Morphological and genetic analysis of Milnesium cf. granulatum (Tardigrada: Milnesiidae) from Northeastern North America Author Jackson, Kathy Jo A. Author Meyer, Harry A. text Zootaxa 2019 2019-05-15 4604 3 497 510 journal article 26814 10.11646/zootaxa.4604.3.6 33a05386-c01c-41fd-9855-3695876e2458 1175-5326 2852665 40A4C93F-300B-46A2-B16F-DCC3B88ABF8A 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 1A ) have dorsal-lateral cuticle covered with a reticular design of polygons, most evident in caudal and medial regions ( Figure 2A ). This cuticular ornamentation is sometimes difficult to see in large specimens viewed with PCM. FIGURE 1. Milnesium cf. granulatum from New Hampshire, USA. Phase contrast microscopy. A, adult female habitus. B, hatchling habitus. Scale bars: A, 100 μm. B, 50 μm. FIGURE 2. Milnesium cf. granulatum from New Hampshire, USA. A, dorsal reticular sculpture of adult female. B, dorsal reticular sculpture of hatchling. C, posterior pseudoplates. A, B, phase contrast microscopy. C, fluorescent microscopy. Scale bars: 10 μm. Pseudoplates, delineated geometric areas of cuticle ( Morek et al . 2016 ), are only clearly evident in the three most caudal segments ( Figure 2C ); 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. FIGURE 3. Milnesium cf. granulatum from New Hampshire, USA. Phase contrast microscopy. A, buccal tube of adult female. B, buccal tube of hatchling. Scale bars: 10 μm. Two lateral and six peribuccal lamellae are present. The buccal tube is wide ( Figure 3A ) in full-size adults, but narrower in younger specimens, especially hatchlings ( Figure 3B ); 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, C ). Thirty out of 38 eggs hatched within 10 days of being placed in water ( Figure 1B ). Hatchlings had the same reticular cuticular ornamentation as adults ( Figure 2B ). Claw configuration in hatchlings was [3-3]-[3-3] ( Figure 4B, D ), identical to their mothers ( Figure 4A, C ). 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 ). 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%	–

......continued on the next page TABLE 2. (Continued )

Character	N	Range	Mean		SD	b	a*	a*pt
μm	pt	μm pt		μm	pt	μm
Claw 3 lengths
External primary branch	19	9.3–17.7	30.0–39.4	12.9	34.2	3.2	2.7	0.82*	13.0	34.0
External base + secondary branch	18	7.4–15.9	24.9–32.4	11.0	28.6	3.0	2.1	0.90	10.9	28.5
External spur	14	3.5–6.5	10.3–14.6	5.0	12.4	1.1	1.3	0.74*	4.8	12.6
External branches length ratio	18	76%–94%	–	84%	–	5%	–	–	84%	–
Internal primary branch	19	9.4–20.2	20.9–39.5	13.0	34.5	3.5	2.8	0.86	13.0	34.0
Internal base + secondary branch	18	7.0–15.0	24.0–32.5	10.8	28.4	2.6	2.6	0.80*	10.8	28.3
Internal spur	16	2.9–8.7	10.4–17.3	5.3	13.2	1.7	2.0	1.00	5.1	13.4
Internal branches length ratio	18	71%–92%	–	82%	–	6%	–	–	83%	–
Claw 4 lengths
Anterior primary branch	18	10.5–21.6	35.9–43.0	14.7	39.2	4.6	2.0	1.06	15.0	39.3
Anterior base + secondary branch	15	8.4–18.4	29.4–35.1	12.4	32.8	3.9	1.9	1.04	12.6	33.0
Anterior spur	14	3.2–7.9	11.1–17.6	5.2	14.3	1.5	1.7	0.86	5.4	14.1
Anterior branches length ratio	15	77%–87%	–	83%	–	3%	–	–	84%	–
Posterior primary branch	18	10.6–24.9	35.0–45.9	15.2	40.5	5.0	3.1	1.06	15.4	40.3
Posterior base + secondary branch	15	8.5–19.2	30.5–38.3	12.8	34.0	4.0	2.3	1.03	13.0	34.0
Posterior spur	12	3.6–8.0	11.3–16.3	5.2	14.6	1.6	1.7	0.92	5.5	14.4
Posterior branches length ratio	15	77%–94%	–	84%	–	5%	–	–	84%	–

TABLE 3. Morphometric data and pt values of selected characters of five females of Milnesium cf. granulatum from Newfoundland, Canada. 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, = 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	6	221–653	1090 –1933	368	1569	152	353	1.13	364.4	1571
Peribuccal papilla length	2	2.6–3.5	14.4–15.1	3.1	14.8	0.6	0.6	1.22	3.5	15.0
Lateral papilla length	5	2.4–5.6	11.2–16.6	3.4	14.3	1.3	2.5	1.06	2.9	12.5
Buccal tube
Length	6	18.1–33.8	–	23.2	–	6.1	–	–	–	–
Stylet support insertion point	5	11.7–21.4	63.0–67.6	15.0	64.6	3.8	1.6	0.49	15.0	64.6
Anterior width	4	8.6–17.8	47.5–52.7	12.4	50.1	4.3	2.4	1.16	11.6	50.0
Standard width	5	7.5–15.8	41.4–50.6	11.0	45.3	3.4	3.7	1.18	10.7	46.1
Posterior width	2	7.5–2.8	40.4–41.4	9.2	40.9	2.3	0.7	0.94	9.5	40.9
Standard width/length ratio	5	41%–51%	–	45%.	–	4%	–	–	46%	–
Posterior/anterior width ratio	2	78%–87%	–	83%	–	6%	–	–	82%	–
Claw 1 lengths
External primary branch	6	6.0–10.9	30.7–34.0	7.5	32.4	1.9*	1.2	0.93	7.6	32.8
External base + secondary branch	6	5.4–9.5	24.33–30.3	6.6	28.5	1.5	2.2	0.80	6.4	27.5
External spur	6	2.0–4.0	10.1–13.3	2.7	11.5	0.7	1.1	0.91	2.6	11.2
External branches length ratio	6	77%–93%	–	88%	–	5%	–	–	84%	–
Internal primary branch	6	5.9–9.2	27.2–34.6	7.1	30.9	1.3	2.6	0.69*	7.1	30.6
Internal base + secondary branch	5	5.4–8.0	23.7–30.4	6.2	27.5	1.1	2.8	0.63	6.3	27.2
Internal spur	5	2.7–4.3	10.9–14.9	3.1	13.5	0.7	1.7	0.64*	3.0	12.9
Internal branches length ratio	5	83%–94%	–	88%	–	4%	–	–	89%	–
Claw 2 lengths
External primary branch	6	6.4–10.8	29.0–39.2	7.6	33.1	1.6	3.9	0.64	7.5	32.3
External base + secondary branch	6	5.0–9.5	26.0–30.3	6.4	27.7	1.7	1.7	0.94	6.4	27.6
External spur	6	2.5–3.9	9.7–16.0	3.0	13.1	0.5	2.2	0.47	2.9	12.5
External branches length ratio	6	73%–91%	–	84%	–	7%	–	–	85%	–
Internal primary branch	6	6.1–10.1	29.0–35.4	7.3	31.6	1.5	2.7	0.74	7.2	31.0
Internal base + secondary branch	6	5.1–8.7	25.1–29.8	6.3	27.2	1.4	1.7	0.80	6.2	26.7
Internal spur	6	2.3–4.2	10.8–14.4	2.9	12.6	0.7	1.2	0.85	2.9	12.5
Internal branches length ratio	6	83%–94%	–	86%	4%	–	–	86%	–

......continued on the next page TABLE 3. (Continued)

Character	N	Range	Mean	SD	b	a*	a*pt
μm	pt	μm	pt	μm	pt	μm
Claw 3 lengths
External primary branch	6	6.2–9.9	29.3–36.7	7.7	33.5	1.5	2.5	0.75*	7.8	33.6
External base + secondary branch	5	5.3–9.2	25.1–30.3	6.8	28.5	1.5	2.5	0.80	6.6	28.4
External spur	5	2.5–4.1	12.1–14.9	3.1	13.1	0.6	1.1	0.73	3.0	12.9
External branches length ratio	5	76%–93%	–	87%	–	7%	–	–	85%	–
Internal primary branch	6	6.1–8.5	25.1–35.6	7.3	32.0	1.0	3.7	0.55*	7.4	31.9
Internal base + secondary branch	6	5.1–8.4	24.9–30.4	6.3	27.64	1.2	2.4	0.70*	6.3	27.2
Internal spur	6	2.5–4.5	12.1–16.0	3.2	13.9	0.8	1.4	0.82	3.2	13.8
Internal branches length ratio	6	821%–99%	–	87%	–	7%	–	–	85%	–
Claw 4 lengths
Anterior primary branch	5	6.8–11.7	33.7–38.1	8.5	35.4	2.0	1.7	0.86	8.2	35.3
Anterior base + secondary branch	5	2.9–4.6	29.3–33.3	7.7	32.2	1.7	1.7	0.85	7.6	32.8
Anterior spur	5	2.9–4.6	13.6–16.0	3.5	14.9	0.7	0.9	0.77*	3.5	15.1
Anterior branches length ratio	5	85%–98%	–	91%	–	6%	–	–	93%	–
Posterior primary branch	5	6.7–12.8	37.0–47.1	9.4	39.3	2.2	4.4	0.84	9.2	39.7
Posterior base + secondary branch	5	5.9–10.4	30.8–34.6	7.8	32.5	1.8	1.4	0.90	7.6	32.8
Posterior spur	4	2.8–4.9	14.5–16.0	3.7	15.3	1.0	0.7	0.92	3.6	15.5
Posterior branches length ratio	5	70%–93%	–	83%	–	9%	–	–	83%	–

FIGURE 4. Milnesium cf. granulatum from New Hampshire, USA. Phase contrast microscopy. A, claw 2 of mother’s exuvia. B, claw 2 of hatchling. C, claw 4 of mother’s exuvia. D, claw 4 of hatchling. Scale bars: 10 μm. 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%

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 5 ). 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

TABLE 6. P-distance in the ITS-2 region between Milnesium cf. granulatum and several Milnesium species. [1] [2] [3] [4]