Grevenius granulifer (Thulin, 1928) revised: a fresh look at one of the most intensively studied water bears (Eutardigrada: Isohypsibioidea)

Abstract

Redescriptions of species established in the incipient stage of tardigrade taxonomy, in the first half of 20th century, have currently become a routine. Especially type species of all genera should undergo an urgent revision to allow more precise diagnoses and unravel species complexes. Herein, an aquatic species Grevenius granulifer is amended based on the topotypic population from a pond in the Botanical Garden of Lund. Detailed qualitative and quantitative characters alongside a full set of standard DNA barcodes (18S rRNA, 28S rRNA, ITS-2, and COI) are provided. This will open a window for systematic works on other representatives of Grevenius and delivers new data on rarely addressed freshwater tardigrades.

Full text

Vol.:(0123456789)
Organisms Diversity & Evolution
https://doi.org/10.1007/s13127-024-00658-7
ORIGINAL ARTICLE
Grevenius granulifer (Thulin, 1928) revised: afresh look atone ofthe
most intensively studied water bears (Eutardigrada: Isohypsibioidea)
PiotrGąsiorek1,2
Received: 12 July 2024 / Accepted: 19 September 2024
© The Author(s) 2024
Abstract
Redescriptions of species established in the incipient stage of tardigrade taxonomy, in the first half of 20thcentury, have
currently become a routine. Especially type species of all genera should undergo an urgent revision to allow more precise
diagnoses and unravel species complexes. Herein, an aquatic species Grevenius granulifer is amended based on the topotypic
population from a pond in the Botanical Garden of Lund. Detailed qualitative and quantitative characters alongside a full set
of standard DNA barcodes (18S rRNA, 28S rRNA, ITS-2, and COI) are provided. This will open a window for systematic
works on other representatives of Grevenius and delivers new data on rarely addressed freshwater tardigrades.
Keywords Cuticle· Freshwater· Integrative taxonomy· Redescription· Sweden· Type locality
* Piotr Gąsiorek
piotr.lukas.gasiorek@gmail.com
1 Natural History Museum ofDenmark, University
ofCopenhagen, Copenhagen, Denmark
2 Department ofInvertebrate Evolution, Faculty ofBiology,
Jagiellonian University, Kraków, Poland
Introduction
Swedish tardigrade fauna has been sporadically studied (Gui-
detti etal., 2015) since the crucial monographs of Thulin
(1911, 1928), which also laid foundations for tardigrade sys-
tematics owing to the impressive observational and analyti-
cal skills of their author. Although Thulin’s descriptions are
particularly detailed for that time, also thanks to the accu-
rate drawings that likely could be compared only to those of
Murray (1907), there is a necessity of providing modern and
complete descriptions for these taxa. Recently, such descrip-
tions were made available for some: Echiniscus lapponicus
Thulin, 1911 (Gąsiorek & Vončina, 2023), Arctodiphascon
tenue (Thulin, 1928) (Tumanov & Tsvetkova, 2023) or Gui-
dettion prorsirostre (Thulin, 1928) (Gąsiorek etal., 2023).
Earlier, redescriptions based on type materials, which are
deposited in the Natural History Museum of Denmark, were
provided for Hypsibius microps Thulin, 1928 and H. pallidus
Thulin, 1911 (Kaczmarek & Michalczyk, 2009). However,
as the condition of slides from the Thulin collection varies
greatly, it is preferable to obtain new populations of respec-
tive taxa, as this allows for a wider range of analyses.
Grevenius granulifer was found for the first time in
mosses in one of the ponds of the Botanical Garden of
Lund (Skåne, Sweden). Thulin (1928) not only drew the
morphology of claws and bucco-pharyngeal apparatus in
lateral view (notably, also accurately reflecting the shape of
the apophyses for the insertion of stylet muscles), but also
outlined some details of internal anatomy: digestive tract
ending with cloaca, ovary, and Malpighian tubules. Further
details of external morphology were added by Pilato and
Binda (1977), who noted the presence of short bars under
internal claws I–III (see drawings in Bertolani, 1982), and
Schuster etal. (1978), who showed the rugged cuticular sur-
face in 3D. Two subspecies were described (Iharos, 1971;
Ramazzotti, 1966), using animals from the Eastern Palae-
arctic (Lake Baikal and Guryong Falls, respectively), and
later elevated to species level (Degma & Guidetti, 2007;
Gąsiorek etal., 2019). Gąsiorek etal. (2019) transferred all
freshwater (limnic) Isohypsibius spp. to Grevenius, and this
taxon wasestablished the type species of the genus. Like
in other aquatic isohypsibioidean taxa, such as Halobiotus,
Pseudobiotus and Thulinius, claw branches of Grevenius
are elongated, and their bases are stalk-shaped (Nelson &
Marley, 2000). Despite all these amendments, G. granulifer
requires a modern depiction and defined genetic characters

P.Gąsiorek
to make species delineation with related spp. (Tumanov,
2003) possible. To achieve this, the type locality in Lund
was visited in March 2024 for the purpose of sample collec-
tion. Limnic tardigrades achieve population peaks in spring
in the temperate climate (Kathman & Nelson, 1987); thus, it
was expected to obtain a number of animals for integrative
analyses. I present the redescription of G. granulifer, discuss
the literature devoted to the species and discoveries pertinent
to it, and finally show that G. granulifer may be a host to
parasitic, kickxellomycotin genus Ballocephala.
Materials andmethods
Sampling andsample processing
Ten sediment, green algae, and duckweed (Lemna) fresh-
water samples were collected in all ponds of the Botanical
Garden of Lund on 16 March 2024. Plastic vials with biologi-
cal material were kept in a fridge and examined in the labo-
ratories of the Natural History Museum of Denmark 2days
later. All animals were extracted from two samples collected
in one pond (55°42′13″N, 13°12′14″E, 46m asl) under a
binocular stereomicroscope using glass pipettes and later
divided for analyses in phase contrast microscopy (PCM:
187 individuals, including 85 adults and 102 juveniles that
hatched from 75 exuviae kept in embryo dishes in a fridge for
about 1week), observations in scanning electron microscopy
(SEM: ca. 50 individuals), DNA barcoding (8 individuals),
and genome sequencing (ca. 50 individuals and exuviae with
eggs). Only individuals extracted alive and with no signs of
fungal infestation were used for the latter two purposes.
Microscopy andimaging
Specimens for light microscopy were mounted on microscope
slides in Hoyer’s medium and secured with cover slips. Slides
were examined under an Olympus BX53 PCM, associated with
an Olympus DP74 digital camera. Tardigrades for SEM were
first subjected to a 60 °C water bath for 20min to obtain fully
extended animals, next to a water/ethanol and an ethanol/ace-
tone series, then to CO2 critical point drying and finally sput-
ter coated with a thin layer of gold. Bucco-pharyngeal appara-
tuses were extracted following the protocol of Eibye-Jacobsen
(2001). Specimens and apparatuses were examined under high
vacuum in a Versa 3D DualBeam scanning electron microscope
at the ATOMIN facility of the Jagiellonian University, Kraków,
Poland. All figures were assembled in Corel Photo-Paint X8.
For structures that could not be fully focused in a single light
microscope photograph, a stack of two to six images was taken
with an equidistance of ca. 0.1μm and assembled manually
into a single deep-focus image in Corel Photo-Paint X8.
Morphometry andterminology
All measurements are given in micrometres (μm). Structures
were measured only when not broken, deformed, or twisted,
and their orientations were suitable. Body length was meas-
ured from the anterior extremity to the end of the body, exclud-
ing the hind legs. Claws were measured following Beasley
etal. (2008). The pt ratio is the ratio of the length of a given
structure to the length of the buccal tube, expressed as a per-
centage (Pilato, 1981). The br ratio is the ratio between the
length of a secondary branch to the length of a primary claw
branch (Gąsiorek etal., 2019). Terminology for the structures
within the bucco-pharyngeal apparatus, including apophyses
for the insertion of stylet muscles (AISMs), and for the claws
follows that of Pilato and Binda (2010) and Gąsiorek etal.
(2019). The oral cavity armature (OCA; former buccal arma-
ture, e.g. Pilato, 1974) denotation system follows Gąsiorek
etal. (2019). Morphometric data were handled using the
“Parachela” template, which is available from the Tardigrada
Register (Michalczyk & Kaczmarek, 2013).
Genotyping andgenetic comparisons
DNA was extracted from individual animals following a
Chelex® 100 resin (Bio-Rad) extraction method by Cas-
quet etal. (2012), with modifications from Stec etal. (2020).
Vouchers (Pleijel etal., 2008) were obtained after extraction
for five out of eight specimens. Four DNA fragments were
sequenced: the small ribosome subunit (18S rRNA) and the
large ribosome subunit (28S rRNA), the internal transcribed
spacer (ITS-2), and the cytochrome oxidase subunit I (COI).
All fragments were amplified and sequenced according to the
protocols described in Stec etal. (2020). Primers and original
references for specific PCR programs are listed in Supple-
mentary Material 1. Sequences were processed in BioEdit
ver. 7.2.5 (Hall, 1999). Sequences were aligned with the
ClustalW Multiple Alignment tool (Thompson etal., 1994)
implemented in BioEdit, and the aligned fragments were
trimmed to the size of the shortest available alignment. Uncor-
rected pairwise distances were calculated for all Grevenius
spp. available in GenBank in MEGA7 (Kumar etal., 2016).
Results
Morphology andtaxonomy
Phylum: Tardigrada Doyère, 1840
Class: Eutardigrada Richters, 1926
Order: Parachela Schuster etal., 1980
Superfamily: Isohypsibioidea Sands etal., 2008
Family: Doryphoribiidae Gąsiorek etal., 2019

Grevenius granulifer (Thulin, 1928) revised: afresh look atone ofthemost intensively studied…
Fig. 1 General habitus of G. granulifer from the type locality (PCM): A gravid adult in dorsolateral view, B adult in ventral view, C hatchling in
dorsolateral view. Scale bars in μm

P.Gąsiorek
Genus: Grevenius Gąsiorek etal., 2019
Species: Grevenius granulifer (Thulin, 1928).
(Figs.1, 2, 3, 4, 5 and 6, Table1 with measurements of
adults).
Adults. Body large and stocky, clearly bent dorsoventrally
(Fig.1A). Cuticular sculpturing well-developed dorsally
throughout dorsum, extending to lateral sides and legs IV;
legs I–III with weak wrinkling, mainly in the centromedian
Fig. 2 Cuticular sculpturing of G. granulifer (A, B PCM, C–G SEM). Black arrows indicate MAPs. Scale bars in μm

Grevenius granulifer (Thulin, 1928) revised: afresh look atone ofthemost intensively studied…
portion (Fig.1A). Ventral cuticle smooth, not sculptured,
only with some wrinkling (Fig.1B). Muscle attachment
points (MAPs)visible dorsolaterally on trunk (Fig.2A, E).
Cephalic region with a different type of sculpturing com-
prising deep wrinkling (Fig.2B), smoothly passing at the
level of legs I into the reticulum covering the rest of dorsum.
Reticulum with thickened edges of polygons (Fig.2A, C, G);
corners of polygons in the form of larger tubercles/granules,
which sometimes may be more evident than reticulum itself
(Fig.2D–F).
Bucco-pharyngeal apparatus of the Isohypsibius type
(Fig.3). OCA visible in light microscope as black dots
(Fig.3, insert); in fact, it comprises two bands: the first con-
taining three to five rows of small conical teeth and the sec-
ond composed of one row of tear-shaped teeth (Fig.4A–D).
AISMs of the Isohypsibius type (Figs.3E and 4E). Buccal
tube narrow and terminated with pharyngeal apophyses.
Pharynx with three macroplacoids, of which the first and the
second are roughly of the same length, and the third is longer
(Fig.3). Macroplacoids are connected by a thin cuticular list,
and usually malformed, with rugged margins (Fig.4F–H).
Especially the terminal portion of the third macroplacoid
can be separated from the remaining macroplacoid by a deep
groove, causing an impression that a microplacoid is present
(Fig.4H). Peribuccal lobes absent, a continuous peribuccal
lamina present (Fig.4A, B).
Claws of the Pseudobiotus type, with secondary and pri-
mary branches similar in height (br 0.81–1.00, 0.93 on aver-
age; this agrees with previous data published in Gąsiorek etal.
(2019): br 0.83–0.99, 0.91 on average). Elongated (chalice-
shaped) basal tracts and prominent humps on primary branches
of internal and anterior claws present (Fig.5). Accessory points
closely adjacent to primary branches. Pseudolunulae present
(Fig.5C, E), but often weakly developed and not observable in
light microscope. Short and faint cuticular bars present under
internal claws I–III (Fig.5B–D), yet usually difficult to iden-
tify. Bars are subcuticular since not visible in SEM (Fig.5A).
Hatchlings. 111–177μm long, 144 μm on average
(N = 20). Body elongated, not arched. The cuticular sculp-
turing in the form of delicate, rugose wrinkling (Fig.1C),
which is uniform throughout the dorsum. At first glance,
the cuticle may even seem to be smooth. The difference in
sculpturing between adults and juveniles has already been
noted (Ramazzotti & Maucci, 1983).
Exuviae. Containing 23–50 oval to roundish eggs; 34
on average (N = 15). Eggs are densely packed in exuvia
(Fig.6A) and sometimes fall out through the anterior open-
ing made by an individual laying eggs and shedding cuticle.
Chorion smooth.
Remarks. Many largest adults, which probably can reach
even up to 600μm (not measured due to unsuitable position
on slides), seemed to be emaciated after egg deposition and
Fig. 3 Bucco-pharyngeal apparatus of G. granulifer (A–C SEM, D, E PCM). Black arrowheads indicate AISMs; insert: OCA (scale bar = 5μm).
Scale bars = 20μm

P.Gąsiorek
Fig. 4 Mouth opening and details of bucco-pharyngeal apparatus of G. granulifer (SEM): A, B peribuccal ring (I, II—the first and second band
of teeth, respectively, white arrowhead—porous area), C oral cavity in frontal view (note distal tips of stylets), D OCA and the porous area in
close-up, E buccal crown, F–H pharynx. Scale bars in μm

Grevenius granulifer (Thulin, 1928) revised: afresh look atone ofthemost intensively studied…
likely die soon after the last ecdysis. This is supported by
analogous behaviour in another freshwater tardigrade, Pseu-
dobiotus megalonyx (pers. observ.). However, it is also pos-
sible that animals were exhausted by massive infection of a
parasitic fungus from the genus Ballocephala (Fig.6B, C), as
unicellular assimilative hyphae developed in many recently
deceased specimens. Ballocephala is a kickxellomycotin pre-
viously reported for eutardigrades dwelling in leaf litter and
sheep dung (Drechsler, 1951; Richardson, 1970).
Genetic characteristics
A single haplotype was recovered for all eight sequenced
specimens in the DNA markers: 18S rRNA (PQ275724,
848bp), 28S rRNA (PQ275726, 800bp), ITS-2 (PQ278648,
422bp), COI (PQ281440, 622bp). Besides some scarce
genetic data for other population of G. granulifer, only three
spp. from the genus have some DNA barcodes: G. asper
(Murray, 1906), G. cryophilus Zawierucha etal., 2020, and
G. pushkini (Tumanov, 2003). After discarding non-homol-
ogous DNA fragments, the uncorrected pairwise distances
were as follows (Supplementary Material 2):
• 18S rRNA (four spp. compared): 0.0% (G. cryophilus, G.
pushkini, and populations of G. granulifer from Denmark
[Møbjerg etal., 2007], Germany [Kiehl etal., 2007], and
Italy [Cesari etal., 2016]) to 3.7% (G. asper)
• 28S rRNA (four spp. compared): 0.1% (G. pushkini) to
10.1% (G. asper)
• ITS-2: 0.7% (population of G. granulifer from Denmark)
• COI (4 spp. compared): 0.0% (population of G. granulifer
from Denmark) to 25.6% (G. asper).
Based on this, the presence of G. granulifer in Denmark
(Møbjerg etal., 2007) is supported by DNA evidence. Since
18S rRNA cannot be confidently used in species delimitation
(Tang etal., 2012), it cannot be determined with certainty
whether the previously sequenced populations from Ger-
many and Italy indeed represent this species. The largest
p-distances with regard to G. asper correspond well with
its biogeographic origin (Antarctica). Only p = 0.6% in
COI between G. granulifer and a population of G. pushkini
(Gąsiorek etal., 2019) signifies either a misidentification or
a conspecificity of these two spp.
Fig. 5 Claws of G. granulifer (A, F SEM, B–E PCM): A–B claws I, C claws II, D claws III, E, F claws IV. White arrowheads indicate pseudolu-
nulae. Scale bars in μm

P.Gąsiorek
Fig. 6 Exuviae and infected individuals of G. granulifer (PCM): A exuvia with over 30 embryonated eggs, B, C individuals parasitized by a
fungus from the genus Ballocephala (Kickxellomycotina). Scale bars = 50μm

Grevenius granulifer (Thulin, 1928) revised: afresh look atone ofthemost intensively studied…
Discussion
Morphotype ofGrevenius granulifer andits
distribution
The short cuticular bars under internal claws I–III were first
reported by Pilato and Binda (1977) and later consolidated
as species-specific (Bertolani, 1982). However, these bars
are often poorly visible or even entirely/asymmetrically lack-
ing in some specimens. It is advisable not to use their pres-
ence as one of the main criteria in identifying G. granulifer.
In contrast with the well-established notion that the ven-
tral side is also sculptured, as the dorsum (Bertolani, 1982;
Ramazzotti & Maucci, 1983), it is in fact smooth besides the
usual wrinkling (Fig.1B). The dorsal sculpturing gradually
disappears laterally and is poorly developed on legs I–III
Table 1 Measurements (in
µm) of selected morphological
structures of topotypic
specimens of Grevenius
granulifer from Lund (Sweden)
mounted in Hoyer’s medium. N
number of specimens/structures
measured, RANGE refers to
the smallest and the largest
structure among all measured
specimens, SD standard
deviation
Character NRange MEAN SD
µm pt µm pt µm pt
Body length 20 328 − 530 832 − 1263 436 1084 59 119
Bucco-pharyngeal tube
Buccal tube length 20 35.3 − 43.4 −40.2 – 2.3 –
Stylet support insertion point 20 24.5 − 30.2 64.0 − 73.9 27.3 68.0 1.8 3.5
Buccal tube external width 20 4.4 − 5.8 11.2 − 14.1 5.0 12.4 0.4 0.8
Buccal tube internal width 20 2.4 − 3.7 6.0 − 9.6 3.0 7.4 0.3 0.8
Placoid lengths
Macroplacoid 1 20 2.6 − 5.9 6.9 − 14.4 4.3 10.7 1.0 2.2
Macroplacoid 2 20 4.0 − 5.7 10.2 − 14.0 4.9 12.1 0.5 1.0
Macroplacoid 3 20 4.8 − 7.5 12.3 − 17.7 5.9 14.7 0.8 1.6
Macroplacoid row 20 14.5 − 20.9 38.4 − 48.3 17.5 43.5 1.7 3.3
Claw 1 heights
External base 14 6.7 − 11.0 19.0 − 26.8 8.9 22.5 1.1 2.1
External primary branch 1 16.1 − 16.1 44.0 − 44.0 16.1 44.0 ??
External secondary branch 14 14.5 − 21.2 37.1 − 50.6 17.9 45.0 2.2 4.0
Internal base 13 5.7 − 9.5 14.8 − 23.2 7.6 19.1 1.1 2.6
Internal primary branch 1 13.0 − 13.0 32.0 − 32.0 13.0 32.0 ??
Internal secondary branch 12 12.0 − 18.3 29.6 − 45.0 16.0 40.0 2.0 4.6
Claw 2 heights
External base 18 6.5 − 11.0 16.0 − 27.5 9.1 22.6 1.3 2.7
External primary branch 5 17.2 − 21.5 44.0 − 56.5 19.4 49.3 2.0 4.7
External secondary branch 18 16.1 − 22.2 41.2 − 54.0 18.9 47.2 2.1 4.2
Internal base 17 6.6 − 10.1 16.5 − 24.6 7.8 19.5 1.1 2.6
Internal primary branch 5 15.5 − 23.8 38.4 − 58.8 19.2 46.2 3.4 8.2
Internal secondary branch 17 13.2 − 20.9 31.3 − 51.6 17.0 42.5 2.0 5.1
Claw 3 heights
External base 17 7.1 − 10.7 19.2 − 25.2 9.0 22.4 1.1 2.1
External primary branch 3 17.1 − 21.4 44.0 − 56.8 18.8 49.2 2.3 6.7
External secondary branch 17 14.8 − 23.0 37.9 − 53.4 18.6 46.5 2.3 4.8
Internal base 14 5.8 − 8.6 14.3 − 22.5 7.2 17.8 0.9 2.1
Internal primary branch 5 14.0 − 18.9 34.4 − 44.3 15.7 39.7 2.1 4.2
Internal secondary branch 14 13.1 − 19.2 31.0 − 46.7 15.7 39.0 2.0 4.9
Claw 4 heights
Anterior base 8 6.7 − 9.1 18.2 − 23.0 8.0 20.4 0.8 1.9
Anterior primary branch 5 16.8 − 21.4 38.9 − 52.1 19.2 47.1 2.0 5.1
Anterior secondary branch 7 14.2 − 19.6 33.8 − 49.4 17.1 43.7 2.3 5.5
Posterior base 11 6.9 − 10.9 19.5 − 26.5 9.6 24.0 1.1 2.2
Posterior primary branch 10 19.6 − 27.4 50.1 − 67.8 23.5 59.4 2.6 5.8
Posterior secondary branch 10 17.5 − 25.0 43.3 − 59.0 20.8 52.2 2.6 5.2

P.Gąsiorek
(Fig.1A). Interestingly, already Bertolani (1975) underlined
that there are differences in dorsal sculpturing between Ital-
ian populations: one exhibited a reticulum, whereas another
classical granular tubercles. Given that the sculpturing of the
topotypic population can be described as a reticulum with
tubercles positioned at the corners of polygons throughout
most of the trunk, except for the cephalic region, which is
wrinkled (Fig.2), it may be assumed that cuticular sculptur-
ing can be variable in the genus. This was also revealed in
the case of G. asper (Dastych, 2016). Such variability could
explain potential misidentifications with species that seemed
to be distinct in the form of sculpturing from G. granulifer
(e.g., Tumanov, 2003) and suggests a need for re-evaluating
their conspecificity. Grevenius baldii (Ramazzotti, 1945)
was proposed as a likely sister species of G. granulifer based
on the similarity of sculpturing (Bertolani, 1982; Ramaz-
zotti & Maucci, 1983), but it attains much smaller body size
(< 200μm in body length) and has sculptured ventral side
(Bertolani & Balsamo, 1989).
Since the year of its description (Thulin, 1928), the spe-
cies was reliably recorded from many Palaearctic localities,
with a detailed drawing/microphotographic documentation
(Cuénot, 1932, Marcus, 1936, Rudescu, 1964, Greven &
Blom, 1977, Betolani, 1982, Ramazzotti & Maucci, 1983,
Maucci, 1986, Dastych, 1988). The Nearctic record by
Schuster etal. (1978) was published with a SEM picture of
the cuticle, which indicated that the identification was cor-
rect. Also, the record from Korea (Moon etal., 1989) seems
to truly represent G. granulifer. Therefore, assuming that
a biogeographic structuring is present among limnic tardi-
grades as in their cryptogam-dwelling relatives (Gąsiorek,
2023), the species should be tentatively regarded as Hol-
arctic until new evidence shows otherwise. Non-Holarctic
records (McInnes, 1994) are thus treated as uncertain and
not considered herein. The first comprehensive DNA bar-
codes for G. granulifer were published in Møbjerg etal.
(2007), and when cross-checked with new data, they posi-
tively verified the presence of this species in Denmark (see
also above).
Recently, Massa etal. (2024) introduced a division of
Grevenius into four morphogroups to ease species identifi-
cation. Grevenius granulifer was included within the asper
morphogroup (three macroplacoids in the pharynx and no
microplacoid), which is clearly separate from the annulatus
morphogroup (two macroplacoids in the pharynx and no
microplacoid). The latter should be probably separated from
Grevenius s.s. as an independent genus. However, the spe-
cies recorded by Massa etal. (2024) from the British Colum-
bia does not belong to Grevenius, but to Isohypsibius s.s.,
as it was found in moss (Grevenius is primarily aquatic) and
represents a classical Isohypsibius prosostomus morphotype
(Dastych, 1988; Thulin, 1928) with sculpturing in the form
of reticulum. A precise diagnosis of I. prosostomus, and the
more precise diagnosis of Isohypsibius s.s., is still lacking
due to the unavailability of modern redescription.
History ofresearch onGrevenius granulifer
The species, thanks to its relative commonness in water
bodies in Europe and rather large body size easing light
microscope analyses was comprehensively researched from
various angles in the past. Bertolani (1975) discovered poly-
ploidy in Italian populations of this species and suggested
that hermaphroditism, generally rare in eutardigrades, may
occur in G. granulifer. This was later confirmed by Ber-
tolani and Manicardi (1986). Wolburg-Buchholz and Gre-
ven (1979) focused on spermiogenesis and morphology of
spermatozoa, noting that spermatozoa of tardigrades and
onychophorans are divergent and do not exhibit a syna-
pomorphy. In a series of publications, Węglarska (1987a,
1989) described in detail the excretory system, which takes
the form of small Malpighian tubules positioned at the bor-
der of midgut and hindgut (Møbjerg etal., 2018); a stage
of oogenesis termed vitellogenesis, or yolk formation, was
also characterised at that time (Węglarska, 1987b). The rel-
atively well-studied anatomy, as per tardigrade standards,
constituted the basis for unravelling the ultrastructure of the
hermaphroditic gonad (Poprawa & Grzywa, 2006) and the
description of chorion formation (Poprawa, 2011). Further-
more, midgut epithelium was disclosed to be involved in
yolk precursor synthesis whilst oogenesis (Rost-Roszkowska
etal., 2011b) in healthy gravid individuals, but also takes
part in immunological response of an organism during par-
asite (microsporidian) infection by exhibiting autophagy
(Rost-Roszkowska etal., 2011a). Finally, coelomocytes,
or body cavity/storage cells, of G. granulifer were demon-
strated to accumulate mostly polysaccharides, and that they
participate in the synthesis of vitellogenins (Hyra etal.,
2016). This account illustrates how important role the spe-
cies played in characterising tardigrade anatomy.
Conclusions
Grevenius granulifer was provided with a modern redescrip-
tion, which will permit confident species identification in the
integrative taxonomy framework (Jørgensen etal., 2018).
The species was frequently used by researchers, mainly to
better understand tardigrade anatomy and development.
This means that G. granulifer is a good candidate for the
next model tardigrade species, if only a culturing technique
is elaborated for it, which should be possible as Grevenius
myrops is currently maintained in laboratory conditions
(Ito etal., 2016). If successfully cultured, the species would

Grevenius granulifer (Thulin, 1928) revised: afresh look atone ofthemost intensively studied…
be suitable for studies of Ballocephala epidemiology and
pathogenesis in tardigrades. Grevenius s.s. most likely cor-
responds with the asper morphogroup (Massa etal., 2024).
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s13127- 024- 00658-7.
Acknowledgements Allison Linnea Perrigo kindly consented on sam-
pling in the Botanical Garden of Lund. Two reviewers improved this
paper with their comments.
Data availability All data is published in the manuscript and its sup-
plementary materials. Sequences are deposited in GenBank.
Code availability Software and programs are cited in the manuscript.
Declarations
Ethics approval No approval of research ethics committees was
required to accomplish the goals of this study because experimental
work was conducted with unprotected invertebrate species.
Consent for publication The permission for the following paper to
be published in Organisms Diversity & Evolution was granted by the
author.
Conflict of interest The author declares no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
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otherwise in a credit line to the material. If material is not included in
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