Echiniscus masculinus, Gąsiorek & Vončina & Michalczyk, 2020

Gasiorek, Piotr, Voncina, Katarzyna & Michalczyk, Lukasz, 2020, An overview of the sexual dimorphism in Echiniscus (Heterotardigrada, Echiniscoidea), with the description of Echiniscus masculinus sp. nov. (the virginicus complex) from Borneo, Zoosystematics and Evolution 96 (1), pp. 103-113 : 103

Echiniscus masculinus sp. nov. Figures 1 , 2 , 3 , Tables 2, 3, 4, 5

Description.

Mature females (i.e. from the third instar onwards; measurements and statistics in Table 2 ). Body cylindrical, orange with minute red eyes present in live specimens; colours disappearing soon after mounting in Hoyer’s medium. Echiniscus -type cephalic papillae (secondary clavae) and (primary) clavae; cirri growing out from bulbous cirrophores (Figure 1A ). The body appendage configuration is A-C-D-Dd-E, with all trunk appendages formed as spines or spicules. All usual trunk appendages always symmetrical and smooth. Spine Cd rudimentarily developed in two females (one with an asymmetrical spicule [2 µm], the other normally formed [8 µm]).

Dorsal plates with the mixed type of sculpturing, with an evident layer of endocuticular pillars visible as black dots under PCM, and an upper layer of greyish epicuticular matrix forming the ornamented pattern together with pseudopores, enhanced as dark belts on the anterior portions of the paired segmental plates (Fig. 1A ). Generally, the epicuticular sculpture is poorly developed and gives way to large pillars, especially on the cephalic and scapular plates, and also on the central portion of the median plate I and centroposterior portions of segmental plates. The cephalic plate is relatively large whereas the cervical (neck) plate is barely demarcated from the scapular plate, formed only as thin grey belt without pillars. The scapular plate large, with additional lateral sutures separating narrow rectangular lateral portions with poorly developed pillars. Paired segmental plates divided into a smaller, much narrower anterior and a dominant posterior part by a smooth, wide transverse stripe (Fig. 1A ). The caudal (terminal) plate with short incisions and fully developed epicuticular layer. Median plate I unipartite, whereas median plate II divided into weakly defined parts, with a wide rhomboidal smooth space between them (Fig. 1A ). Median plate III small but with a well-developed epicuticular layer. Ventral cuticle with minute endocuticular pillars distributed throughout the whole venter, and a pair of oval subcephalic (Fig. 1C ) and trapezoid genital plates. Sexpartite gonopore placed between genital plates, and a trilobed anus between legs IV.

Pedal plates I-III absent, pedal plate IV developed as a dark matrix without pillars, bearing a typical dentate collar (Figure 1A ). Distinct pulvini on all legs (Fig. 1A ). A small spine on leg I (Fig. 1E ) and a papilla on leg IV present. Claws IV slightly higher than claws I-III (Table 2 ). External claws on all legs smooth (Figure 1E ). Internal claws with large spurs positioned at circa 1/3 of the claw height and bent downwards.

Buccal apparatus short, with a rigid, stout tube and a spherical pharynx. Stylet supports absent.

Mature males and sexually dimorphic traits (i.e. from the third instar onwards; measurements and statistics in Tables 3 , 4 ). Generally resembling females, but a closer observation reveals two qualitative differences (body appendage configuration and dorsal plate sculpturing) and numerous morphometric dissimilarities between males and females (all summarised in Table 4 ). Densely punctuated areas in the central leg portions present (Fig. 2A ). Male genital plates are always clearly visible (of identical shape as female plates), and dark densely arranged pillars are present in the entire genital zone, extending between the plates (Fig. 1D ).

Juveniles (i.e. the second instar, measurements and statistics in Table 5 ). Clearly smaller than adult females and males, with the body appendage configuration A-C-D-Dd-E. Endocuticular pillars well developed in all plates, the largest pillars present in the posterior portion of the scapular plate and in the central part of the caudal (terminal) plate. Epicuticular ornamented pattern absent, although lighter and darker parts of the scapular plate can be distinguished under PCM (Fig. 1B ), constituting presumably the developing epicuticular layer.

Larvae. Unknown.

Eggs. Up to two round, yellow eggs per exuvia were found.

Genetic markers and phylogenetic position. The 18S rRNA, 28S rRNA and ITS-2 were characterised by single haplotypes (GenBank accession numbers: MT106621, MT106620, MT106622, respectively), but three haplotypes were detected in the case of ITS-1 (MT106623-5), and five in COI (MT106223-7). All three DNA-based phylogenetic reconstructions revealed E. masculinus sp. nov. as the sister species to the clade E. lineatus + E. virginicus with a maximum support (Fig. 4 ). The divergence between the new species and the other two congeners was notably larger in COI compared to the ITS markers (compare Fig. 4A and 4B, C ). The differences are congruent with the p -distances (see SM.2).

Type material.

Holotype (mature female, slide MY.026.05), allotype (mature male, slide MY.026.07) and 42 paratypes on slides MY.026.01-09. Moreover, one voucher specimen (hologenophore) mounted on the slide MY.026.14. In total: 21 females, 14 males, and nine juveniles. Slides MY.026.01-07 are deposited in the Institute of Zoology and Biomedical Research, Jagiellonian University, Poland; slide MY.026.08 (4♀♀, 3♂♂, one juvenile) is deposited in the Natural History Museum of Denmark, University of Copenhagen, Denmark; slide MY.026.09 (4♀♀, 2♂♂, 2 juveniles) is deposited in the Catania University, Sicily, Italy. Found together with a new species of Echiniscus and a new species of Pseudechiniscus (descriptions in preparation).

Type locality.

Ca 6°05'N, 116°32'E, ca 3500 m a.s.l.: Malaysia, Borneo, Sabah, Gunung Kinabalu; subalpine vegetation zone with single Leptospermum and Rhododendron ericoides bushes, moss on a stunted tree trunk.

Etymology.

From Latin Echiniscus masculinus = male (an adjective in the nominative singular). The name underlines the presence of males in the new species, in contrast to closely related parthenogenetic E. lineatus and E. virginicus .

Differential diagnosis.

There are four known members of the E. virginicus complex: E. clevelandi Beasley, 1999, E. hoonsooi Moon & Kim, 1990, E. lineatus Pilato et al., 2008, and E. virginicus Riggin, 1962 ( Gąsiorek et al. 2019a). Echiniscus masculinus sp. nov. can be differentiated from (body appendage configuration given collectively for both sexes):

E. clevelandi, recorded from China, the only other dioecious representative of this group, by the body appendage configuration (A-C-D- (D d) -E in E. masculinus sp. nov. vs A-B-C-C d-D-D d-E in E. clevelandi) and dorsal sculpturing (faint and poorly visible epicuticular layer with pseudopores in E. masculinus sp. nov. vs well-developed epicuticular layer with bright and large pores in E. clevelandi; see Pilato et al. 2008).

E. hoonsooi, recorded from Korea, by the body appendage configuration (A-C-D- (D d) -E in E. masculinus sp. nov. vs A- (C) - (D) -E in E. hoonsooi), homomorphic spurs on all legs (heteromorphic spurs I-III and IV in E. hoonsooi; see Abe et al. 2000), and by the presence of males.

E. lineatus, distributed widely in the tropical and subtropical zone, by the body appendage configuration (A-C-D- (D d) -E in E. masculinus sp. nov. vs A- (B) -C-C d-D-D d-E in E. lineatus), and by the presence of males.

E. virginicus, native to the eastern Nearctic realm, by the body appendage configuration (A-C-D- (D d) -E in E. masculinus sp. nov. vs A- (B) -C-C d-D-D d-E in E. virginicus), dorsal plate sculpturing (pseudopores in E. masculinus sp. nov. vs pores in E. virginicus), and by the presence of males.

Figures

Figure 1. Morphology of Echiniscus masculinus sp. nov. (PCM). A. Adult female (holotype, dorsolateral view); B. Juvenile (paratype, dorsolateral view); C. Subcephalic plates; D. Genital plates enclosing male gonopore; E. First leg pair with claws and spine I. All scale bars in µm.

Figure 2. Morphology of males of E. masculinus sp. nov. (PCM). A. allotype (dorsolateral view, arrowheads indicate areas with densely packed pillars in legs); B. paratype with fully developed sculpturing (dorsal view); C. paratype with poorly developed epicuticular layer of sculpturing (dorsal view). See Table 4 for the phenotypic comparison between females and males. All scale bars in µm.

Figure 3. Close-up on the details of sculpturing of E. masculinus sp. nov. (PCM). A. evident epicuticular layer, endocuticular pillars of various sizes; B. remnants of epicuticular layer on the scapular and caudal (terminal) plates, endocuticular pillars densely packed and of equal, minute size. All scale bars in µm.

Figure 4. Bayesian phylogenetic trees showing the relationships between members of the E. virginicus complex; E. succineus was used as an outgroup, and branches within species-specific clades were collapsed. Bayesian posterior probability values are given above tree branches. Phylogenetic analyses were run on the subsequent DNA markers to assure that the tree topology was congruent: COI, ITS- 1, and ITS- 2.