Current knowledge on Mexican tardigrades with a description of Milnesium cassandrae sp. nov. (Eutardigrada: Milnesiidae) and discussion on the taxonomic value of dorsal pseudoplates in the genus Milnesium Doyère, 1840 Author Moreno, Antonio Author Roszkowska, Milena Author García, Mario Alberto Author Flores, José Juan Author Kaczmarek, Łukasz text Zootaxa 2019 2019-11-04 4691 5 501 524 journal article 24986 10.11646/zootaxa.4691.5.5 864e2b8d-c2da-4a20-9fbf-02bcb20be24a 1175-5326 3527562 0684AE18-0510-4F7B-B75D-AE5177FBF2A2 Milnesium cassandrae sp. nov. ( Table 2 , Table 3 , Figure 1 a–e, Figure 2 a–e) Type material: Holotype (female) and 47 paratypes ( 32 adult females, 13 hatchlings, two juveniles of indeterminate sex), 25 eggs and nine exuviae from piedmont of Sierra Las Mitras ( 25°44′55.57′′N , 100°24′11.19′′W , ca. 632 m asl ), Monterrey, Nuevo León , Mexico . Lichen ( Physcia stellaris ) on trees ( Parkinsonia aculeta and Celtis pallida ), coll. Antonio Moreno - Talamantes, 7 August 2014 . Additional material: Thirteen females from a lichen sample and five eggs in exuviae from San Agustin Park ( 25°39′18.63′′N , 100°20′34.51′′W , ca. 605 m asl), San Pedro Garza García Municipality, Nuevo Leon . Mexico . Lichen ( Nephroma sp.) on trees ( Fraxinus sp. and Quercus sp.), coll. Antonio Moreno - Talamantes, 6 November 2013 . Four females from a piedmont of Sierra Las Mitras ( 25°44′45.98′′N , 100°25′27.68′′W , ca. 700 m asl), Monterrey, Nuevo Leon , Mexico . Lichen ( Physcia stellaris ) on tree ( Quercus virginiana ), coll. Antonio Moreno - Talamantes, 8 November 2014 . Six females from piedmont of Sierra Las Mitras ( 25°44′38.55′′N , 100°25′31.65′′W , ca. 719 m asl), Monterrey, Nuevo Leon , Mexico . Lichen ( Physcia stellaris ) on tree ( Quercus virginiana ), coll. Antonio Moreno - Talamantes, 8 November 2014 . Four females from La Posta ( 26°00′46.21′′N , 97°58′59.00′′W , ca. 22 m asl), 2.2 km N of Buenavista, Rio Bravo, Tamaulipas , Mexico . Lichen on mesquite tree ( Prosopis glandulosa ) coll. Antonio Moreno - Talamantes, 19 December 2016 . Species description. Body white or transparent with light yellow to brownish tones before fixation ( Fig. 1a ). Eyes present in all living and mounted adults. Hatchling and juveniles without eyes. Adults cuticle sculptured with pseudopores (0.6 – 1.4 μm in diameter), sparsely distributed and not forming a reticular design. Hatchling and juve- niles with reticular design ( Figs 1b and 2b ). In adults, dorsal side with delineated geometric areas i.e. pseudoplates clearly visible only in FM ( Figs 3 – 4 ). The row I of pseudoplates situated anteriorly to legs I with four (two small and two large) pseudoplates. The row II situated in line with legs I with two triangular pseudoplates connected in the middle. The row III situated between legs I and II with four connected rectangular pseudoplates forming a large rectangle in the midline of the body. The row IV situated in the line of legs II with six rectangular pseudoplates forming a large rectangle in the midline of the body and two pairs of small oval dorsolateral pseudoplates. The row V situated between legs II and III with two rectangular pseudoplates connected in the middle, and two pairs of small oval dorsolateral pseudoplates. The row VI situated in line with legs III with six rectangular pseudoplates forming a large rectangle in the midline of the body and two pairs of small oval dorsolateral pseudoplates. The rows VII to IX situated between legs III and IV (row VII just behind legs III , row VIII in the middle between legs III and IV , row IX just before legs IV ). The row VII with a double rectangular paired pseudoplate (the caudal pair poorly developed) and two pairs of small oval dorsolateral pseudoplates. The row VIII with a complex of ten pseudoplates, eight clustered on the dorsal side and two oval placed dorso - laterally. The row IX with a pair of pseudoplates connected in the middle. The pseudoplates arrangements see Remarks below. For pseudoplates in row I and II poorly visible under PCM , but clearly visible in FM . ( Fig. 4 ). The pseudoplates in the hatchlings and juveniles poorly developed. Buccal apparatus of the Milnesium type ( Figs 1c and 2c ). Buccal tube in adults wide and short (standard width on average 56% of its length) and funnel - shaped, wider anteriorly (posterior diameter on average 89% of the anterior diameter); buccal tube in hatchling and juveniles is more slender (standard width on average 35% of its length) and funnel - shaped, wider anteriorly (posterior diameter on average 80% of the anterior diameter). Six peribuccal papillae and six peribuccal lamellae (of unequal size, 4+2) around the mouth opening present. Two cephalic papillae positioned laterally. The peribuccal papillae longer than the cephalic one. Pharyngeal bulb elongated and pear - shaped, without placoids or septulum. FIGURE 1. Adult of Milnesium cassandrae sp. nov. [holotipe]. (a) habitus (dorsolateral view); (b) details of dorsal cuticle with pseudopores; (c) buccal tube apparatus; (d) claws I; (e) claws IV (all PCM). Scale bar in a = 100 µm, b = 2 µm, c, d and e = 10 µm. Claws of the Milnesiidae type ( Figs 1 d–e and 2d–e). Primary branches on all claws with small accessory points. Secondary branches of all claws with rounded basal thickenings. Transverse cuticular bars present under claws I – III . In adult specimens, internal secondary branches on legs I–III with three points, external with two points. Secondary branches of anterior claw on legs IV with three points, posterior claws with two points. In hatchling and juvenile specimens all secondary branches on all legs with two points. Milnesium cassandrae sp. nov. exhibits an early positive internal CC (claw configuration [2 - 2/3] - [2/3 - 2]); in other words, changes in claw configuration ( CC ) occur between the hatchling/juvenile stages [2 - 2] - [2 - 2], and adults [2 - 3] - [3 - 2]. FIGURE 2. Hatchling of Milnesium cassandrae sp. nov. (a) habitus (ventral view); (b) details of dorsal cuticle with pseudopores; (c) buccal apparatus; (d) claws I; (e) claws IV (all PCM). Scale bar in a = 100 µm, b = 2 µm, c, d and e = 10 µm. Males : not observed. Eggs : Oval, smooth and deposited in exuvium as in all other known Milnesium species. Type depositories: The holotype (slide ELITE - 1/053) and 11 paratypes (slide: ELITE - 1/052, ELITE - 2/054 - 055 - 056 - 057, ELITE - 4/089, ELITE - 5/091 - 092 - 093, ELITE - 30/ 1068 - 1069 ), one exuviae with four eggs (slide ELITE - 30/1067) are deposited at Colección Carcinológica - FCB - UANL at Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León , San Nicolás de los Garza, Nuevo León , Mexico , nine paratypes (slides ELITE - 12/878, ELITE - 14/881, ELITE - 43/ 1097 - 1098 - 1099, ELITE - 44/ 1100 - 1101 - 1102, ELITE - 52/1115), one exuviae and eight eggs ( ELITE - 52/1114) are preserved at the Department of Animal Taxonomy and Ecology, Adam Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, Poznań, Poland and 27 paratypes (slides: ELITE - 3/058 - 060, ELITE - 13/879 - 880, ELITE - 28/1060, ELITE - 29/ 1065 - 1066 , ELITE - 35/ 1084 - 1085 , ELITE - 36/ 1086 - 1087 , ELITE - 37/1089, ELITE - 40/1092, ELITE - 41/1094, ELITE - 42/1096. ELITE - 45/1103, ELITE - 47/1106, ELITE - 49/ 1108 - 1109 , ELITE - 50/ 1110 - 1111 , ELITE - 53/1116, ELITE - 54/ 1119 - 1120 , ELITE - 58/1139, ELITE - 59/ 1140 - 1141 ), eight exuviae with 13 eggs (slides: ELITE - 27/987, ELITE - 29/1064, ELITE - 36/1088, ELITE - 38/1090, ELITE - 39/1091, ELITE - 53/1117, ELITE - 54/1118) are deposited in the collection of first author. Etymology: The new species is dedicated to Cassandra Moreno Perales, the first author’s youngest daughter. Remarks: To clarify the nomenclature of the pseudoplates arrangement we propose the system which is similar to that used for the numbering of gibbosities in Doryphoribius and Isohypsibius species by Michalczyk & Kaczmarek (2010) . In this system all rows of pseudoplates are named and assigned to a specific place on the dorsal cuticle and the number of pseudoplates is counted: row I of pseudoplates situated anteriorly to legs I, row II of pseudoplates situated in line with legs I, row III situated between legs I and II, row IV situated in line with legs II, row V situated between legs II and III, row VI situated in line with legs III, row VII situated between legs III and IV (just behind legs III), row VIII situated between legs III and IV (in the middle between legs III and IV), row IX situated between legs III and IV (just before legs IV). The row numbers are always in Roman numerals and the number of pseudoplates is always listed in Arabic numerals. For example, in this system a configuration of pseudoplates (CP) in Mil. cassandrae sp. nov. will be: (CP: I:4; II:2; III:4; IV:10; V:6; VI:10; VII:8; VIII:10; IX:2, Figs 3 – 4 ). FIGURE 3. Milnesium cassandrae sp. nov . under UV fluorescence, showing dorsal pseudoplates. Scale bar 100 µm. FIGURE 4. Semi - schematic drawing of the dorsal pseudoplates of adult Milnesium cassandrae sp. nov. , based on PCM and UV fluorescence observation (pseudoplates in anterior segment of leg I and leg I are poorly visible under PCM, but clearly visible under fluorescence microscopy). Roman numbers indicate rows of pseudoplates. Contour of Milnesium redrawn and adapted from Morek et al. 2016 . Differential diagnosis. Based on having a sculptured dorsal cuticle, Mil. cassandrae sp. nov. belongs to the granulatum group ( Michalczyk et al . 2012a , b). The new species with claw configuration [2 - 3] - [3 - 2] in adults, is most similar to Mil. krzysztofi Kaczmarek & Michalczyk, 2007 and Mil. beasleyi Kaczmarek et al ., 2012 , but it differs from: 1. Milnesium krzysztofi by: different dorsal sculpture (very dense pseudopores, but not forming a reticular design in adults of Mil. cassandrae sp. nov. vs. a fine reticular design in Mil. krzysztofi ), presence of eyes, a higher pt of the standard buccal tube width ([ 41.6–67.2 ] in Mil. cassandrae sp. nov. and [ 33.1–38.4 ] in Mil. krzysztofi ), and the presence of pseudoplates (but see Discussion, below). 2. Milnesium beasleyi by: presence of eyes, having higher pt buccal tube width ([ 47.4–66.2 ], [ 41.6–67.2 ], [ 39.9– 61.1 ] anterior, standard and posterior, respectively in Mil. cassandrae sp. nov. vs. [ 35.3–41.8 ], [ 31.2–39.8 ], [ 33.2–39.6 ] anterior, standard and posterior, respectively in Mil. beasleyi ), higher standard width/ length ratio of buccal tube (42% – 67% in Mil. cassandrae sp. nov. vs. 31% – 40% in Mil. beasleyi ), smaller lateral papillae (4.0 – 6.5 in Mil. cassandrae sp. nov. vs. 7.5 – 10.3 in Mil. beasleyi ), and presence of pseudoplates (but see Discussion, below). In addition, Mil. cassandrae sp. nov. is similar to other species with the claw configuration [2 - 3] - [3 - 2] i.e. Mil. lagniappe Meyer et al., 2013 , Mil. reductum Tumanov, 2006 , Mil. reticulatum Pilato et al., 2002 , Mil. tardigradum tardigradum Doyère, 1840 , Mil. tetralamellatum Pilato & Binda, 1991 and Mil. vorax Pilato et al., 2016 , but it differs from: 3. Milnesium lagniappe by: larger number of peribuccal lamellae (six in Mil. cassandrae sp. nov. vs. four in Mil. lagniappe ), different dorsal sculpture (very dense pseudopores, but not forming a reticular design adults of Mil. cassandrae sp. nov. vs. reticulated pattern of irregular polygons in Mil. lagniappe ), presence of eyes, stylet supports inserted in less caudal position ([ 58.7–67.6 ] in Mil. cassandrae sp. nov. vs. [ 69.7–73.4 ] in Mil. lagniappe ), and lower pt of buccal tube width ([ 47.4–66.2 ], [ 39.9–61.1 ] anterior and posterior, respectively, in Mil. cassandrae sp. nov. vs. [ 68.0–77.5 ], [ 61.8–70.8 ] anterior and posterior, respectively, in Mil. lagniappe ). 4. Milnesium reductum by: sculptured dorsal cuticle, presence of accessory points on primary branches of claws, stylet supports inserted in more anterior position (16.5 – 26.0 in Mil. cassandrae sp. nov. vs. 29.2 – 28.1 in Mil. reductum ), smaller claws IV (see Table 2 in this work and Table 5 in Tumanov (2006) for the exact differences in dimensions of claws), and presence of pseudoplates (but see Discussion, below). TABLE 2. Measurements and pt values of selected morphological structures of 30 selected adult specimens of Milnesium cassandrae sp. nov. mounted in PVA medium. Range refers to the smallest and the largest structure among all measured specimens. The pt values are provided in italics. N, number of specimens or structures measured; SD, standard deviation.

Character	N	Range		Mean		SD		Holotype
µm	pt	µm	pt	µm	pt	µm	pt
Body length	30	344	–	693	–	560	97	550
Peribuccal papillae length	25	5.1	–	10.2	19.0	–	29.3	8.5	23.5	1.3	2.9	9.4	27.2
Lateral papillae length	28	4.0	–	6.5	9.3	–	17.2	5.0	13.9	0.6	1.7	4.6	13.2
Buccal tube
Length	30	26.2	–	42.5	–	36.0	–	4.1	–	34.6	–
Stylet support insertion point	30	16.5	–	26.0	58.7	–	67.6	22.8	63.5	2.4	2.3	22.5	64.9
Anterior width	30	12.3	–	24.8	47.1	–	66.2	21.0	58.4	2.7	4.9	20.3	58.5
Standard width	30	10.9	–	25.2	41.6	–	67.2	20.0	55.5	2.9	5.6	19.4	56.0
Posterior width	30	10.4	–	22.9	39.9	–	61.1	18.7	51.9	2.6	5.3	18.2	52.6
Standard width/length ratio	30	42%	–	67%	–	55%	–	6%	–	56%	–
Posterior/anterior width ratio	30	81%	–	96%	–	89%	–	4%	–	90%	–
Claw 1 lengths
External primary branch	30	9.3	–	19.0	35.6	–	57.0	15.9	44.4	1.9	4.1	14.8	42.6
External base + secondary branch	30	8.8	–	15.8	33.6	–	52.1	13.9	38.8	1.5	3.8	13.3	38.4
External branches length ratio	29	75%	–	97%	–	87%	–	6%	–	90%	–
Internal primary branch	30	9.1	–	17.2	34.7	–	52.3	15.3	42.6	1.7	3.5	14.0	40.6
Internal base + secondary branch	30	8.2	–	15.4	31.5	–	50.8	13.0	36.5	1.5	4.1	13.1	37.8
Internal spur	30	2.6	–	5.0	8.6	–	15.8	4.0	11.2	0.6	1.7	4.0	11.5
Internal branches length ratio	30	78%	–	97%	–	86%	–	5%	–	93%	–
Claw 2 lengths
External primary branch	29	11.5	–	19.0	40.6	–	59.0	16.8	47.1	1.6	4.0	16.3	47.2
External base + secondary branch	30	8.8	–	17.6	33.6	–	53.9	14.4	40.2	1.7	3.9	13.9	40.2
External branches length ratio	29	77%	–	96%	–	85%	–	5%	–	85%	–
Internal primary branch	30	11.1	–	18.8	37.8	–	55.6	15.8	44.0	1.8	3.9	14.9	43.2
Internal base + secondary branch	30	8.5	–	15.8	32.2	–	52.3	13.2	36.7	1.5	3.9	12.1	34.9
Internal spur	29	2.2	–	5.8	8.3	–	16.2	4.2	11.8	0.7	1.8	3.9	11.3
Internal branches length ratio	30	76%	–	94%	–	84%	–	5%	–	81%	–
Claw 3 lengths
External primary branch	30	10.7	–	23.8	39.3	–	61.2	17.3	48.2	2.2	4.8	16.4	47.5
External base + secondary branch	29	9.2	–	16.4	33.5	–	54.3	14.1	39.3	1.6	4.2	13.8	39.9
External branches length ratio	29	63%	–	98%	–	82%	–	6%	–	84%	–
Internal primary branch	30	10.9	–	19.2	37.6	–	54.0	16.2	45.1	1.7	3.5	15.3	44.3
Internal base + secondary branch	30	8.8	–	15.6	31.9	–	51.6	13.1	36.6	1.4	3.7	12.4	35.7
Internal spur	30	2.7	–	5.6	7.8	–	15.0	4.1	11.5	0.7	1.5	3.7	10.7
Internal branches length ratio	30	67%	–	96%	–	81%	–	6%	–	81%	–
Claw 4 lengths
Anterior primary branch	30	12.6	–	25.0	48.2	–	69.7	20.8	58.1	2.3	5.3	21.0	60.6
Anterior base + secondary branch	30	9.1	–	19.1	34.8	–	51.8	14.8	41.3	1.8	4.2	13.8	39.8
Anterior spur	25	1.2	–	4.6	3.0	–	11.7	2.8	7.6	0.9	2.5	2.1	6.0
Anterior branches length ratio	30	64%	–	83%	–	71%	–	5%	–	66%	–
Posterior primary branch	30	15.0	–	26.8	53.4	–	77.7	21.9	61.3	2.3	5.6	20.1	58.0
Posterior base + secondary branch	30	9.8	–	19.6	37.2	–	53.2	15.6	43.6	1.9	4.2	15.3	44.3
Posterior branches length ratio	30	64%	–	84%	–	71%	–	5%	–	76%	–

5. Milnesium reticulatum by: larger number of peribuccal lamellae (six in Mil. cassandrae sp. nov. vs. four in Mil. reticulatum ), absence of cuticular gibbosities, different dorsal sculpture (very dense pseudopores, but not forming a reticular design in adults of Mil. cassandrae sp. nov. vs. pseudopores arranged in nine sculptured bands, forming a reticular design in Mil. reticulatum ), higher pt of the standard buccal tube width ([ 45.6–67.2 ] in Mil. cassandrae sp. nov. vs. [ 30.4–37.4 ] in Mil. reticulatum ), and higher pt of claws II, III and IV (see Table 2 in this work and Table 2 in Pilato et al . (2002) for the exact differences in dimensions of claws). 6. Milnesium tardigradum tardigradum by: having sculptured dorsal cuticle and presence of pseudoplates (but see Discussion, below). TABLE 3. Measurements and pt values of selected morphological structures of 13 hatchling and juvenile specimens (instar I and II) of Milnesium cassandrae sp. nov. mounted in PVA medium. Range refers to the smallest and the largest structure among all measured specimens. The pt values are provided in italics; N, number of specimens or structures measured; SD, standard deviation.

Character	N	Range µm		pt	Mean µm	pt	SD µm	pt
Body length	13	131	–	202	–	179	20
Peribuccal papillae length	3	3.3	–	4.2	16.2	–	19.7	3.7	17.9	0.4	1.7
Lateral papillae length	5	2.3	–	3.4	11.3	–	16.5	3.0	14.6	0.4	2.1
Buccal tube
Length	13	18.2	–	22.6	–	20.7	–	1.2	–
Stylet support insertion point	13	10.3	–	14.8	56.5	–	70.5	13.8	66.3	1.1	3.4
Anterior width	13	7.2	–	9.6	38.1	–	47.1	8.8	42.3	0.7	3.2
Standard width	13	6.1	–	7.8	28.2	–	39.4	7.2	34.6	0.5	3.0
Posterior width	13	6.3	–	7.3	29.6	–	36.1	7.0	33.6	0.3	2.0
Standard width/length ratio	13	28%	–	39%	–	35%	–	3%	–
Posterior/anterior width ratio	13	74%	–	88%	–	80%	–	4%	–
Claw 1 lengths
External primary branch	13	7.4	–	10.4	35.1	–	52.8	8.9	42.9	0.9	5.7
External base + secondary branch	12	6.5	–	7.9	30.1	–	39.7	7.3	35.1	0.4	2.6
External branches length ratio	12	72%	–	96%	–	84%	–	10%	–
Internal primary branch	13	7.6	–	9.8	35.8	–	48.4	8.7	42.1	0.7	4.1
Internal base + secondary branch	13	6.2	–	8.2	30.2	–	39.6	7.1	34.3	0.5	2.9
Internal branches length ratio	13	74%	–	94%	–	82%	–	6%	–
Claw 2 lengths
External primary branch	13	7.6	–	11.0	35.4	–	60.5	9.3	45.2	0.9	6.5
External base + secondary branch	13	6.7	–	8.1	30.9	–	40.9	7.5	36.3	0.5	3.5
External branches length ratio	13	65%	–	94%	–	81%	–	7%	–
Internal primary branch	12	7.6	–	9.7	37.4	–	53.5	9.0	43.3	0.6	4.3
Internal base + secondary branch	13	6.4	–	8.9	29.9	–	40.9	7.2	34.7	0.7	3.4
Internal spur	1	1.8	–	1.8	10.1	–	10.1	1.8	10.1	?	?
Internal branches length ratio	12	70%	–	96%	–	80%	–	9%	–
Claw 3 lengths
External primary branch	12	8.5	–	10.3	39.4	–	48.9	9.4	44.7	0.5	3.2
External base + secondary branch	13	6.0	–	8.3	31.3	–	38.5	7.3	35.1	0.6	2.3
External branches length ratio	12	71%	–	90%	–	79%	–	6%	–
Internal primary branch	12	8.6	–	9.7	39.0	–	47.2	9.3	44.4	0.4	2.8
Internal base + secondary branch	13	6.7	–	7.6	30.4	–	40.0	7.1	34.2	0.3	2.7
Internal branches length ratio	12	70%	–	83%	–	76%	–	4%	–
Claw 4 lengths
Anterior primary branch	11	8.4	–	11.2	41.5	–	54.9	10.2	49.7	0.8	3.6
Anterior base + secondary branch	13	5.9	–	8.1	26.1	–	40.7	7.1	34.4	0.5	3.7
Anterior branches length ratio	11	61%	–	80%	–	71%	–	6%	–
Posterior primary branch	11	9.5	–	11.8	45.8	–	56.7	10.7	52.3	0.7	3.3
Posterior base + secondary branch	13	6.6	–	8.2	30.6	–	40.9	7.5	36.1	0.5	3.1
Posterior branches length ratio	11	65%	–	77%	–	70%	–	4%	–

7. Milnesium tetralamellatum by: larger number of peribuccal lamellae (six in Mil. cassandrae sp. nov. vs. four in Mil. tetralamellatum ), sculptured dorsal cuticle, and presence of pseudoplates (but see Discussion, below). 8. Milnesium vorax by: sculptured dorsal cuticle, longer % external base + secondary branch/external primary branch I to III in Mil. cassandrae sp. nov. (see Table 2 in this work and Table 1 in Pilato et al . (2016) for the exact differences), and presence of pseudoplates (but see Discussion, below).