Macrobiotus noongaris, Coughlan & Stec, 2019

Coughlan, Kyle & Stec, Daniel, 2019, Two new species of the Macrobiotus hufelandi complex (Tardigrada: Eutardigrada: Macrobiotidae) from Australia and India, with notes on their phylogenetic position, European Journal of Taxonomy 573, pp. 1-38 : 6-15

Macrobiotus noongaris sp. nov.

urn:lsid:zoobank.org:act:A7BF7FF6-451F-4382-A842-CAC1EF51E6B8

Figs 1–7

Etymology

The name refers to the indigenous Australians who live in the region where the new species was found. These are the Noongar peoples, 14 different but related language groups that occupied these lands before western settlement, including the modern city of Perth where the sample was collected. In their languages, the term Noongar means ‘a person of the southwest of Western Australia’.

Material examined

86 animals (including 31 simplex) and 57 eggs. Specimens mounted on microscope slides in Hoyer’s medium (72 animals + 47 eggs), fixed on SEM stubs (10+10) and processed for DNA sequencing (4+0).

Holotype

AUSTRALIA – Western Australia • ♀; Perth, Kings Park ; 31°57′30″ S, 115°350′09″ E; 46 m a.s.l.; moss on soil in an urban park; IZiBB AU.031.12.

Paratypes

AUSTRALIA – Western Australia • 62 paratypes; same collection data as for holotype; IZiBB AU.031.06 to AU.031.14 • 32 eggs; same collection data as for holotype; IZiBB AU.031.02–05 .

Description

Animals (measurements and statistics in Table 2 )

Body transparent in juveniles and white in adults but transparent after fixation in Hoyer’s medium ( Fig. 1A ). Eyes present in live animals as well as in specimens mounted in Hoyer’s medium. Small round and oval cuticular pores (0.3–0.8 μm in diameter), visible under both PCM and SEM, scattered randomly on entire body ( Fig. 1 B–C). Granulation present on all legs ( Fig. 2 A–F). A patch of clearly visible granulation present on external surface of legs I–III ( Fig. 2 A–B).A cuticular bulge/fold (pulvinus) present on internal surface of legs I–III, with a faint cuticular fold covered with faint granulation and paired muscles attachments just above the claws ( Fig. 2 C–D). Both structures are visible only if legs are fully extended and properly oriented on slide (particularly in the case of the pulvinus and cuticular fold). Granulation on legs IV always clearly visible and consists of a single large granulation patch on each leg ( Fig. 2 E–F).

Claws stout, of hufelandi type ( Fig. 3 A–D). Primary branches with distinct accessory points, a common tract, and with an evident stalk connecting claw to lunula ( Fig. 3 A–D). Lunulae I–III smooth ( Fig. 3A, C ), whereas lunulae IV clearly dentate ( Fig. 3B, D ). Cuticular bars under claws absent. Double muscle attachments faintly marked under PCM but clearly visible under SEM ( Fig. 3A, C ).

Mouth antero-ventral followed by ten peribuccal lamellae and a circular sensory lobe ( Figs 4A , 5A ). Bucco-pharyngeal apparatus of Macrobiotus type ( Fig. 4A ). Under PCM, oral cavity armature of the patagonicus type, i.e., with only 2 nd and 3 rd bands of teeth visible ( Fig. 4 B–C). However, in SEM all three bands of teeth visible, with first band being situated at base of peribuccal lamellae and composed of a single row of small fused cone-shaped teeth connected to form a continuous, slightly serrated ring ridge around oral cavity ( Fig. 5 B–C). Second band of teeth situated between ring fold and third band of teeth and comprises 3–6 rows of small cone-shaped teeth ( Figs 4 B–C, 5B–C). Teeth of third band located within posterior portion of oral cavity, between second band of teeth and buccal tube opening ( Figs 4 B–C, 5B–C). Third band of teeth discontinuous and divided into dorsal and ventral portions. Under PCM, dorsal teeth appear as three distinct transverse ridges, whereas ventral teeth appear as two separate lateral transverse ridges and a median tooth ( Fig. 4 B–C). In SEM, both dorsal and ventral teeth also clearly distinct ( Fig. 5 B–C). Under SEM, margins of medio-dorsal tooth slightly serrated ( Fig. 5B ), whereas the medio-ventral tooth slightly anterior to lateral teeth ( Fig. 5C ). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a small triangular microplacoid ( Fig. 4A , D–E). Macroplacoid length sequence 2 <1. First macroplacoid exhibits central constriction, whereas second macroplacoid sub-terminally constricted ( Fig. 4A , D–E).

Eggs (measurements and statistics in Table 3 )

Laid freely, white, spherical or slightly ovoid ( Fig. 6A ). Surface between processes is of the hufelandi type, i.e., covered with a reticulum ( Figs 6E , 7 B–D, F). Meshes of reticulum small (0.1–0.6 µm) and rounded, regular in size and with blurred rims in PCM ( Fig. 6E ), irregular in size and with thick borders in SEM (meshes in SEM appear as pores; Figs 7 B–D, F). Interbasal meshes larger than peribasal meshes, but peribasal meshes do not form rings around process bases ( Figs 6E , 7 B–D, F). Eggs have 22–30 processes on circumference, 26 on average ( Fig. 6A ). Processes are of inverted goblet shape, with slightly concave trunks and concave terminal discs ( Figs 6 C–E, 7B–E). Terminal discs are round and strongly serrated ( Fig. 7 C–E). Each terminal disc has a distinct concave central area which may contain some scattered granulation within, which is also always present on the margin (visible only under SEM; Fig. 7 C–E).

Reproduction

The new species is dioecious. No spermathecae filled with sperm have been found in gravid females on the freshly prepared slides. However, the testis in males, filled with spermatozoa, is clearly visible under PCM up to 24 hours after mounting in Hoyer’s medium ( Fig. 6F ). The new species does not exhibit male secondary sexual dimorphism traits such as lateral gibbosities on legs IV.

DNA sequences

We obtained sequences for all four of the above mentioned DNA markers. All sequenced fragments were represented by single haplotypes except the ITS-2, in which two distinct haplotypes were present:

The 18S rRNA sequence (GenBank: MK737069 ), 1010 bp long.

The 28S rRNA sequence (GenBank: MK737063 ), 786 bp long.

The ITS-2 haplotype 1 sequence (GenBank: MK737065 ), 418 bp long.

The ITS-2 haplotype 2 sequence (GenBank: MK737066 ), 418 bp long.

The COI sequence (GenBank: MK737919 ), 658 bp long.

Figures

Fig. 1. Macrobiotus noongaris sp. nov., habitus. A. Dorso-ventral projection (holotype, Hoyer’s medium, PCM, IZiBB AU.031.12). B–C. Cuticular pores on the dorsal part of the body seen in PCM (B: holotype) and in SEM (C: paratype, IZiBB). Scale bars in μm.

Fig. 2. Macrobiotus noongaris sp. nov., cuticular structures on legs (paratypes, IZiBB). A–B. External granulation on legs II and III seen in PCM (A) and SEM (B), respectively. C–D. A cuticular bulge (pulvinus) and a faint cuticular fold, covered by granulation, on the internal surface of legs I and III seen in PCM (C) and SEM (D), respectively. E–F. Granulation on leg IV seen in PCM (E) and SEM (F). Filled indented arrowheads indicate the cuticular bulge and empty indented arrowheads indicate the faint cuticular fold under the claws. Scale bars in μm.

Fig. 3. Macrobiotus noongaris sp. nov., claws (paratypes, IZiBB). A–B. Claws II and IV seen in PCM, with smooth and dentate lunules, respectively. C–D. Claws I and IV seen in SEM, with smooth and dentate lunules, respectively. Filled indented arrowheads indicate double muscle attachments under the claws. Scale bars in μm.

Fig. 4. Macrobiotus noongaris sp. nov., buccal apparatus and the oral cavity armature seen in PCM (paratypes, IZiBB). A. Dorso-ventral projection of the entire buccal apparatus. B–C. Oral cavity armature visible in dorsal (B) and ventral (C) views, respectively. D–E. Placoid morphology visible in dorsal (D) and ventral (E) views, respectively. Empty indented arrowheads indicate the second band of teeth in the oral cavity, filled indented arrowheads indicate the third band of teeth in the oral cavity, empty flat arrowheads indicate central constrictions in first macroplacoids and subterminal constriction in second macroplacoids. Scale bars in μm.

Fig. 5. Macrobiotus noongaris sp. nov., mouth opening and the oral cavity armature seen in SEM (paratype, IZiBB). A. Mouth opening with peribuccal sensory lobes and ten peribuccal lamellae. B–C. The oral cavity armature of a single paratype seen in SEM from different angles, in dorsal (B) and ventral (C) views, respectively. Empty flat arrowheads indicate the first band of teeth in the oral cavity, empty indented arrowheads indicate the second band of teeth in the oral cavity, filled indented arrowheads indicate the third band of teeth in the oral cavity. Scale bars in μm.

Fig. 6. Macrobiotus noongaris sp. nov. A–E. Egg, seen in PCM (IZiBB). A. Midsection under 400 × magnification. B. Surface under 400× magnification. C–D. Midsection under 1000× magnification. E. Surface and terminal discs under 1000× magnification. F. Testis seen in PCM, with visible spermatozoa in male freshly mounted in Hoyer’s medium (paratype, IZiBB). Scale bars in μm.

Fig. 7. Macrobiotus noongaris sp. nov., egg chorion morphology seen in SEM (IZiBB). A. Entire egg. B. Magnification of the egg surface. C–D. Details of the egg processes. E. Terminal disc. F. Details of the reticulation between egg processes. Arrowheads indicate scattered granulation on the terminal disc surface. Scale bars in μm.

Tables

Table 2. Measurements (in μm) and pt values of selected morphological structures of the holotype and paratypes of Macrobiotus noongaris sp. nov. mounted in Hoyer’s medium (N = number of specimens/ structures measured; Range = the smallest and the largest structure among all measured specimens; SD = standard deviation).

Character	N	Range µm pt		Mean µm pt		SD µm pt		Holotype µm pt
Body length	30	333–557	1039 – 1332	461	1201	56	71	443	1205
Buccopharyngeal tube
Buccal tube length	30	31.8–43.5		38.3	–	3.4	–	36.8	–
Stylet support insertion point	30	24.7–34.6	76.7–81.6	30.2	78.7	2.8	1.0	28.8	78.3
Buccal tube external width	30	4.4–7.4	13.8–17.9	5.9	15.5	0.7	1.0	5.7	15.4
Buccal tube internal width	30	2.9–5.0	8.0–11.9	4.1	10.6	0.6	0.9	3.7	10.1
Ventral lamina length	30	19.4–27.8	55.4–70.2	23.4	61.1	2.1	3.2	22.0	59.7
Placoid lengths
Macroplacoid 1	30	8.0–12.7	24.5–3.6	10.6	27.6	1.4	1.7	10.5	28.5
Macroplacoid 2	30	5.2–8.8	15.0–21.0	7.0	18.3	1.1	1.6	6.1	16.5
Microplacoid	30	2.0–4.2	6.2–9.7	3.1	8.0	0.5	1.0	2.9	7.9
Macroplacoid row	30	13.9–22.7	41.9–57.7	18.4	47.9	2.6	3.5	17.1	46.5
Placoid row	30	16.4–26.1	51.3–63.7	22.1	57.5	2.8	3.3	21.2	57.6
Claw 1 heights
External primary branch	30	7.5–10.9	20.8–28.7	9.6	25.2	0.9	2.1	10.4	28.3
External secondary branch	30	5.5–9.4	14.8–24.3	7.5	19.6	1.1	2.5	8.4	22.9
Internal primary branch	30	6.7–10.2	19.2–29.6	9.0	23.7	0.8	2.3	9.2	24.9
Internal secondary branch	30	5.4–8.2	16.6–22.1	7.3	19.1	0.7	1.7	7.8	21.1
Claw 2 heights
External primary branch	30	8.5–12.1	22.9–32.9	10.5	27.6	0.8	2.5	12.1	32.9
External secondary branch	30	6.1–9.9	16.6–26.5	8.4	22.0	0.9	2.3	9.1	24.7
Internal primary branch	30	7.9–10.3	20.9–29.3	9.5	24.9	0.6	1.9	9.4	25.6
Internal secondary branch	30	6.5–8.8	16.4–24.9	7.7	20.3	0.7	2.0	8.8	23.9
Claw 3 heights
External primary branch	30	8.8–11.9	23.6–32.7	10.5	27.4	0.7	2.1	11.3	30.6
External secondary branch	30	6.1–9.7	17.9–25.8	8.3	21.8	0.8	2.1	8.9	24.2
Internal primary branch	30	7.4–10.7	22.4–29.6	9.5	24.8	0.7	1.8	9.5	25.8
Internal secondary branch	30	5.5–9.3	16.6–25.2	7.5	19.6	0.9	2.0	9.3	25.2
Claw 4 lengths
Anterior primary branch	30	8.2–12.3	22.7–32.2	10.5	27.5	1.1	2.6	11.8	32.2
Anterior secondary branch	30	5.4–9.7	15.7–25.1	8.0	21.0	1.0	2.4	9.2	25.1
Posterior primary branch	30	8.6–12.0	23.1–32.7	10.5	27.5	0.8	2.6	11.1	30.3
Posterior secondary branch	30	5.6–9.5	16.3–26.5	8.0	21.0	1.1	2.6	8.9	24.1