Milnesium inceptum, Morek & Suzuki & Schill & Georgiev & Yankova & Marley & Michalczyk, 2019

Morek, Witold, Suzuki, Atsushi C., Schill, Ralph O., Georgiev, Dilian, Yankova, Maria, Marley, Nigel J. & Michalczyk, Łukasz, 2019, Redescription of Milnesium alpigenum Ehrenberg, 1853 (Tardigrada: Apochela) and a description of Milnesium inceptum sp. nov., a tardigrade laboratory model, Zootaxa 4586 (1), pp. 35-64 : 54-55

Delineation of M. alpigenum and M. inceptum sp. nov.

The two species are genetically distinct but morphologically very similar, although not identical. Therefore, they could be classified as pseudocryptic species, i.e. species that can be differentiated morphologically but only with a detailed analysis; in this case—with the use of statistical testing of morphometric traits measured in a number of specimens, since the classical identification based on qualitative traits and non-overlapping morphometric ranges is not sufficient to tell the species apart.

The PCA analysis indicated three components comprising 59.3% of the total variance in the pt ratios. The three factors were as follows: PC1: the pt of the primary and secondary branches of all claws (38.2%), PC2: the pt of external and posterior spurs (11.3%), and PC3: the pt of buccal tube widths and stylet support insertion point (9.8%). The relationships between the principal components are shown in Fig. 3 . The two species did not differ in PC1 and PC3 but in contrast, they differed in PC2, thus comparisons of the first three principal components did not result in congruent conclusions. Specifically, when PC1 and PC3 were compared ( Fig. 3B ), ranges for the two species largely overlapped. In the PC1 vs PC2 comparison ( Fig. 3A ), the overlap between species was smaller. Finally, when PC2 and PC3 were compared, the ranges barely overlapped ( Fig. 3C ). Thus, we compared only the traits constituting PC2 (i.e. the pt values of claw spurs) with a series of t -tests and adjusted α-levels. Student’s t - tests revealed significant differences in three of the eight compared traits (mean values, ± SD, and [ranges] for M. alpigenum vs M. inceptum sp. nov.):

• spur on external claw I: 13.3 ± 1.5 [11.3–17.8] vs 11.3 ±1.3 [7.6–14.8], t 46 =4.002, p <0.001;

• spur on external claw II: 15.3 ±1.4 [12.7–17.5] vs 12.9 ±2.0 [9.2–16.6], t 54 =4.630, p <0.001;

• spur on external claw III: 15.2 ±1.4 [12.4–17.8] vs 12.4 ±1.8 [8.9–16.8], t 48 =5.758, p <0.001.

In other words, the analysis showed that M. alpigenum has statistically longer (relatively to the buccal tube length) external spurs than M. inceptum sp. nov. (compare also Fig. 1 G–H and 2H–I).

In contrast to subtle morphometric differences, the two species exhibit considerable genetic distances in all four analysed markers. Specifically, they differ by 1.0% in 18S rRNA, 5.2% in 28S rRNA, 21.6% in ITS-2, and by 18.1% in COI. Most importantly, however, the two species are not immediately related to each other (see Fig. 4 for the positions of both species on the Milnesium phylogenetic tree), which is the strongest evidence that the two species represent separate phylogenetic lineages.

To conclude, differences both in phenotypic and genetic traits unequivocally show that M. inceptum sp. nov. is a bona species. Nevertheless, an extreme care must be taken when identifying these species using solely phenotypic data.

A B C 0.2 0.2

0.2

0.1 0.1 0.1

))) % % % 9 0 0... 2 0 0 1 0.0 1 0.0 1 0.0 (((2 3 3 PC PC PC

-0.1 -0.1 -0.1

-0.2

-0.2 -0.2

-0.2 0.0 0.2 -0.2 0.0 0.2 -0.1 0.0 -0.1 PC1 (3 5.8%) PC1 (3 5.8%) PC2 (1 2.9%)

Figures

FIGURE 1. Milnesium alpigenum Ehrenberg, 1853. A—habitus, ventral view (neotype, PCM). B—habitus, dorsal view (SEM). C—dorsal cuticle, the arrow indicates area with visible pseudopores (neotype, PCM). D—dorsal cuticle with the barely visible outline of a pseudoplate (neoparatype, SEM). E—buccal apparatus (neotype, PCM). F—six peribuccal lamellae with the 4+2 configuration (neoparatype, SEM). G—claws III with the cuticular bar below (neoparatype, PCM). H—claws IV (neotype, PCM). All scale bars in µm.

FIGURE 3. Graphs illustrating the relationships between the first three principal components revealed by the PCA for the single population of M. alpigenum Ehrenberg, 1853 and the three pooled populations of M. inceptum sp. nov.

FIGURE 4. The positions of M. alpigenum Ehrenberg, 1853 and M. inceptum sp. nov. on the Milnesium phylogenetic tree based on the concatenated data set of ITS-2 and COI sequences. Branch support values are BI posterior probabilities. Species names in square brackets and in grey font are GenBank labels that are incorrect species identifications, uncertain identifications, or invalid names (correct identifications are provided in black font, before the incorrect labels). Filled circles represent both ITS-2 and COI sequences, empty circles indicate COI sequences only. Scale bar shows the number of substitutions per site.