Milnesiidae Ramazzotti,

Jackson, Kathy Jo A. & Meyer, Harry A., 2019, Morphological and genetic analysis of Milnesium cf. granulatum (Tardigrada: Milnesiidae) from Northeastern North America, Zootaxa 4604 (3), pp. 497-510: 499-507

publication ID

https://doi.org/10.11646/zootaxa.4604.3.6

publication LSID

lsid:zoobank.org:pub:40A4C93F-300B-46A2-B16F-DCC3B88ABF8A

persistent identifier

http://treatment.plazi.org/id/03C03821-DF13-FFD7-3AEB-8B8E1A12CE89

treatment provided by

Plazi

scientific name

Milnesiidae Ramazzotti
status

 

Family Milnesiidae Ramazzotti  , 1 962

Genus Milnesium Doyère  , 1840

Milnesium cf. granulatum Ramazzotti, 1962  (elevated to species level by Michalczyk et al. 2012a)

Morphology and Morphometrics. Morphometric data for 13 adult female Milnesium cf. granulatum  and six hatchlings from New Hampshire are shown in Table 2, and for five adult females from Newfoundland in Table 3. No males were found at either site. No exuviae or eggs were found in Newfoundland. New Hampshire adults ( Figure 1AView FIGURE 1) have dorsal-lateral cuticle covered with a reticular design of polygons, most evident in caudal and medial regions ( Figure 2AView FIGURE 2). This cuticular ornamentation is sometimes difficult to see in large specimens viewed with PCM.View FigureView Figure

Pseudoplates, delineated geometric areas of cuticle ( Morek et al. 2016), are only clearly evident in the three most caudal segments ( Figure 2CView FIGURE 2); the pattern is the same in all individuals observed. The pattern is similar to Mil- nesium variefidum ( Morek et al. 2016)  on the first segment immediately posterior to legs III (a singular rectangular paired pseudoplate flanked on each side by a smaller dorso-lateral pseudoplate), and on the second pseudoplate posterior to legs III (a cluster of eight dorsal pseudoplates flanked on each side by a dorso-lateral pseudoplate), but differs on the segment immediately above legs IV, where in Milnesium cf. granulatum  the pseudoplate is not paired, but rather divided longitudinally into four parts.

View Figure

Two lateral and six peribuccal lamellae are present. The buccal tube is wide ( Figure 3AView FIGURE 3) in full-size adults, but narrower in younger specimens, especially hatchlings ( Figure 3BView FIGURE 3); i.e., buccal tube width is significantly allometric ( Table 2). In Newfoundland significant allometry was not detected in buccal width ( Table 3). Fewer specimens, representing a narrower range of body lengths, were available in the Newfoundland samples, making detection of allometry less likely. The adult claw formula is [3-3]-[3-3] ( Figure 4A, CView FIGURE 4).

Thirty out of 38 eggs hatched within 10 days of being placed in water ( Figure 1BView FIGURE 1). Hatchlings had the same reticular cuticular ornamentation as adults ( Figure 2BView FIGURE 2). Claw configuration in hatchlings was [3-3]-[3-3] ( Figure 4B, DView FIGURE 4), identical to their mothers ( Figure 4A, CView FIGURE 4).

Genetic analysis. The COI gene was amplified from 11 New Hampshire specimens and 7 Newfoundland specimens. New Hampshire adults exhibited a genetic diversity of 6 haplotypes with p-distances of 0.2–0.7% over a 581 bp region while Newfoundland adults displayed a genetic diversity of 3 haplotypes with p-distances of 0.2–0.3% over a 604 bp region of the COI gene (see Table 1 for accession numbers). Milnesium cf. granulatum  from New Hampshire and Newfoundland exhibited a total of 6 COI haplotypes and the two populations had a p-distance of 0.2% further suggesting that they represented the same species.

Genetic distance of the COI gene was compared between Milnesium cf. granulatum  and other Milnesium  spp. and were as follows: Milnesium berladnicorum  ( Ciobanu et al. 2014; from Romania)—18%, Milnesium  tardi- gradum (Michalczyk et al. 2012a; from Germany)—18%, Milnesium variefidum  ( Morek et al. 2016; from Scotland)—18%, and Milnesium lagniappe  (Meyer et al. 2013; from Louisiana, USA)—21%. The overall mean distance in the COI gene for Milnesium  spp. was 17%. Among Milnesium  spp., Milnesium berladnicorum  and Milnesium variefidum  had the lowest p-distance (13%) while several species had p-distances of 21% ( Table 4).

)

View Figure

A 564 bp fragment of the ITS-2 region was also amplified from 20 adult Milnesium cf. granulatum  specimens from New Hampshire. Molecular analysis was performed on a 407 bp region of the fragment using MEGA7. Tajima’s Neutrality Test indicated population expansion occurring in the animals collected for analysis. The number of identified ITS-2 haplotypes was 13, with p-distances ranging from 0.2–3.7% with an overall mean distance of 1.6% (see Table 1 for accession numbers). The ITS-2 region of siblings from 8 clutches was also analyzed. Each clutch contained between 2 and 5 siblings. Genetic data demonstrated that 2–5 haplotypes were present in siblings from a single clutch ( Table 5). DNA sequencing for most hatchlings resulted in shorter DNA sequences varying in length between 111 bp and 440 bp (269 bp on average) allowing for fewer bases to be analyzed. Genetic distance of ITS- 2 sequences was compared between Milnesium cf. granulatum  and Milnesium  spp. available in GenBank ( Table 6). The p-distances were as follows: Milnesium berladnicorum  —22 %, Milnesium tardigradum  —20%, Milnesium variefidum  —21%, and Milnesium lagniappe  —27%. The overall mean distance in the ITS-2 region for Milnesium  spp. was 26%. Among Milnesium  spp., Milnesium berladnicorum  and Milnesium variefidum  had the lowest p-distance (9%) while Milnesium lagniappe  and Milnesium tardigradum  had the highest p-distance (28%). A phyloge- netic tree of Milnesium  spp. and two Echiniscus  spp. was constructed using the neighbor-joining method. Bootstrap values were high for all branches (95–100%) except one (50%), which was located at the tip of the phylogenetic tree ( Figure 5View FIGURE 5).

[1] [2] [3] [4]

TABLE 2. Morphometric data and pt values of selected characters of 13 adult females and six hatchlings of Milnesium cf. granulatum from New Hampshire, USA. Range refers to the smallest and largest structure found among all measured specimens. All lengths in micrometers. Abbreviations: N = number of specimens measured, SD = standard deviation, pt = pt index, b = allometric exponent and a* = Y-intercept of the regression line of the Thorpe normalized trait versus buccal tube length. Values of b followed by an asterisk indicate that the character was significantly allometric (P <0.05). a* pt was calculated using the mean value of buccal tube length.

Character N   Range Mean   SD b a* a*pt
    μm pt μm pt μm pt μm    
Body length 19 245–773 819 –1486 455 1161 188 213 1.32* 453 1185
Peribuccal papilla length 13 4.5–11.9 15.5–28.6 8.2 21.2 2.8 3.8 1.06 8.4 22.0
Lateral papilla length 10 4.5–12.4 11.7–23.7 7.4 17.8 2.4 3.3 0.82 6.6 17.3
Buccal tube                    
Length 19 27.7–54.2 38.2 11.2
Stylet support insertion point 18 17.8–34.5 61.9–65.2 24.7 63.7 7.0 1.0 0.97* 24.3 63.6
Anterior width 19 13.5–30.1 45.6–55.7 19.7 51.1 6.5 3.0 1.11* 19.6 51.3
Standard width 18 11.9–28.3 40.2–52.2 18.6 47.0 6.6 3.7 1.23* 18.0 47.1
Posterior width 17 11.6–21.9 37.7–43.4 15.6 41.3 6.6 3.7 0.92* 15.7 41.1
Standard width/length ratio 18 40%–52% 47%. 4% 47%
Posterior/anterior width ratio 17 68%–93% 81% 6% 80%
Claw 1 lengths
External primary branch 18 8.4–16.0 28.5–40.7 12.2 33.2 2.8 3.7 0.76* 12.6 33.0
External base + secondary branch 19 7.6–14.3 22.3–32.5 10.4 27.5 2.6 2.3 0.82* 10.4 27.2
External spur 19 2.7–7.0 9.4–15.2 4.4 11.6 1.2 1.5 0.84 4.4 11.5
External branches length ratio 18 70%–97% 83% 7% 83%
Internal primary branch 19 8.9–16.0 27.9–37.9 12.1 32.4 2.7 2.8 0.75* 12.2 31.9
Internal base + secondary branch 19 7.8–14.6 22.9–33.9 10.5 27.9 2.6 2.4 0.81* 10.6 27.7
Internal spur 18 2.8–8.0 9.7–16.2 4.9 12.4 1.7 1.6 1.12 4.8 12.6
Internal branches length ratio 19 74%–96% 86% 6% 87%
Claw 2 lengths                    
External primary branch 18 9.2–17.9 30.0–40.8 13.1 34.7 3.0 3.6 0.75* 13.1 34.3
External base + secondary branch 15 7.6–14.5 24.4–32.7 10.7 27.8 2.5 2.7 0.78* 10.6 27.7
External spur 12 3.3–6.6 11.1–14.3 5.1 12.4 1.3 1.0 0.91 4.8 12.6
External branches length ratio 15 73%–88% 81% 5% 81%
Internal primary branch 18 9.4–20.2 30.4–39.9 13.2 34.6 3.5 2.7 0.85* 13.1 34.3
Internal base + secondary branch 17 7.7–14.4 21.0–32.8 10.9 28.0 2.6 2.8 0.77* 10.6 27.7
Internal spur 16 2.8–7.9 10.0–15.7 5.3 13.3 1.7 1.7 1.09 5.2 13.6
Internal branches length ratio 16 69%–90% 80%   6% 81%

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TABLE 4. P-distances in the COI gene between Milnesium cf. granulatum and several Milnesium species.

  [1] [2] [3] [4]
[1] Milnesium cf. granulatum         
[2] Milnesium lagniappe  20.5%      
[3] Milnesium berladnicorum  17.7% 20.7%    
[4] Milnesium variefidum  18.1% 18.9% 12.6%  
[5] Milnesium tardigradum  18.1% 20.5% 14.3% 13.8%

TABLE 5. Genetic diversity of Milnesium cf. granulatum siblings from 8 clutches expressed as the number of identified ITS-2 haplotypes.

Clutch Number of individuals per clutch Number of haplotypes Sequence read length
1 3 3 111 bp
2 5 4 280 bp
3 2 2 440 bp
4 5 4 176 bp
5 4 3 314 bp
6 2 1 259 bp
7 3 2 185 bp
8 4 4 387 bp

Kingdom

Animalia

Phylum

Tardigrada

Class

Eutardigrada

Order

Apochela

Family

Milnesiidae

Loc

Milnesiidae Ramazzotti

Jackson, Kathy Jo A. & Meyer, Harry A. 2019
2019
Loc

variefidum (

Morek 2016
2016
Loc

Milnesium berladnicorum

Ciobanu, Zawierucha, Moglan & Kaczmarek 2014
2014
Loc

Milnesium berladnicorum

Ciobanu, Zawierucha, Moglan & Kaczmarek 2014
2014
Loc

Milnesium berladnicorum

Ciobanu, Zawierucha, Moglan & Kaczmarek 2014
2014
Loc

Milnesium berladnicorum

Ciobanu, Zawierucha, Moglan & Kaczmarek 2014
2014
Loc

Milnesium berladnicorum

Ciobanu, Zawierucha, Moglan & Kaczmarek 2014
2014
Loc

Milnesium lagniappe

Meyer, Hinton & Dupré 2013
2013
Loc

Milnesium lagniappe

Meyer, Hinton & Dupré 2013
2013
Loc

Milnesium lagniappe

Meyer, Hinton & Dupré 2013
2013
Loc

Milnesium lagniappe

Meyer, Hinton & Dupré 2013
2013
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium cf. granulatum

Ramazzotti 1962
1962
Loc

Milnesium

Doyere 1840
1840
Loc

Milnesium

Doyere 1840
1840
Loc

Milnesium

Doyere 1840
1840
Loc

Milnesium

Doyere 1840
1840
Loc

Milnesium

Doyere 1840
1840
Loc

Milnesium tardigradum

Doyere 1840
1840
Loc

Milnesium

Doyere 1840
1840
Loc

Milnesium tardigradum

Doyere 1840
1840
Loc

Milnesium

Doyere 1840
1840
Loc

Milnesium

Doyere 1840
1840
Loc

Milnesium tardigradum

Doyere 1840
1840
Loc

Milnesium

Doyere 1840
1840
Loc

Milnesium

Doyere 1840
1840