Macrobiotus kamilae sp. nov.

urn:lsid:zoobank.org:act: AA314AF2-9A60-47E3-9EB3-B8B249FC1580

Figs 8–15

Etymology

We take great pleasure in dedicating this new species to the friend of the second author, Kamila Zając, who is a young malacologist and a PhD student at the Institute of Environmental Sciences, Jagiellonian University, Kraków, Poland.

Material examined

77 animals (including 19 simplex) and 42 eggs. Specimens mounted on microscope slides in Hoyer’s medium (63 animals + 32 eggs), fixed on SEM stubs (10+10) and processed for DNA sequencing (4+0).

Holotype INDIA – Chuy Province • ♀; Camel’s Back Road, Mussoorie, Dehradin District, Uttarakhand State; 30°27′28″ N, 78°04′41″E; 2001 m a.s.l.; moss on rock; IZiBB IN.030.08.

Paratypes INDIA – Chuy Province • 71 paratypes; same collection data as for holotype; IZiBB IN.030.01– 06, IN.030.08–12, IN.030.14–15, IN.030.18–19 • 47 eggs; same collection data as for holotype; IZiBB IN.030.13, IN.030.16–17, IN.030.20 .

Description

Animals (measurements and statistics in Table 4)

Body transparent in juveniles and yellowish in adults, but transparent after fixation in Hoyer’s medium (Fig. 8A). 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. 8 B–C). Granulation present on all legs (Fig. 9 A–F). A patch of clearly visible granulation present on external surface of legs I–III (Fig. 9 A–B).A cuticular bulge/fold (pulvinus) present on internal surface of legs I–III, with a faint cuticular fold and a patch of granulation between them (Fig. 9 C–D). Both structures visible only if legs fully extended and properly oriented on slide. Cuticular granulation on legs IV always clearly visible and consisting of a single large granulation patch on each leg (Fig. 9 E–F). In addition to granulation on legs, three patches of granulation on body located dorso-laterally between legs III and IV, with granule size and density increasing from 1 st to 3 rd patch (Fig. 10 A–E).

Claws long and slender, of the hufelandi type (Fig. 11 A–D). Primary branches with distinct accessory points, a long common tract and with an evident stalk connecting the claw to the lunula (Fig. 11 A–D). Lunulae I–III smooth (Fig. 11A, C), whereas lunulae IV clearly dentate (Fig. 11B, D). Cuticular bars under claws are absent. Double muscle attachments are faintly marked under PCM but clearly visible under SEM (Fig. 11A, C, respectively). A faintly marked horseshoe structure connecting the anterior and the posterior claw is visible only in PCM (Fig. 11B, D).

Mouth antero-ventral with ten peribuccal lamellae and a circular sensory lobe (Figs 12A, 13A). Buccopharyngeal apparatus of the Macrobiotus type (Fig. 12A). Under PCM, the oral cavity armature is of the patagonicus type, i.e., with only the 2 nd and 3 rd bands of teeth visible (Fig. 12 B–E). However, in SEM all three bands of teeth are visible, with the first band being situated at the base of peribuccal lamellae and composed of a single row of small cone-shaped teeth. The second band of teeth is situated between the ring fold and the third band of teeth and comprises 2–4 rows of small cone-shaped teeth, slightly larger than those in the first band (Figs 12 B–E, 13B–C). Under PCM the second band is faintly visible in large as well as small specimens (Fig. 12 B–E). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the buccal tube opening (Figs 12 B–E, 13B–C). The third band of teeth is discontinuous and divided into the dorsal and ventral portions. Under PCM, the dorsal teeth are fused and seen as one distinct transverse ridge, whereas the ventral teeth appear as two separate lateral transverse ridges and a median tooth which is sometimes divided into two roundish teeth (Fig. 12 B–E). In SEM, both dorsal and ventral teeth are also clearly distinct (Fig. 13 BC). Under SEM, the margins of the dorsal portion of the third band are slightly serrated with two clearly visible peaks (Fig. 13B), whereas the ventral teeth are separated with a medio-ventral tooth slightly anterior to the lateral teeth (Fig. 13C). Pharyngeal bulb spherical, with triangular apophyses, two rodshaped macroplacoids and a small triangular microplacoid (Fig. 12A, F–G). The macroplacoid length sequence 2 <1. The first macroplacoid exhibits a central constriction, whereas the second macroplacoid is faintly sub-terminally constricted (Fig. 12 F–G).

Eggs (measurements and statistics in Table 5)

Laid freely, yellowish, spherical or slightly ovoid (Figs 14A, 15A). The surface between processes is of the hufelandi type, i.e., covered with a reticulum (Figs 14E, 15 B–F). Meshes of the reticulum small and rounded, irregular in size (mesh diameter 0.3–0.8 µm), with interbasal meshes slightly larger than peribasal meshes but peribasal meshes do not form rings around process bases (Figs 14E, 15 B–F). The nodes of reticulum are often narrower than the mesh diameters visible in PCM and SEM (Figs 14E, 15F). Eggs have 26–32 processes on the circumference, 29 on average (Fig. 14A). Processes are of the inverted goblet shape with slightly concave trunks and concave terminal discs (Figs 14 C–D, 15B–E). Terminal discs round, with faintly indented margins (Fig. 15 B–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. 15E).

Reproduction

The new species is dioecious. No spermathecae filled with sperm have been found in gravid females on the freshly prepared slides. However, in males the testis, filled with spermatozoa, is clearly visible under PCM up to 24 hours after mounting in Hoyer’s medium (Fig. 14F). 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 COI, in which two distinct haplotypes were present:

The 18S rRNA sequence (GenBank: MK737070), 1015 bp long.

The 28S rRNA sequence (GenBank: MK737064), 793 bp long.

The ITS-2 sequence (GenBank: MK737067), 381 bp long.

The COI haplotype 1 sequence (GenBank: MK737920), 658 bp long.

The COI haplotype 2 sequence (GenBank: MK737921), 658 bp long.

Phylogenetic analysis

The phylogenetic analysis, based on available COI sequences of M. hufelandi spp., conducted in our study showed that M. noongaris sp. nov. and M. kamilae sp. nov. indeed belong to this group. The analysis recovered two highly supported clades (Fig. 16). The first grouping (blue nodes) is of species with typical processes of inverted goblet shape and whitish body (with the only exception being M. cf. recens, which has processes in the shape of thin cones devoid of terminal discs). In contrast to the first clade, the second group (red nodes) is of species with yellowish body and morphological modifications of egg processes (flexible filaments on the terminal discs or processes without terminal discs). Interestingly, the two new species described within this study, which both exhibit typical inverted goblet-shaped processes, have been found to cluster together with the species which have modified egg processes. However, M. kamilae sp. nov. has a yellowish body, which conforms to the second characteristic of this clade, whereas M. noongaris sp. nov. has a whitish body.