New Echiniscidae (Heterotardigrada) from Amber Mountain (Northern Madagascar)

A moss sample from the local biodiversity hotspot in lowland rainforest in the vicinity of Amber Mountain, Madagascar, yielded the discovery of two Echiniscus C.A.S. Schultze, 1840 species, of which one is new to science. Echiniscus succineus sp. nov. is related to other members of the spinulosus group, but differs from them by the highly complicated structure of the dorsal plates, with intricately thickened parts of the armour forming orna mented pattern. The validity of the intraporal dark rings as a taxonomic trait is discussed in the context of the recovered intraspecific variability for the new taxon. Besides, rare Echiniscus africanus Murray, 1907 is reported for the first time from the island.


Introduction
Madagascan fauna is widely recognised among biologists for its unprecedented level of endemism and notable species diversity (Myers et al. 2000;Goodman andBenstead 2003, 2005;Holt et al. 2013).However, such enormous biodiversity, like in the majority of the tropical regions of the globe, is in great danger due to massive extirpation of rainforests (Brown and Gurevitch 2004).As an immense fraction of the world's biodiversity remains unexplored (Mora et al. 2011), especially within the taxonomic groups belonging to aquatic meiofauna and limno-terrestrial microfauna, degradation of so unique ecosystems threatens many organisms.Tardigrades are micrometazoans that can be found both in sea and land habitats (Nelson et al. 2015), constituting an important portion of local species  (Maucci 1993, Pilato andLisi 2003).As a consequence, only 13 species were recorded from Madagascar.In the present contribution, we present the results of systematic study on two Echiniscus C.A.S. Schultze, 1840 species found in the Diana Region located in the Northern Madagascar.Echiniscus succineus sp.nov. is described by the means of morphological and genetic analyses and is included within the spinulosus group as it exhibits evident pores on all dorsal plates and appendages in the form of spines.Echiniscus africanus Murray, 1907, previously reported from South Africa, Angola, Tanzania, and Lesotho (da Cunha and do Nascimento Ribeiro 1964, Binda and Pilato 1995, Middleton 2003, Gąsiorek and Kristensen 2018), is also recorded and illustrated.

Sample processing and microscopy
Twenty-two specimens of the new species and a single juvenile of E. africanus were extracted from one moss sample, collected from a tree at the edge of lowland rainforest in the vicinity of Amber Mountain (see the subsection Material examined for precise location) in December 2018.Dry material was placed and maintained in distilled water for 12 hours, approximately two weeks after collection.Tardigrade extraction procedure followed Stec et al. (2015).Hoyer's medium was chosen for mounting the animals on permanent slides.Fifteen representatives of the new species and the individual of E. africanus were examined and photographed under a Nikon Eclipse 50i phase contrast microscope (PCM) associated with a Nikon Digital Sight DS-L2 digital camera.Three specimens of the new species were processed for scanning electron microscopy (SEM) according to the protocol by Stec et al. (2015).All figures were assembled in Corel Photo-Paint X8 software.For deep structures that could not be fully focused in a single photograph, a series of 5-8 images were taken every ca.0.2 μm and then assembled into a single deep-focus image.

Morphometrics and terminology
All measurements are given in micrometres (μm) and were taken under PCM with Nikon Digital Sight DS-L2 software.Structures were measured only if their orientations were suitable, and structures were not twisted or broken.Body length was measured from the anterior to the posterior end of the body, excluding the hind legs.The sc ratio is the ratio of the length of a given structure to the length of the scapular plate (Fontoura and Morais 2011; values italicised in the tables).Morphometric data were handled using the Echiniscoidea ver.1.2 template available from Tardigrada Register, www.tardigrada.net/register(Michalczyk and Kaczmarek 2013).Detailed measurements are additionally provided as Suppl.material 1.General taxonomy and morphological terminology follow Kristensen (1987), with the further amendments introduced by Gąsiorek et al. (2017Gąsiorek et al. ( , 2019)).

Genetic data
DNA was extracted from four individuals of the new species (all animals were examined under 400× magnifica-tion in PCM prior to DNA extraction) following a Chel-ex® 100 resin (Bio-Rad) extraction method by Casquet et al. (2012) with modifications described in detail by Stec et al. (2015).Four molecular markers were sequenced (18S rRNA, 28S rRNA, ITS-2 and cox1); see Table 1 for primers and their source details.All fragments were amplified and sequenced according to the protocols described in Stec et al. (2015).Available 18S rRNA and 28S rRNA (dataset identical as in Gąsiorek et al. 2019) + ITS-2 and cox1 Echiniscus sequences were uploaded from GenBank to be aligned using default settings of MAFFT version 7 (Katoh et al. 2002;Katoh and Toh 2008).Uncorrected pairwise distances for trimmed alignments (898 bp -18S rRNA, 670 bp -28S rRNA, 427 bp -ITS-2, 535 bp -cox1) were calculated using MEGA7 (Kumar et al. 2016) and are presented in the Suppl.material 2.  Synthetic description.Body yellow and plump, 140 μm long.Cephalic appendages lengths: cirrus internus 12.7, cephalic papilla (secondary clava) 5.8, cirrus externus 14.7, primary clava 4.1, cirrus A 30.3.Trunk appendage formula C-C d -D-D d -D cd -E, most spines of similar lengths (16.1-19.0),but spines C d and D cd much shorter (7.5-9.4), and two additional spicules (2.5-3.1)present at the posterior edge of the scapular plate (29.6).The dorsal plate sculpture of the mixed type (sensu Gąsiorek et al. 2019), with large pores surrounded by polygonal edges on the scapular and caudal plates, and endocuticular pillars visible as densely arranged dark dots on the remaining plates, sometimes covered by thick epicuticular ornamentation (Fig. 1).* -All PCR programmes are also provided in Stec et al. (2015).Leg appendages and claw lengths: spine on the first leg pair 2.6, papilla on the fourth leg pair 3.9, claws I-IV 7.5-9.3.Serrated fringe on the fourth leg pair consisting of nine teeth.

Taxonomic account
Distribution.This elusive species has been reported several times only from Southern and Eastern Africa since its description over a century ago (McInnes et al. 2017, Gąsiorek andKristensen 2018).The record from Vietnam (Węglarska 1962) suggests either disjunctive range or misidentification with E. semifoveolatus Ito, 1993, which, however, is not properly delimited from the former species (Qiao et al. 2013).
Remarks.The specimen lacks lateral spines B and centrodorsal (mediodorsal) spines C cd , which are characteristic for this species (Murray 1907(Murray , 1913)).However, both positions are highly instable in terms of the presence/absence of appendages, which was demonstrated for E. lapponicus Thulin, 1911 with similar appendage configuration (Dastych 1980).

Echiniscus succineus sp. nov.
http://zoobank.org/D0F1B3CA-D1E6-49C3-8E2F-77973244E9E6Figs 2-5, Tables 2, 3 Material examined.Holotype (adult female on the slide MG.005.05) and sixteen paratypes (slides MG.005.04-7,including two voucher exoskeletons preserved after DNA extraction on the slides MG.005.28-29 and two specimens on the SEM stub no.17  elements of sponge-like endocuticular layer visible under SEM (Fig. 3D, 4B).The level of development of the rings is not associated with life stage, and some individuals do not exhibit intraporal rings.Cephalic plate large, halved, with scarce and minute pores (Figs 2, 3A-C).Cervical (neck) plate present, poreless and developed as grey rectangular belt adjacent to the anterior margin of the scapular plate (Fig. 3A-C).Scapular plate with the system of thick epicuticular extensions, dividing its surface into clearly defined areas of thinner cuticle, being lighter under PCM and slightly concave under SEM (Fig. 3A-C).Median plates I-III large and uniformly dark under PCM, the first and the third plate are unipartite, whereas the second one is bipartite, with its anterior portion being a poorly developed, narrow triangle (Figs 2, 3A-B).At the posterior margins of median plates I-II and paired plates, irregular epicuticular thickenings may be present, especially in larger animals (compare Figs 3A-B, D).Each of paired plates indistinctly divided by a thin smooth band into a narrow anterior portion with condensed epicuticular matrix, and a larger posterior portion with more complex ornamentation pattern.The proximal part of each posterior portion is thick similarly to the anterior portion, but more distal one is thinner, with reduced and less numerous pores (Figs 3A-B, D).Marginally, a single dark epicuticular belt is present, and the second belt appears more centrally (Fig. 3A), however, sometimes it is not discernible (Fig. 3B).Caudal (terminal) plate with typical incisions, rarely sclerotised as if being a prolonged extensions of spine E (Fig. 3B).Its epicuticular ornamentation is similar in form to that occurring on the scapular plate (Figs 2, 3A), but may be less developed (Fig. 3B, 3E).
Ventral plates absent, but simple granulation covers the entire venter from the subcephalic to genital zone.Endocuticular pillars minute and not-differentiated in size.Pedal plates and pulvini absent.Spine on the first leg pair minuscule (Figs 2A, 2C-D, 5A), either in the form of usual triangle or blunt (Fig. 2A).Dentate collar on the fourth leg pair present, with short teeth similar in shape (Figs 2, 3B).Papilla on the fourth leg pair present (Figs 2, 3B).External claws spurless, but internal ones bear acute spurs inserted at ca. 20-25% of the claw branch and directed downwards (Figs 2C, 5).Claws IV longer than claws I-III.
Larvae and eggs.Unknown.DNA barcodes.Four genetic markers were represented by single haplotypes.The 18S rRNA sequence (898 bp long, GenBank accession no.MK675903):

Etymology.
From Latin succineus = amber, referring to the locus typicus near Amber Mountain.An adjective in the nominative singular.

Discussion
The knowledge on the Madagascan tardigrade fauna is limited.Most of the taxa recorded by Maucci (1993) are now recognised as species complexes, thus the presence of type species being typical Palearctic elements on Madagascar is highly dubious (e.g.Guidetti et al. 2019, Morek et al. 2019).In result of the paucity of studies, its microfauna is of unknown origin at present (Yoder and Nowak 2006).E. africanus inhabits unchanged lowland rainforest on the island, which suggests some influence of Afrotropical fauna on Madagascan biota.Interestingly, species most similar in terms of morphology to E. succineus sp.nov.also occur in the tropical and subtropical zone.
Traditional species delineation in many Echiniscidae relied on the appendage configuration, however the spinulosus group poses a significant problem in this context as characterised by high variability in symmetry and presence of trunk spines.Pilato et al. (2008) introduced the presence of intraporal rings as a specific trait and an additional criterion in the taxonomy of the spinulosus group.Nevertheless, the variability in the development of these structures, shown for the first time to be endocuticular elements of the sponge-like layer, in E. succineus sp.nov., suggests the need for re-assessment of the validity of this feature.Considering the fact of explicitly emphasised significance of the dorsal plate sculpturing for classification and understanding the phylogeny of the Echiniscus lineage (Gąsiorek et al. 2019), the clarification of this issue would be desirable.There is a possibility that some species within the spinulosus complex always exhibit or do not exhibit dark rings, whereas other taxa are more inconstant in that respect.
.11).Except for two paratypes (slide MG.005.04)deposited in the Natural History Museum of Denmark, University of Copenhagen, the en-tire type series deposited in the Institute of Zoology and Biomedical Research, Jagiellonian University, Poland.Locus typicus.Lowland rainforest close to the road from Joffreville (Diana Region, Antsiranana Province, Northern Madgascar); coordinates and altitude: 12°30'49"S, 49°10'56"E; 993 m asl.Substratum: moss growing on a tree branch (ca.two metres above ground level); collection: December 2018 by W. Witaliński.Diagnosis.Small representative of the Echiniscus spinulosus group with peculiarly complex dorsal plate sculpturing developed as thick epicuticular ridges on scapular, paired segmental and caudal plates.Spines in almost all lateral and dorsal trunk positions.Parthenogenetic.Description.Adult females and juveniles.Body dark yellow and plump.Red eyes present, dissolved after mounting.External cirri not markedly longer than internal cirri, with swollen cirrophores (Fig. 3C).Primary and secondary clavae (cephalic papillae) of similar lengths.Cirrus A short (cirrus A/body length ratio below 20%), with short and poorly marked cirrophore.Trunk appendage configuration (B)-C-C d -D-D d -E in adults (Figs 2, 3A-B), reduced to (C d )-D d -E in juveniles.All appendages in form of spines of similar lengths, spines D d and E more robust, and sometimes gently serrated or rough (Figs 3A-B).Asymmetry in the development of appendages frequent, one of the spines B almost always absent, more rarely one of the spines C and D absent.Dorsal plates with rather irregularly distributed, large to very large pores (spinulosus type; Figs 2, 3).Dark endocuticular rings variously developed: from barely visible on the central portions of median plates (Fig. 3A) to well-developed rings present in pores from different plates (Figs 3B, 4A); they are the
from: • E. marginatus, reported from Hawaii Archipelago, by the appendage configuration (A-(B)-C-C d -D-D d -E in E. succineus sp.nov.vs A-(C)-(D)-D d -E in E. marginatus), and the morphology of posterior portions of me-

Figure 5 .
Figure 5. Claws of Echiniscus succineus sp.nov.(SEM): A -first leg pair (small spine I visible in the upper right corner); B -fourth leg pair.Scale bars: in μm.

Table 1 .
Primers used for sequencing of DNA fragments (one mitochondrial and four nuclear) of Echiniscus succineus sp.nov.
the location of epicuticular ornamentation on the dorsal armour (except for the median plates, all trunk plates ornamented in E. succineus sp.nov.vs only scapular and caudal plates ornamented in E. ornamentatus), and the pore morphology (very large pores, sometimes with endocuticular dark rings in E. succineus sp.nov.vs minute pores, always without endocuticular dark rings in E. ornamentatus).The claws II-IV and all claw spurs seem to be relatively longer in E. succineus sp.nov.with respect to E. ornamentatus (compare values from Table 2 with table 4 from

Table 2 .
Measurements [in µm]of selected morphological structures of adult females (the 3 rd and older instars) of Echiniscus succineus sp.nov.mounted in Hoyer's medium.N -number of specimens/structures measured, Range refers to the smallest and the largest structure among all measured specimens; SD -standard deviation.

Table 3 .
Measurements [in µm] of selected morphological structures of juveniles (the 2 nd instar) of Echiniscus succineus sp.nov.mounted in Hoyer's medium.N -number of specimens/structures measured, Range refers to the smallest and the largest structure among all measured specimens; SD -standard deviation.