Dactylobiotus, Schuster, 1980

Doubtful Dactylobiotus View in CoL taxa

Several explanations could account for the discrepancies in egg ornamentation morphology observed between the newly discovered Greenlandic population and D. octavi . Specifically: (i) the eggs in the type material may have had undeveloped (not fully extended) processes, (ii) the processes may have been distorted by environmental factors or during preparation, or (iii) the varying morphologies could reflect intraspecific variability.

Intraspecific variability in egg morphology has been previously documented in several other tardigrade genera, such as Bertolanius Özdikmen, 2008 (primarily variability in the apical portion of the processes; Dastych 1983), Ramazzottius Binda and Pilato, 1986 (mainly in process length and shape; Stec et al. 2016 2017; Vecchi and Stec 2024), and Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar and Wolf, 2009 (notably in minor differences in the shape of the processes, sometimes even within the same egg; Guidetti et al. 2019). However, an extreme case was recently reported in the latter genus for Paramacrobiotus bifrons Brandoli, Cesari, Massa, Vecchi, Rebecchi and Guidetti, 2024 , which exhibits two morphologically distinct egg forms. Regarding the considerable intraspecific variability of egg ornamentation in Dactylobiotus , this phenomenon has so far been reported only for D. ovimutans . Eggs of this species, thoroughly examined in a culture maintained under stable laboratory conditions by Kihm et al. (2020), exhibited variability in the number, size, and inflation of egg processes. This variability was unlikely to result from seasonality or the production of dormant or active eggs, given the consistent conditions under which the culture was maintained ( Kihm et al. 2020). Thus, it cannot be excluded that a similar variability may also occur in D. octavi , given the correspondence in all egg characters between the type population and the newly examined population, except for the number and inflation of processes. Specifically, fewer and less inflated processes were observed in the type population from Greenland ( Guidetti et al. 2006), whereas eggs with more processes, which were always well extended, were found in the Greenlandic population studied here. Interestingly, differences in the shape of egg processes, attributed to developmental stages, have also been reported for Paramacrobiotus derkai ( Degma, Michalczyk and Kaczmarek 2008) and P. bifrons ( Brandoli et al. 2024; Degma et al. 2008). This suggests that similar situations might occur in other genera as well. Given these uncertainties and the inability to compare DNA sequences of variable markers from the population studied here with those of D. octavi , we classify our population as D. cf. octavi until further data become available.

After examining the type material of D. caldarellai and reviewing its original description, we concluded that this species is insufficiently diagnosed. Pilato and Binda (1994) described D. caldarellai based on two specimens collected from two different locations in Tierra del Fuego, without finding any eggs. The authors considered a morphologically identical population of D. ambiguus reported by Dastych (1984) on King George Island as conspecific with D. caldarellai . Their rationale was based on Dastych’s (1984) observation that the eggs of the newly found population of D. ambiguus differed from those of the population from the species’ type locality in Europe. Notably, the two locations (Tierra del Fuego (type locality of D. caldarellai ) and King George Island) are more than 300 km apart. Given the morphological uniformity among animals of different species within the genus and the absence of egg description for D. caldarellai , it cannot be confidently determined whether these populations represent the same or different species. Furthermore, the suboptimal condition of the holotype and paratype of D. caldarellai hinders a detailed examination (SM. 3). Therefore, until further analyses and a potential integrative redescription based on material from the locus typicus are conducted, D. caldarellai should be considered a nomen dubium, as designated in the results section.

For the second dubious species, D. lombardoi , the original description was based on two specimens also collected in Tierra del Fuego. In their study, Binda and Pilato (1999) provided a table of pt values derived from the measurements of a single specimen of D. lombardoi . These values fall within the pt range of the most similar species, D. parthenogeneticus , as reported in the same paper, with the exception of buccal tube width and ventral lamina length. However, it is likely that these small differences are the result of an insufficient number of measured specimens or variations in measurement techniques used by different authors. Importantly, when the original measurements from Binda and Pilato (1999) are compared with those provided for D. parthenogeneticus by Pogwizd and Stec (2020), the specimen falls perfectly within the newly reported measurement ranges. Although dorsal papillae are not mentioned in the original description of D. lombardoi , it has been suggested that this species can be distinguished from D. parthenogeneticus by the absence ( D. lombardoi ) or presence ( D. parthenogeneticus ) of these structures. However, despite the poor condition of the type series, it appears that dorsal papillae may indeed be present in specimens of D. lombardoi (SM. 3). Given the vague diagnosis of this species, which prevents its clear distinction from other congeners, the lack of a population with a sufficient number of specimens, the unknown egg morphology, and the poor condition of the type material, D. lombardoi should be considered a nomen dubium. This designation, as outlined in the results section, will remain until further analyses confirm whether it represents a distinct species.

As a result, all species for which eggs have never been found ( i.e., D. aquatilis , D. caldarellai , D. henanensis , D. kansae , D. lombardoi , D. macronyx ), with the exception of D. haplonyx Maucci, 1981 , are considered nomina dubia. Regarding D. haplonyx , although no specimens from the type series have been examined, it is important to note that the type series comprises individuals collected from multiple locations, with the holotype originating from a different locality than the paratypes (a total of 33 paratypes from five distinct locations, all different from that of the holotype). Furthermore, the species description lacks not only an account of the eggs but also distinctive diagnostic traits that clearly differentiate it from other species. Consequently, further analyses are necessary to assess its validity. Therefore, we consider it appropriate to designate this species as a nomen inquirendum with the following combination: Dactylobiotus haplonyx Maucci, 1981 nom. inq.

Dichotomous key

Given that egg ornamentation often comprises fundamental diagnostic characters for distinguishing Dactylobiotus taxa, the presented key includes only valid species for which eggs have been described (12 species), excluding from the key all the species designated as nomina dubia or nomina inquirenda.

1. Two dorso-lateral papillae present ............................................. 2

- two dorso-lateral papillae absent ................................................ 6

2. (1). Accessory points not visible with LM, eggs with truncoconical (crater-like) processes ................ Dactylobiotus selenicus View in CoL

- Eggs with conical processes ....................................................... 3

3. (2). Secondary branch of each claw less than one-third the length of the primary branch; distal portion of egg processes not divided into multiple apices .................................................................... 4

- Secondary branch of each claw more than one-third the length of the primary branch; distal portion of egg processes divided into multiple apices ............................................................................ 5

4. (3). Pt value of the IV claws <55, width of the egg processes lower than its height, ca. 40 processes present on the egg circumference .............................................. Dactylobiotus dispar View in CoL

- Pt value of the IV claws> 70, width of the egg processes similar to its height, ≥ 50 processes present on the egg circumference .... ................................................................ Dactylobiotus grandipes View in CoL

5. (3). Egg process bases surrounded by a line of around 25 pores faintly visible with PCM .................. Dactylobiotus taiwanensis *

- Few pores, randomly distributed around the bases of egg processes, not visible with PCM ................................................... ................................................ Dactylobiotus parthenogeneticus View in CoL *

6. (1). Egg processes clearly spaced from each other .................... 7

- Egg processes in contact with each other, with almost no space left between them ....................................................................... 8

7. (6). Delicate reticulation on the egg surface between processes present ..................................................... Dactylobiotus vulcanus

- Delicate reticulation on the egg surface between processes absent ..................................................................................................... 9

8. (6). Width of egg processes <15 µm, more than 20 processes on the egg circumference ....................... Dactylobiotus ambiguus View in CoL **

- Width of egg processes> 15 µm, less than 20 processes on the egg circumference .................................................................... 10

9. (7). Egg process bases width> 10 µm, pores around egg processes bases regularly distributed and visible under light microscope ........................................... Dactylobiotus ovimutans

- Egg process bases width <10 µm, egg surface between processes with irregularly distributed pores or pores absent/not visible under light microscope ............................................................. 11

10. (8). Large, conical egg processes, pores around egg processes not visible under light microscope, width of egg processes bases ≤ 23 µm ............................................. Dactylobiotus ampullaceus View in CoL

- Large, dome-like (or conical) processes, pores around each process well visible under light microscope, width of egg processes bases ≥ 27 µm ....................... Dactylobiotus octavi View in CoL ***

11. (9). Distal portion of egg processes occasionally bi- or trifurcated with short tips; width of egg process bases <8 µm, egg surface between processes with irregularly distributed pores, 31–36 processes on the egg circumference ................................... ..................................................................... Dactylobiotus dervizi View in CoL

- Distal portion of egg processes bi-, tri- or multifurcated and often divided into short branches; width of egg processes bases about 9 µm, egg surface between processes without pores or pores not visible under light microscope, 37–41 processes on the egg circumference .................................................. Dactylobiotus luci

*There are also two minor differences in egg morphology between these species, as their ranges slightly overlap. Dactylobiotus taiwanensis has 38–42 processes on the egg circumference, with process bases measuring 5–6.5 µm, whereas Dactylobiotus parthenogeneticus View in CoL has 34–38 processes, with process bases ranging from 3 to 5.5 µm. Notably, differences in egg morphometrics clearly distinguish the two species ( Fig. 10 View Fig ).

**The measurement was obtained by proportionally scaling a drawing from the species description by Murray (1907), which reported the egg diameter, including processes, as 130 µm. Additionally, data from Thulin (1911) indicated that the process bases had a diameter of 9.5 µm.

***It must be stressed that further investigations are necessary to confirm whether the processes can also have a conical shape. For more details, please refer to the section on Dactylobiotus cf. octavi in this study, which discusses the shape of egg processes in Dactylobiotus octavi .