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Research Article
Milnesium minutum and Milnesium sandrae, two new species of Milnesiidae (Tardigrada, Eutardigrada, Apochela)
expand article infoGiovanni Pilato, Oscar Lisi
‡ University of Catania, Catania, Italy
Open Access

Abstract

Two new species of Milnesium are described, Milnesium minutum sp. n. from Sicily and Milnesium sandrae sp. n. from the Hawaiian Archipelago. The body size of Milnesium minutum is the smallest of the known species of the genus. The stylet supports are inserted on the buccal tube at 63–66% of its length and the claws have a [3-3]-[3-3] configuration. Milnesium sandrae has stylet supports inserted on the buccal tube at 58–60.5% of its length, a [3-3]-[3-3] claw configuration, and the percent ratio between the secondary claw and primary claw length on legs I–III (78.6%–85.5%) clearly higher than on legs IV (70.5%–71.4%). With the description of these two new species, the number of species in the genus is increased to 31.

Keywords

Tardigrada , Milnesiidae , new species, Sicily, Hawaiian Archipelago

Introduction

For 150 years, the genus Milnesium was considered monospecific. Realizing that the individual variability of some characters of Eutardigrada was not as wide as believed for a long time, Binda and Pilato (1990) described a second species of the genus: Milnesium brachyungue Binda & Pilato, 1990. Subsequently, various authors described many more species.

In this paper, two new species are described: one, Milnesium minutum sp. n., from two Sicilian localities and the other, Milnesium sandrae sp. n., from Hawai’i Island (Hawaiian Archipelago).

Material and methods

All studied specimens were mounted in polyvinyl lactophenol. Measurements, in micrometers (µm), and photomicrographs were made under x100 oil immersion, using a Leica Phase Contrast Microscope equipped with “Canon S40” digital camera and Adobe Photoshop Elements 2.0 digital imaging software. The pt index (Pilato 1981) is the percent ratio between the length of a structure and the length of the buccal tube. In Milnesiidae, the length of the buccal tube is measured from the anterior margin of the stylet sheaths to the caudal end, including the flexible portion (Tumanov 2006). We measured only specimens that were aligned to provide accurate morphometric measurements; for this reason, when only a small population is available, only few specimens are suitable for measurement. Though this prevents the assessment of statistical analyses, provided the morphological characters are clearly indicative of speciation, this method avoids the sometime questionably large ranges within statistical analyses caused by imprecise measurements. Claw length refers to the maximum length of the external, primary claws correctly oriented with neither bent nor abnormally straight apices. Configuration of the number of claw points on secondary claws (claw configuration) is given according to Michalczyk et al. (2012b).

In addition to the literature descriptions of many species, the following species (deposited in the Binda & Pilato collection) have been examined for comparison: Milnesium brachyungue Binda & Pilato, 1990; Milnesium eurystomum Maucci, 1991; Milnesium antarcticum Tumanov, 2006; Milnesium asiaticum Tumanov, 2006; Milnesium longiungue Tumanov, 2006.

Results

Milnesium minutum sp. n.

Fig. 1, Table 1

Type locality

Sicily, Moio Alcantara, Contrada Rinazzo 37°54'04"N, 15°03'08"E.

Material examined

Moio Alcantara: Contrada Rinazzo (holotype and one paratype: (slide No. 4127) from a moss sample on rock collected by Dr. R. Catanzaro (Catania) (April 1986); Noto: Contrada Volpiglia, (one paratype, slide No. 3238) from a moss sample collected on a dry wall by Mr. S. Di Stefano (Catania) (February 1980).

Type repository

Holotype and two paratypes are deposited in the Binda and Pilato Collection (slides Nos. 4127 and 3238), Museum of the Department of Biological, Geological and Environmental Sciences, University of Catania, Sicily.

Specific diagnosis

Body of small size (up about 300 µm in the specimens found); colourless; cuticle smooth; eye spots present; six peribuccal and two lateral papillae present; mouth terminal with six triangular peribuccal lamellae with basal stripes; stylet supports inserted on the buccal tube at about 63–66% of its length; claws of the Milnesium type with a [3-3]-[3-3] configuration; primary claws with thin accessory points; secondary claw bases each with a rounded basal thickening (lunule); a long cuticular bar present under claws I–III.

Description of the holotype

Body colourless, 288 µm long; cuticle smooth without pseudopores, reticulum, tubercles or gibbosities; eye spots present. Six peribuccal and two lateral papillae present. Bucco-pharyngeal apparatus of the Milnesium type (Fig. 1A) (rigid buccal tube without ventral lamina, apophyses for the insertion of the stylet muscles in the shape of very short and flat ridges symmetrical with respect to the frontal plane and without caudal processes; pharyngeal bulb elongated, pear-shaped and without apophyses, placoids or septulum); six triangular peribuccal lamellae present with basal stripes. Wide stylet furcae triangular in shape (Fig. 1A).

Figure 1. 

A–D Milnesium minutum sp. n. (holotype). A bucco-pharyngeal apparatus B Claws of the second pair of legs; arrow ‘a’ indicates a claw basal thickening (lunule); arrow ‘b’ indicates the long cuticular thickening C Claws of the third pair of legs D claws of the hind legs E Claws of the hind legs of a paratype (slide No. 3238) where the accessory points are visible (arrow). Scale bars: 10 µm.

Buccal tube cylindrical, 25.7 µm long; the external width at the level of the stylet supports insertion point is 10.9 µm (pt = 42.4). Stylet supports short, inserted on the buccal tube at 65.9% of its length.

Claws of the Milnesium type (Fig. 1), secondary claw branches with three points: configuration [3-3]-[3-3]. Primary claws on legs II, 11.3 µm long (pt = 44.0) and secondary claw, 8.0 µm (pt = 31.1); primary claws on legs III, 11.8 µm long (pt = 45.9); secondary claw, 8.5 µm long (pt = 33.1); primary claws on legs IV, 13.1 µm long (pt = 51.0), secondary claw, 8.6 µm (pt = 33.5). The secondary claw length is 70.8% of the primary claw length on legs II, 72.0% on legs III and 65.6% on legs IV.

Primary claws with thin accessory points (Fig. 1E arrow); each secondary claw base with rounded basal thickening (lunule) (Fig. 1B, arrow a); a long cuticular bar is present under the claws I–III (Fig. 1B arrow b).

Eggs not found.

Remarks

The paratypes are similar to the holotype in both qualitative and quantitative characters (Table 1).

Table 1.

Measurements in µm, pt index values relative to some structures, and percent ratio between secondary claw and primary claw lengths of the holotype and two paratypes of Milnesium minutum sp. n. Also the differences between maximum and minimum values of some characters are given.

Slide number


Measurements
Milnesium minutum sp. n.
4127
Moio Alcantara
Contrada Rinazzo
paratype
4127
Moio Alcantara
Contrada Rinazzo
holotype
3238
Noto
Contrada Volpiglia
paratype
Difference between Max.-Min.
values
µm pt µm pt µm pt
Body length 284 - 288 - ? -
Buccal tube length 25.8 - 25.7 - 26.4 -
Buccal tube width 10.9 42.2 10.9 42.4 10.2 38.6
Stylet supports insertion point 65.5 65.9 63.0 2.9
Primary claw I 10.1 39.1 ? ? ? ?
Secondary claw I 7.3 28.3 ? ? ? ?
Secondary: primary claw I ratio 72.3% ? ? ?
Primary claw II 10.9 42.2 11.3 44.0 11.7 44.3
Secondary claw II 7.6 29.5 8.0 31.1 8.3 31.4
Secondary: primary claw II ratio 69.7% 70.8% 70.9% 1.2
Primary claw III 11.7 45.3 11.8 45.9 11.7 44.3
Secondary claw III 8.2 31.8 8.5 33.1 8.5 32.2
Secondary: primary claw III ratio 70.1% 72.0% 72.6% 2.5
Primary claw IV 13.1 50.8 13.1 51.0 13.2 50.0
Secondary claw IV 8.7 33.7 8.6 33.5 9.1 34.5
Secondary: primary claw IV ratio 66.4% 65.6% 68.9% 3.3

Etymology

The specific name minutum (minutus = small) refers to the small body size.

Differential diagnosis

Eight species of Milnesium with six peribuccal lamellae and a [3-3]-[3-3] claw configuration are known with a smooth cuticle: Milnesium brachyungue Binda & Pilato, 1990; Milnesium eurystomum Maucci, 1991; Milnesium asiaticum Tumanov, 2006; Milnesium antarcticum Tumanov, 2006; Milnesium longiungue Tumanov, 2006; Milnesium zsalakoae Meyer & Hinton, 2010; Milnesium barbadosense Meyer & Hinton, 2012 and Milnesium bohleberi Bartels, Nelson, Kaczmarek & Michalczyk, 2014.

Milnesium minutum sp. n. differs from all these species in having a smaller body size, and other character detail indicated in the following comparisons. We noticed that the three specimens we attributed to Milnesium minutum sp. n. are in particular very similar to Milnesium asiaticum and, considering the body size, it was necessary to determine whether they were three young specimens of Milnesium asiaticum or belonged to a different species. Three facts have to be stressed: a) we collected the specimens attributed to the new species in two different localities. b) We examined and measured specimens of the 15 species of Milnesium present in the collection of Binda & Pilato, and we noticed that for each species in all cases the buccal tube width pt index values for smaller specimens were lower than larger specimens. Specimens of the new Sicilian species with 300 µm body length have buccal tube width pt values that are similar to (or slightly higher than) those of Milnesium asiaticum, which have a body length more than twice as long (Tables 1 and 2). c) Milnesium minutum sp. n. differs from Milnesium asiaticum in having wider buccal tube with respect to the body length; a lower posterior primary claw pt ratio, and a slightly higher percent ratio between the secondary claw and primary claw lengths on legs III and IV (Tables 12; Figs 1C, D and 2A). These facts led us to conclude that the three Milnesium minutum sp. n. specimens were not young examples of Milnesium asiaticum but, independent of body size, belonged to a distinct species.

Figure 2. 

A Claws of the third pair of legs of Milnesium asiaticum. B Claws of the first pair of legs of Milnesium brachyungue C Claws of the first pair of legs of Milnesium longiungue D Claws of the second pair of legs of Milnesium antarcticum, Scale bars: 10 µm.

Table 2.

Measurements in µm, pt index values relative to some structures, and percent ratio between secondary claw and primary claw lengths of a paratype of Milnesium asiaticum, the holotype of Milnesium brachyungue, and a paratype of Milnesium longiungue.

Slide number
Measurements
M. asiaticum M. brachyungue M. longiungue
5105
paratype
3940
holotype
5103
paratype
µm pt µm pt µm pt
Body length 685 - 801 - 747 -
Buccal tube length 54.0 - 59.8 - 46.6 -
Buccal tube width 22.1 40.9 23.7 39.6 22.1 47.4
Stylet supports insertion
point
63.8 69.8 62.3
Primary claw I 21.8 40.4 13.9 23.2 22.5 48.3
Secondary claw I 15.1 28.0 12.4 20.7 14.2 30.5
Secondary: primary claw I ratio 69.3% 89.2% 63.1%
Primary claw II 24.5 45.4 15.4 25.8 25.4 55.5
Secondary claw II 16.0 29.6 13.3 22.2 15.2 32.6
Secondary: primary claw II ratio 65.3% 86.4% 59.8%
Primary claw III 26.3 48.7 16.5 27.6 27.2 57.7
Secondary claw III 16.4 30.4 14.2 23.8 16.4 35.2
Secondary: primary claw III ratio 62.4% 86.1% 60.3%
Primary claw IV 33.6 62.2 18.9 31.6 36.5 78.3
Secondary claw IV 20.5 38.0 15.4 25.8 21.5 46.2
Secondary: primary claw IV ratio 61.0% 81.5% 58.9%

In addition to the body size, the new species differs from Milnesium eurystomum and Milnesium bohleberi by having a cylindrical (not funnel-shaped) buccal tube; from Milnesium eurystomum by having a higher pt of the insertion point of the stylet supports (pt = 63–66 in Milnesium minutum sp. n. vs 58–61 in Milnesium eurystomum); and from Milnesium bohleberi in having lower percent ratio between the secondary claw and the primary claw lengths on all legs (the percent ratio is 69.7–72.6 in the claws I–III of Milnesium minutum sp. n. and 77.9–84.9, for Milnesium bohleberi (according to Bartels et al. 2014); in claw IV the values are 65.6–68.9 in Milnesium minutum sp. n. and, 78.9–80.4 for Milnesium bohleberi (see: Bartels et al. 2014)).

Milnesium minutum sp. n. differs from Milnesium brachyungue by having slightly lower pt of the stylet supports insertion point (63–66 in the new species vs 67–70 in Milnesium brachyungue), by higher pt of the primary and the secondary claw lengths, and by higher values of the percent ratio between the secondary claw and primary claw lengths (Tables 1 and 2, Figs 1 and 2B).

The new species differs from Milnesium longiungue by having accessory points as well as lower pt of the primary claw lengths and higher values of the percent ratio between the secondary claw and primary claw lengths (Tables 1 and 2, Figs 1 and 2C).

The new species differs from Milnesium antarcticum by having a higher pt of the buccal tube width (38.6–42.4 in Milnesium minutum sp. n., 25.9-31.8 in Milnesium antarcticum according to Tumanov 2006); lower pt of the insertion point of the stylet supports on the buccal tube (63.0–66.0 in the new species, 70.0-73.7 in Milnesium antarcticum according to Tumanov 2006); higher pt of the primary claw lengths on legs I-III (Tables 1 and 4, Figs 1 and 2D).

Milnesium minutum sp. n. differs from Milnesium zsalakoae by the more anterior insertion of the stylet supports on the buccal tube (pt = 63–66 in Milnesium minutum sp. n., 68.2–71.1 in Milnesium zsalakoae, according to Meyer and Hinton 2010). The new species also differs by having accessory points and by having a higher percent ratio between the secondary claw and primary claw lengths on legs IV where the values are 65.6–68.9 in Milnesium minutum and 47.2–48.6 for Milnesium zsalakoae (see: Meyer and Hinton 2010).

The new species clearly differs from Milnesium barbadosense by having eyes and by having the stylet supports inserted on the buccal tube in a more anterior position (pt = 63–66 in the new species, about 73 for Milnesium barbadosense according to Meyer and Hinton 2012) (Tables 1 and 4).

Milnesium sandrae sp. n.

Fig. 3, Table 3

Locus typicus

Hawaiian Archipelago: Hawai’i Island.

Material examined

Hawaiian Archipelago: Hawai’i Island (holotype, slide 4290) and 16 paratypes (slides Nos. 4268, 4288–4290; 4293) collected in 1994 by Dr. D.S. Horning (Sydney).

The precise geographic coordinates relative to the type locality in which the specimens were found in 1994 are not available. The specimens were erroneously considered as Milnesium tardigradum by Binda and Pilato (1994).

Type repository

Holotype and paratypes are deposited in the Binda and Pilato Collection, Museum of the Department of Biological, Geological and Environmental Sciences, University of Catania, Sicily.

Specific diagnosis

Colourless; cuticle smooth; eye spots present; six peribuccal and two lateral papillae present; bucco-pharyngeal apparatus of the Milnesium type. Buccal tube wide; mouth terminal with six peribuccal lamellae. Stylet supports inserted on the buccal tube at 58.0–60.5 % of its length. Claws of the Milnesium type with [3-3]-[3-3] configuration; primary claws with thin accessory points; secondary claws each with a rounded basal thickening (lunule); a long cuticular bar present under the claws I–III.

Description of the holotype

Body 567 µm long, colourless, cuticle smooth without pseudopores, reticulum, tubercles or gibbosities; eye spots present. Six peribuccal and two lateral papillae present. Bucco-pharyngeal apparatus of the Milnesium type (Fig. 3A) (rigid buccal tube without ventral lamina, apophyses for the insertion of the stylet muscles in the shape of very short and flat ridges symmetrical with respect to the frontal plane and without caudal processes; pharyngeal bulb elongated, pear-shaped, without apophyses, placoids or septulum); mouth terminal with six triangular peribuccal lamellae with basal stripes. Stylet furcae triangular in shape (Fig. 3A). Buccal tube cylindrical, 35.0 µm long; the external width at the level of the stylet supports insertion point is 15.7 µm (pt = 44.9). Stylet supports inserted on the buccal tube at 58.0% of its length.

Figure 3. 

A–D, Milnesium sandrae sp. n. A Bucco-pharyngeal apparatus (holotype) B Claws of the first pair of legs; the arrow indicates the long cuticular thickening (holotype) C Claws of the hind legs; the arrow indicates a claw basal thickening (lunule) (slide No. 1028) D Detail of one claw of the hind legs with an arrow that indicates one accessory point (holotype). Scale bars: 10 µm.

Claws of the Milnesium type (Fig. 3B–D), secondary claws with three points: configuration [3-3]-[3-3]. Primary claws on legs I, 14.5 µm long (pt = 41.4), and secondary claw, 12.4 µm (pt = 35.4); primary claws on legs II, 15.2 µm long (pt = 43.4) and secondary claw, 12.4 µm (pt = 35.4); primary claws on legs III, 15.2 µm long (pt = 43.4) and secondary claw, 12.2 µm (pt =34.9); primary claws on legs IV, 19.2 µm long (pt = 54.9) and secondary claw, 13.7 µm (pt = 39.1). The secondary claw length is 85.5% of the primary claw length on legs I, 81.6% on legs II, 80.3% on legs III and 71.4% on legs IV.

Thin accessory points present on the primary claws (Fig. 3C, D); secondary claws each with rounded basal thickening (lunule) (Fig. 3C); a long cuticular bar is present under the claws I–III (Fig. 3B).

Remarks

The paratypes are similar to the holotype in both qualitative and quantitative characters (Table 3).

Table 3.

Measurements in µm, pt index values relative to some structures, and percent ratio between secondary claw and primary claw lengths of the holotype, and three paratypes of Milnesium sandrae sp. n. Also the differences between maximum and minimum values of some characters are given.

Slide number


Measurements
4290
Hawai’i Island
paratype
4290
Hawai’i Island
paratype
4293
Hawai’i Island
paratype
4290
Hawai’i Island
holotype
Difference between Max.-Min.
values
µm pt µm pt µm pt µm pt
Body length 401 - 504 - 522 - 567 - 166
Buccal tube length 28.6 - 33.3 - 36.3 - 35.0 -
Buccal tube width 13.7 47.9 16 48.0 16.4 45.2 15.7 44.9
Stylet supports insertion point 60.5 58.6 58.5 58.0 2.5
Primary claw I ? ? 14.5 43.5 14.1 38.8 14.5 41.4
Secondary claw I 9.7 33.9 ? ? 11.9 32.8 12.4 35.4
Secondary: primary claw I ratio ? ? 84.4% 85.5% 1.1
Primary claw II 12.5 43.7 15.5 46.6 15.4 42.4 15.2 43.4
Secondary claw II 10.3 36.0 12.6 37.8 12.1 33.3 12.4 35.4
Secondary: primary claw II ratio 82.4% 81.3% 78.6% 81.6% 3.8
Primary claw III 13.2 46.1 15.0 45.0 15.8 43.5 15.2 43.4
Secondary claw III 10.5 36.7 12.2 36.6 12.6 34.7 12.2 34.9
Secondary: primary claw III ratio 79.5% 81.3% 79.7% 80.3% 1.8
Primary claw IV ? ? 19.0 57.1 19.6 54.0 19.2 54.9
Secondary claw IV ? ? 13.4 40.2 13.8 38.0 13.7 39.1
Secondary: primary claw IV ratio ? 70.5% 70.4% 71.4% 1.0

Etymology

The specific name sandrae is in honour of Dr. Sandra J. McInnes (Cambridge, United Kingdom), who kindly improved the English of many of our papers.

Differential diagnosis

Milnesium sandrae sp. n. is compared with other species of the genus having six peribuccal lamellae, smooth cuticle (without pseudopores, reticulum, tubercles or gibbosities), and the [3-3]-[3-3] claw configuration. The new species differs from all these species, except Milnesium eurystomum, by having a different value of the pt index of the stylet supports insertion point (58.0–60.5 in the new species, over 62 in the remaining taxa) and other characters, which are indicated in detail in the following comparisons.

Milnesium sandrae sp. n. differs from Milnesium eurystomum and Milnesium bohleberi by having a cylindrical instead of a funnel-shaped buccal tube.

Milnesium sandrae sp. n. differs from Milnesium brachyungue by a higher buccal tube width pt index; a higher pt of both the primary and secondary claw lengths, and lower percent ratio values between the secondary claw and primary claw lengths (Tables 3 and 2, and Figs 3BC and 2B); this ratio difference is particularly marked for legs IV where the ratio values of 70.4–71.4 for Milnesium sandrae sp. n. compare with 81 in Milnesium brachyungue (Tables 2 and 3).

The new species differs from Milnesium asiaticum by having a higher pt of the buccal tube width; a higher pt of the secondary claw lengths (particularly on the legs I–III), and a higher percent ratio between the secondary claw and primary claw lengths on all legs (Tables 2 and 3).

Milnesium sandrae sp. n. differs from Milnesium antarcticum by having a shorter buccal tube with respect to the body length; a higher pt index of the buccal tube width; higher pt of the insertion point of the stylet supports on the buccal tube (58.0-60.5 in Milnesium sandrae sp. n., 70.0–73.7 in Milnesium antarcticum according to Tumanov 2006); higher pt indices of the secondary claws, and higher values of the percent ratio between the secondary claw and primary claw lengths (Tables 3 and 4, Figs 3B, C and 2D).

Table 4.

Measurements in µm, pt index values relative to some structures, and percent ratio between secondary claw and primary claw lengths of the holotype of Milnesium barbadosense (*According to Meyer and Hinton 2012) and the holotype of Milnesium antarcticum (** according to Tumanov 2006).

Species


Measurements
M. barbadosense M. antarcticum
holotype * holotype **
µm pt µm pt
Body length 686.4 - ? -
Buccal tube length 44.0 - 74.7 -
Buccal tube width 21.7 49.3 27.4 36.7
Stylet supports insertion point 72.8 71.3
Primary claw I 17.8 40.5 26.3 35.2
Secondary claw I 12.3 28.0 17.8 23.8
Secondary: primary claw I ratio 69.1% 67.7%
Primary claw II 21.6 49.1 ? ?
Secondary claw II 14 31.8 ? ?
Secondary: primary claw II ratio 64.8% ?
Primary claw III 21.1 48.0 ? ?
Secondary claw III 12.3 28.0 ? ?
Secondary: primary claw III ratio 58.3% ?
Primary claw IV 23.3 53.0 39.2 52.5
Secondary claw IV 16.0 36.4 23.7 31.7
Secondary: primary claw IV ratio 68.7% 60.5

The new species differs from Milnesium longiungue by having accessory points; by having lower pt values of the primary claw, and by a higher percent ratio between the secondary claw and primary claw lengths on all legs (Tables 3 and 4).

The new species differs from Milnesium zsalakoae in having accessory points and a higher percent ratio between the secondary claw and primary claw lengths on all legs. The difference is particularly marked in claws IV where the pt ratios are 70.4–71.4 in Milnesium sandrae sp. n. and 47.2–48.6 in Milnesium zsalakoae (see: Meyer and Hinton 2010).

Milnesium sandrae sp. n. differs from Milnesium barbadosense by higher pt of the secondary claw lengths and by higher values of the percent ratio between the secondary claw and the primary claw lengths on legs I–III (Tables 3 and 4).

Milnesium sandrae sp. n. differs from Milnesium minutum by having a larger body size; shorter buccal tube with respect to the body length; a higher pt of the secondary claw lengths and higher values of the percent ratio between the secondary claw and primary claw lengths. This difference is less marked in legs IV (Tables 1 and 3; Figs 1 and 3).

Conclusions

The description of two new species, Milnesium minutum sp. n. and Milnesium sandrae sp. n., raises the number of species ascribed to the genus Milnesium to 31 (30 living and one fossil). Therefore, this tardigrade genus, considered monospecific for 150 years (1840–1990), today is among the 10 most species rich genera. The first species described, Milnesium tardigradum Doyère, 1840, was considered cosmopolitan, but it is evident that specimens of many species have been erroneously attributed to Milnesium tardigradum and, therefore, its geographic distribution must be re-examined and it is probable that the distribution of Milnesium tardigradum is much smaller than formerly believed (Michalczyk et al. 2012a). Many of the newly described species of Milnesium have been reported from only one locality, but it is possible that some of them will be recognized in the future in other geographic areas. Therefore the actual geographic distribution of many species of Milnesium has to be considered provisional.

Acknowledgements

We are very grateful to Dr. Denis Tumanov (St. Petersburg, Russia), who sent us specimens of some studied species, and Prof. Dr. Diane Nelson (Johnson City, Tennessee) for reviewing the English of the text.

References

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