1 from the Angola Basin, southeastern Atlantic Ocean at 5400 m depth (Table 1, Fig. 1). The egg capsule measures about 20 × 15 mm and shows highly irregular structuring and a muddy greenish color (Fig. 4e). Particularly noteworthy is the fact, that the chorion seemingly has swollen, thus rupturing the surrounding, much thicker and tougher egg capsule. While this could represent an artifact caused by the changes of the physico-chemical conditions occurring during sampling from a deep-sea habitat, swollen chorions rupturing their enclosing egg capsules were recently shown to occur in situ as well (Vecchione, 2019). Following extraction and artificial opening of the chorion, a large external yolk sac and an embryo are exposed (Fig. 4f). The embryo provides the following measurements: 13 mm TL, 10 mm ML, 6 mm MW, 9 mm FL, 3.5 mm FW, 3 mm AL, and 1.5 mm FuL. Through its semi-transparent mantle and fin tissues, large and elongated fin cartilages with pointed ends (Fig. 4g) as well as circular-shaped gills and the distal part of the intestine can be seen (Fig. 4h). The arm crown seems to be only basally connected by a small primary web, while the arms with their rounded arm tips show no cirri, but sucker rudiments are present (Fig. 4h). A small funnel can be seen, but no funnel organs are yet discernible.
Remarks
Egg capsule dimensions and structuring as well as color in addition to bathymetry suggest that this specimen constitutes a representative of the genus Grimpoteuthis. In fact, during the same cruise an adult specimen identified as G. wuelkeri was obtained in the vicinity (Piatkowski & Diekmann, 2005).
Specimens N and O
Description
Both specimens were collected in 1996 during cruise ANTARKTIS XIII/3 off Brunt Ice Shelf, Southern Ocean at 620–640 m depth (Table 1, Fig. 1). This specimen lot comprises two chorions and two embryos, but lacks egg capsules as well as external yolk sacs. The smaller embryo (Specimen N) measures 9 mm TL, 5 mm ML, 5 mm MW, 3 mm FL, 2 mm FW, 4 mm AL, 1 mm ED, and 1.5 mm FuL. Its subterminal fins are relatively short, almost square, and their posterior edges merge with the posterior end of the mantle (Fig. 5a). The primary web is well developed and the arms possess suckers as well as rudimentary cirri. Areolar spots are present on the dorsal side of the mantle (Fig. 5a, c). The chorion is egg-shaped and relatively inconspicuous (Fig. 5b). A large part of the internal volume of this embryo is filled with the simple (i.e., unilobular) internal yolk sac (Fig. 5a, d). The elongated fin cartilages with pointed ends attach to an open U-shaped shell that shows a central depression, giving it a shallow W-shaped appearance (Fig. 5d). It is unclear how many optic nerve bundles exactly penetrate the white body, but there appear to be at least three. The larger embryo (Specimen O) measures 16 mm TL, 10 mm ML, 9 mm MW, 7 mm FL, 4 mm FW, 6 mm AL, 2.5 mm ED, and 2.5 mm FuL. The fins are long and situated laterally (Fig. 5e). The primary web is deep and the arms possess well-developed suckers and cirri (Fig. 5h), while a line of areolar spots can be barely made out on the dorsal side of the mantle (Fig. 5e). As in the smaller embryo, the chorion is relatively inconspicuous as well (Fig. 5f). A large part of the internal volume of this embryo is filled with the simple (i.e., unilobular) internal yolk sac (Fig. 5e). Due to the relatively bad state of preservation of this latter specimen, several aspects of its internal anatomy cannot be analyzed.
Remarks
Based on sampling location and depth as well as selected embryonic features (presence of areolar spots, fin and shell shape, number of optic nerve bundles), these two embryos are identified as representatives of the genus Cirroctopus.
Specimen P
Description
Egg capsule collected in 2005 on the Corner Rise Seamounts at 2068 m depth during cruise Deep Atlantic Stepping Stones (Table 1, Fig. 1). This egg capsule measuring 19 × 14 mm was found attached to a large colony of the octocoral species Chrysogorgia tricaulis and has an irregular exterior structuring with beige color—note how part of the egg capsule is wrapped around one of the stems of the octocoral colony (Fig. 6a). Upon arrival on deck the egg capsule ruptured, releasing an embryo (or premature hatchling) with a small external yolk sac still within its arm crown (Fig. 6b). The embryo measures 23 mm TL, 12 mm ML, 10 mm MW, 13 mm FL, 7 mm FW, 11 mm AL, 2 mm ED, and 3 mm FuL. The primary web is relatively shallow, while cirri and suckers are well-developed (Fig. 6b). MRI data reveal the presence of a simple (i.e., unilobular) internal yolk sac that at its posterior end is partially engulfed by an open U-shaped shell (Fig. 6c). An MRI-based 3D reconstruction of selected internal structures illustrates the large size of the internal yolk sac in relation to other organ systems (Fig. 6d, e). The shell bears elongate fin cartilages with pointed ends (Fig. 6f). The white bodies are each penetrated by a single optic nerve bundle.
Remarks
The combined data on egg capsule and embryo dimensions as well as characters permit an identification of this specimen as a member of the genus Grimpoteuthis.
Specimen Q
Description
Another single egg capsule was collected in 2005 during cruise Deep Atlantic Stepping Stones on Kelvin Seamount, northwestern Atlantic Ocean at 1965 m depth (Table 1, Fig. 1). Similar to Specimen P, this egg capsule was found attached to an octocoral host, but in this case Chrysogorgia artospira (Shea et al., 2018). The egg capsule measures 19 × 14 mm as well, but shows a highly irregular structuring with muddy brownish color (Fig. 6g). This egg capsule also ruptured upon arrival on deck (Fig. 6h), releasing another embryo (or premature hatchling). The embryo measures about 29 mm TL, 13 mm ML, 11 mm MW, 17 mm FL, 9 mm FW, 16 mm AL, 2.7 mm ED, and 3.5 mm FuL. A small external yolk sac within the arm crown with its relatively shallow primary web is visible as are the funnel organs at the base of the relatively long funnel (Fig. 6i). Suckers can be clearly differentiated into acetabulum and infidibulum, alternating with relatively long cirri (Fig. 6j). MRI data reveal the presence of a simple (i.e., unilobular) internal yolk sac that at its posterior end is partially surrounded by a U-shaped shell (Fig. 6k). An MRI-based 3D reconstruction of selected internal structures illustrates the large size of the internal yolk sac in relation to other organ systems (Fig. 6l, m). The shell attaches to elongate fin cartilages with pointed ends (Fig. 6n). The white bodies are each penetrated by a single optic nerve bundle.
Remarks
As for the previous specimen and as previously shown (Shea et al., 2018), this egg capsule and the associated embryo can unequivocally be identified as a representative of the genus Grimpoteuthis.
Specimen R
Description
Several specimens were collected in 1884 during a cruise by the research vessel Albatross at Hudson Canyon, northwestern Atlantic Ocean at 1975 m depth (Table 1, Fig. 1). In his report on multiple cirrate eggs collected in this part of the Atlantic Ocean at similar depths, Verrill (1885: 410–411) specifically mentions that some of the eggs collected at Hudson Canyon contained well-formed embryos. He provided the following description: "These embryos have a well-developed body, rounded behind, with relatively large, rather broad lateral fins, having the outer ends broadly rounded, situated far forward and as long as the breadth of the body. The eyes are relatively large and prominent, or some-what stalked. The arms are slender, rounded, with a simple close median row of small suckers. The web is but little developed, the arms being free nearly to the base. The siphon-tube is prominent and the gill-opening is simple and small, but relatively larger than in Stauroteuthis syrtensis. It is probable, therefore, that this embryo belongs to one of the species of Cirrhoteuthis described above."
Remarks
The two new species he assigned to the now invalid genus Cirrhoteuthis are presently considered to be species of Grimpoteuthis (Collins & Villanueva, 2006). In combination with his description of the relevant egg capsules (20 × 14 and 26 × 17 mm with a chorion size of 15 × 11 and 16 × 12 mm, respectively, as well as irregular exterior structuring with a muddy greenish color and egg capsules found attached to octocoral hosts), the information indeed permit identifying these specimens as pertaining to the genus Grimpoteuthis. Unfortunately, the whereabouts of this material are unknown.
Methodological approach
Sigurd von Boletzky based his descriptions of cirrate egg capsules and embryos on material he had obtained from the collections at the Laboratoire Arago in Banyuls, France as well as the Muséum National d'Histoire Naturelle (MNHN) in Paris, France (Boletzky, 1982). Apart from observing and describing the external characteristics of egg capsules as previous authors had done (Table 2), von Boletzky was the first to also histologically study cirrate embryos (Boletzky, 1982). Given the small dimensions of embryos as well as the kind of anatomical information desired, histology obviously constitutes the method of choice for analysis. However, the scarceness of early cirrate life stage material dictates the application of alternative, ideally non-invasive techniques prior to any form of destructive analysis. Such an approach—based on MRI—was recently shown to be effective in studying the anatomy of a prematurely hatched cirrate embryo (Shea et al., 2018), but was later also used to gather anatomical data suitable for a full species description using an adult cirrate specimen collected in the northwestern Pacific Ocean (Werner et al., 2016; Ziegler, 2021; Ziegler & Sagorny, 2021). The present study extends this approach by applying MRI to intact cirrate egg capsules as well as contrast-enhanced µCT to cirrate embryos (Ziegler, 2019; Ziegler et al., 2018). However, while the isotropic voxel resolution achieved here both using MRI and µCT may be sufficient to differentiate between major egg capsule structures as well as embryonic organ systems (Figs. 3, 4, 5, 6), conventional histology obviously offers the advantage of specific staining (Ziegler & Menze, 2013; Ziegler et al., 2018) and as such would be better suited for a detailed anatomical description of cirrate embryonic material. Although the exact whereabouts of the histological sections of cirrate embryos made by von Boletzky remain unknown at present, it is likely that they are still part of his belongings as he had intended to share them with one of us (AZ, personal communication). Finally, given the fact that molecular sequence data are now available for all cirrate genera and have successfully been used for phylogenetic inferences (Ziegler & Sagorny, 2021), DNA barcoding of early cirrate life stage specimens would obviously be one of the most straightforward approaches to identify such material. However, this requires access to appropriately fixed or fresh tissue material as most historical cirrate museum specimens have presumably been fixed in formalin. In addition, DNA sampling requires use of tissues with relevant amounts of DNA, which might not be the case for the tough external egg capsule. However, direct access to the embryo, chorion, or external yolk sac still enclosed in an intact egg capsule necessitates the application of invasive techniques, which in theory should be avoided given the overall scarcity of early cirrate life stage specimens.