Zoosyst. Evol. 96 (1) 2020, 103-113 | DOI 10.3897/zse.96.49989 eee Ee BERLIN An overview of the sexual dimorphism in Echiniscus (Heterotardigrada, Echiniscoidea), with the description of Echiniscus masculinus sp. nov. (the virginicus complex) from Borneo Piotr Gasiorek!, Katarzyna Vonéina!, Lukasz Michalcezyk* 1 Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387 Krakéw, Poland http://zoobank.org/48BDE4B7-B052-4A 00-AF36-BF2F5C7E7285 Corresponding author: Piotr Gasiorek (piotr.lukas.gasiorek@gmail.com) Academic editor: Martin Husemann ¢ Received 8 January 2020 Accepted 25 February 2020 # Published 20 March 2020 Abstract Members of the genus Echiniscus C.A.S. Schultze, 1840 are mostly unisexual, with thelytokously reproducing females. Therefore, every newly described dioecious species in the genus is particularly interesting. Here, we describe Echiniscus masculinus sp. nov. from Gunung Kinabalu, the highest peak of Borneo and the entire Southeast Asia. The new species belongs in the predominantly par- thenogenetic EF. virginicus complex, and its females are confusingly similar to females of the pantropical E. /ineatus Pilato et al., 2008, another member of this group. However, genetic evidence and noticeable sexual dimorphism clearly delineate the new species. Males of EF. masculinus sp. nov. are unlike females in the body proportions, cuticular sculpturing, and appendage configuration. The new dis- coveries provide a justification to review the current knowledge about evolution and forms of sexual dimorphism within Echiniscus. Key Words bisexual, clavae, dioecious, Echiniscidae, endemic, Gunung Kinabalu, limno-terrestrial life cycle, tropics Introduction A swiftly increasing number of tardigrade species is cur- rently at circa 1300 species (Guidetti and Bertolani 2005; Degma and Guidetti 2007; Degma et al. 2009-2019), which have already approached the conservative estimate of Bartels et al. (2016). This number has also exceeded the mean estimate of circa 1150 (upper 95% CI >2100) limno-terrestrial tardigrade species based on a protocol by Mora et al. (2011). Recent works have included DNA barcoding in modern tardigrade taxonomy, disclosing numerous species complexes in various phylogenetic lin- eages of the phylum (e.g. Stec et al. 2018; Guidetti et al. 2019; Cesari et al. 2020). On the other hand, many tardi- grade groups contain a significant number of dubious or synonymic taxa (e.g. Gasiorek et al. 2019b). Within the class Heterotardigrada, the greatest progress in solving taxonomic and phylogenetic problems has been made re- garding a fascinating group of armoured limno-terrestrial tardigrades, the family Echiniscidae (Kristensen 1987; Jorgensen et al. 2011, 2018; Vicente et al. 2013; Vecchi et al. 2016; Gasiorek et al. 2018a, 2018b, 2019b; Cesari et al. 2020). Recently, Gasiorek et al. (2019a) demonstrat- ed synonymy within the Echiniscus virginicus complex, reducing the number of valid species from five to four, and for the first time presenting an integrative evidence for a pantropical tardigrade species. Currently, only one member of this group, E. clevelandi (Beasley 1999), is dioecious. Gunung Kinabalu, together with the Crocker Range located farther south, constitute the highest prominence in the northern part of Borneo. Due to the remarkable ge- ological and climatic conditions, an altitudinal zonation of flora is present on Gunung Kinabalu (Kitayama 1992), which is a characteristic of many high mountain peaks in the Indomalayan region (van Steenis 1984; Ohsawa et al. 1985). Consequently, these mountains harbour unparal- leled animal diversity associated with rich plant vegeta- Copyright Piotr Gasiorek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 104 Piotr Gasiorek et al.: Dioecious Echiniscus masculinus sp. nov. from Borneo tion, even for the extraordinarily speciose faunae of the Malay Archipelago (Lohman et al. 2011; de Bruyn et al. 2014). On the other hand, as in many tropical areas, some animal groups remain barely known. This 1s the case with tardigrades, the subject of only two Bornean papers (Pila- to et al. 2004; Gasiorek 2018). Given the recent explosion of hidden species diversity in several tardigrade genera, it is more than likely that Bornean rainforests hold numer- ous undescribed tardigrade species. In this contribution, by using morphological and phy- logenetic methods, we describe Echiniscus masculinus sp. nov. from a high elevation in Gunung Kinabalu. The new species sheds light on the evolution of the E. vir- ginicus complex and raises questions about the prevalent type of speciation (sympatric vs allopatric) in this group. Finally, the sexual dimorphism within Echiniscus is com- pared to that of other echiniscids, and the apparent mor- phological stasis in females of the E. virginicus complex is discussed. Methods Sample collection and specimen preparation A total of 52 animals representing the new species was extracted from a moss sample collected in Northern Bor- neo by Maciej Barczyk on 29 June 2016 (sample code MY.026). The air-dried sample, stored in a paper enve- lope, was rehydrated in water for several hours, and the obtained sediment was poured into Petri dishes to search for microfauna under a stereomicroscope with dark field illumination. Individuals isolated from the sample were used for two types of analysis: imaging in light micros- copy (morphology and morphometry; 44 specimens) and DNA sequencing + phylogenetics (eight specimens). Imaging, morphometrics, and terminology Individuals for light microscopy and morphometry were first air-dried on microscope slides, and then mounted in a small drop of Hoyer’s medium and examined under a Nikon Eclipse 501 phase contrast microscope (PCM) as- sociated with a Nikon Digital Sight DS-L2 digital cam- era. All figures were assembled in Corel Photo-Paint X6, ver. 16.4.1.1281. For deep structures that could not be fully focused in a single light microscope photograph, a series of 2-12 images was taken every circa 0.1 um and then assembled into a single deep-focus image. All measurements are given in micrometres (um) and were performed under PCM. Structures were measured only if they were not damaged and if their orientations were suitable. Body length was measured from the anterior to the posterior end of the body, excluding the hind legs. The sp ratio 1s the ratio of the length of a given structure to the length of the scapular plate (Dastych 1999). Morpho- metric data were handled using the Echiniscoidea ver. 1.3 zse.pensoft.net template available from the Tardigrada Register, http:// www.tardigrada.net/register (Michalczyk and Kaczmarek 2013). The terminology follows Kristensen (1987) and subsequent changes proposed in Gasiorek et al. (2019b). For qualitative differential diagnoses, species descrip- tions and amendments of the four taxa constituting the Echiniscus virginicus group were studied (Riggin 1962; Moon and Kim 1990; Beasley 1999; Abe et al. 2000; Pi- lato et al. 2008; Kaczmarek and Michalczyk 2010; Gasi- orek et al. 2019a). Genotyping and phylogenetics The DNA was extracted from eight individual animals following a Chelex 100 resin (Bio-Rad) extraction meth- od by Casquet et al. (2012) with modifications described in detail in Stec et al. (2015). All specimens were mount- ed in water on temporary slides and examined under PCM before DNA extraction to ensure correct taxonomic identifications. One hologenophore cuticle (Pleiyel et al. 2008) was retrieved from an Eppendorf tube, mounted on a permanent slide, and deposited in the Institute of Zool- ogy and Biomedical Research in Krakow. We sequenced four nuclear and one mitochondrial DNA fragments: the small and the large ribosome subunit 18S rRNA and 28S tRNA (918 bp and 728 bp, respectively), the internal tran- scribed spacers ITS-1 and ITS-2 (642 and 484 bp, respec- tively), and the cytochrome oxidase subunit I COI (632 bp). All fragments were amplified and sequenced accord- ing to the protocols described in Stec et al. (2015); prim- ers and original references for specific PCR programmes are listed in Table 1. Sequences were aligned using de- fault settings of MAFFT7 (Katoh et al. 2002; Katoh and Toh 2008) under G-INS-i strategy. Uncorrected pairwise distances were calculated using MEGA7 (Kumar et al. 2016) and are included as the Suppl. material 2. To ensure that the topologies of the trees reconstructed on the basis of genetic markers were identical, we calcu- lated Bayesian inference (BI) marginal posterior proba- bilities using MrBayes ver. 3.2 (Ronquist and Huelsen- beck 2003) for each of the three markers (COI, ITS-1, and ITS-2) separately. Random starting trees were used, and the analysis was run for ten million generations, sam- pling the Markov chain every 1000 generations. An aver- age standard deviation of split frequencies of <0.01 was used as a guide to ensure that the two independent analy- ses had converged. The program Tracer ver. 1.3 (Rambaut et al. 2014) was then used to ensure that Markov chains had reached stationarity and to determine the correct ‘burn-in’ for the analysis, which was the first 10% of gen- erations. The ESS values were >200, and a consensus tree was obtained after summarizing the resulting topologies and discarding the ‘burn-in’. Trees were rooted on Echi- niscus succineus. Clades recovered with a posterior prob- ability (PP) between 0.95 and 1.00 were considered well supported, those with a PP between 0.90 and 0.94 were considered moderately supported, and those with a low- Zoosyst. Evol. 96 (1) 2020, 103-113 105 Table 1. Primers and references for specific protocols for amplification of the five DNA fragments sequenced in the study. DNA fragment Primername Primer Primer sequence (5’—3’) direction 18S rRNA 18S_Tar_Ffl forward AGGCGAAACCGCGAATGGCTC 18S Tar_Rr2 reverse CTGATCGCCTTCGAACCTCTAACTTTCG 28S rRNA 28S _Eutar_F forward ACCCGCTGAACTTAAGCATAT 28SRO0990 reverse CCTTGGTCCGTGTTTCAAGAC ITS-1 ITS1_Echi_F — forward CCGTCGCTACTACCGATTGG ITS1_Echi_R reverse GTTCAGAAAACCCTGCAATTCACG ITS-2 ITS3 forward GCATCGATGAAGAACGCAGC ITS4 reverse TCCTCCGCTTATTGATATGC Col bedFO1 forward CATTTTCHACTAAYCATAARGATATTGG bedRO4 reverse TATAAACY TCDGGATGNCCAAAAAA Primer source PCR programme* Stec et al. (2018) Gasiorek et al. (2017) Gasiorek et al. (2018a) Mironov et al. (2012) Gasiorek et al. (2019a) Zeller (2010) Mironov et al. (2012) Wetnicz et al. (2011) White et al. (1990) Dabert et al. (2008) * All PCR programmes are also provided in Stec et al. (2015). er PP were considered unsupported. All final consensus trees were viewed and visualized using FigTree ver. 1.4.3 (available at: https://tree.bio.ed.ac.uk/software/figtree). Data deposition Raw morphometric data are placed as the Suppl. mate- rial 1 and in the Tardigrada Register under http://www. tardigrada.net/register/0062.htm. Type DNA sequences are deposited in GenBank. Results Taxonomic account Phylum Tardigrada Doyere, 1840 Class Heterotardigrada Marcus, 1927 Order Echiniscoidea Richters, 1926 Family Echiniscidae Thulin, 1928 Genus Echiniscus C.A.S. Schultze, 1840 Echiniscus masculinus sp. nov. http://zoobank.org/99CA96E7-D111-4E07-A0A2-4133F54755C9 Figures 1-3, Tables 2—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 (sec- ondary clavae) and (primary) clavae; cirri growing out from bulbous cirrophores (Figure 1A). The body append- age configuration is A~-C-D-D*-E, with all trunk appendag- es formed as spines or spicules. All usual trunk appendages always symmetrical and smooth. Spine C’ rudimentarily developed in two females (one with an asymmetrical spic- ule [2 um], the other normally formed [8 um]). 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 epicutic- ular matrix forming the ornamented pattern together with pseudopores, enhanced as dark belts on the anterior por- Figure 1. Morphology of Echiniscus masculinus sp. nov. (PCM). A. Adult female (holotype, dorsolateral view); B. Ju- venile (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 um. tions 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 scap- ular 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, zse.pensoft.net 106 Piotr Gasiorek et al.: Dioecious Echiniscus masculinus sp. nov. from Borneo Table 2. Measurements [in um] of selected morphological structures of the adult females of Echiniscus masculinus sp. nov. mounted in Hoyer’s medium. N — number of specimens/structures measured, RANGE refers to the smallest and the largest structure among all mea- sured specimens; SD — standard deviation; sp—the proportion between the length of a given structure and the length of the scapular plate. Character N yum Body length 10 159-192 Scapular plate length 10 32.6-43.7 Head appendages lengths Cirrus internus 9 9.7-15.5 Cephalic papilla 10 5.9-7.8 Cirrus externus 8 12.3-18.8 Clava 10 4.7-6.4 Cirrus A 10 23.3-42.3 Cirrus A/Body length ratio 10 15%-24% Body appendages lengths Spine C 10 10.9-21.6 Spine D 10 11.2-21.6 Spine D? 10 2.9-16.8 Spine E 10 13.6-23.3 Spine on leg | length 10 3.0-3.9 Papilla on leg IV length 10 3.6-5.3 Number of teeth on the collar iS! 8-12 Claw | heights Branch 8 8.8-10.7 Spur 8 2.2-3.2 Spur/branch height ratio 8 24%-33% Claw II heights Branch 9 8.4-10.4 Spur 9 2.1-3.1 Spur/branch height ratio g 25%-33% Claw III heights Branch 10 8.4-10.2 Spur 10 2.0-3.1 Spur/branch height ratio 10 24%-31% Claw IV heights Branch 7 9.4-12.1 Spur 7 2.3-3.2 Spur/branch height ratio 7 24%-29% formed only as thin grey belt without pillars. The scapu- lar 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 II small but with a well-developed epicu- ticular 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 I'V. 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 1). Inter- nal claws with large spurs positioned at circa 1/3 of the claw height and bent downwards. zse.pensoft.net Range Mean SD Holotype sp ym sp ym sp ym sp 432-492 At 453 dnl pea 178 444 - 38.8 - 4 - 40.1 — 25.1-38.8 12.6 32.4 2.3 4.9 14.6 36.4 15.1-19.2 6.7 TP 0.5 3 6.4 16.0 37.7-47.0 16.6 42.7 Loa Toy el S36 46.9 11.4-17.1 5.5 14.2 0.6 1.6 5.6 14.0 69.6-105.5 32.8 Ck eat” oO. Ame 33.1 8275 - 19% - 3% - 19% - 33.4-56.3 16.6 43.0 Jol Sno PST Cr aere 29.5-57.9 16.0 41.3 ef 83 13:8 34.4 8.9-45.0 Lb 3031 A wt OR Oh 24.2 33.9-60.7 18.6 48.3 25y (82) 13.6 33.4 8.0-11.0 3.4 8.8 0.3 0.9 333 8.2 9.9-12.9 4.4 Lie 0.6 Lxd 4.6 11.5 - 10.1 - lass - ) - 23.5-27.6 OF 258 0.6 iJ. son 24.2 6.7-8.5 2.8 ee 0.3 0.6 ea 6.7 - 29% - 2% - 28% — 21.5-25.9 9.4 24.4 0.6 1.4 10.0 24.9 6.4-8.2 2.8 ft 0.3. "Oss 2.6 6.5 - 29% - 3% - 26% - 22./-26.2 9 24.5 0.6 12 99 24.7 6.1-7.2 2.6 6.6 0.3 O04 rage) Lie - 27% - 2% - 29% ~ 24,9-30.3 10.9 27.4 09 2.4 ? f 6.1-8.6 3.0 7.4 0.3 2029 ? ? - 27% - 1% - ? - Buccal apparatus short, with a rigid, stout tube and a spherical pharynx. Stylet supports absent. Mature males and sexually dimorphic traits (1.e. from the third instar onwards; measurements and statistics in Tables 3, 4). Generally resembling females, but a closer observa- tion 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-D‘-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 pre- sumably the developing epicuticular layer. Larvae. Unknown. Zoosyst. Evol. 96 (1) 2020, 103-113 107 Table 3. Measurements [in um] of selected morphological structures of the adult males of Echiniscus masculinus 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; sp — the proportion between the length of a given structure and the length of the scapular plate. Character N Range Mean SD Allotype ym sp ym sp ym sp ym sp Body length 10 142-170 464-527 161 493 9 ae 167 527 Scapular plate length 10 30.3-35.7 - 32.6 - 15 - all a - Head appendages lengths Cirrus internus 10 10.2-19.2 31.0-58.9 15.3. 47.2 2.3 7.7 15.0 47.3 Cephalic papilla 10 7.7-9.3 23.4-30.0 8.6 26.6 0.6 2a 8.6 271 Cirrus externus 10 16.0-21.0 47,3-67.3 18.8 57.8 1.6 6.0 l7- Bdy2 Clava 10 6.1-7.5 19.2-22.8 6.8 20.8 0.4 hat 6.1 19:2 Cirrus A 8 28.4-36.2 &84.6-111.0 31.9 98.0 2.8 91 30.0 94.6 Cirrus A/Body length ratio 8 18%-24% - 20% - 2% - 18% - Body appendages lengths Spine C 10 19.9-26.9 63.7-77.9 23.4 70.9 2:3 5.5 24.7 77.9 Spine D 10 17.6-29.7 54.0-83.2 23.0 70.4 3.4 8.5 25:0 78.9 Spine E 10 19.4-30.5 59.1-92.7 2A5 F5ul 4.1 12.0 27.7 87.4 Spine on leg | length 10 2.0-3.7 6.5-11.3 oa 9.6 O5 1.4 28 &.8 Papilla on leg IV length 10 3.8-5.3 12.4-16.2 4.6 14.2 0.5 il 4,1 12.9 Number of teeth on the collar S 7-12 - 9.4 - Lief - ay - Claw | heights Branch 10 8.4-10.7 26.5-33.0 9.4 28.9 Oe ame | 8.4 26.5 Spur 10 2.2-3.1 6.9-9.9 eae § 8.3 0.3 0.8 22 6:9 Spur/branch height ratio 10 23%-32% - 29% - 3% - 26% - Claw Il heights Branch is) 8.4-10.4 24,9-32.1 92 28.4 0.6 2.5 8.6 oF ad Spur 9 1.9-2.7 5.8-8.9 2.4 Yana 0.3 1.0 2.6 8.2 Spur/branch height ratio 9 20%-31% - 26% - 3% - 30% - Claw III heights Branch 8 8.5-10.1 25.8-31.4 9.2 28.3 0.6 270) 8.7 27.4 Spur 8 2.3-2.8 7.0-8.5 rae Pl Oe 0.5 23 yg: Spur/branch height ratio 8 24%-30% - 27% - 2% - 26% - Claw IV heights Branch 4 9.5-10.4 28.1-34.0 hea ind 0.4 3.2 2 ? Spur 4 2./-3.1 8.3-9.2 vo &.8 0.2 0.4 ? ? Spur/branch height ratio 4 26%-30% - 28% - 2% - ? - Table 4. Sexual dimorphism in qualitative and quantitative traits in Echiniscus masculinus sp. nov., with results of one-tailed Stu- dent’s ¢-tests in case of overlapping ranges in morphometric traits (all statistically significant at the a-level of p,,,< 0.05 adjusted with the Benjamini-Hochberg correction). Remarks a single male had an asymmetrically developed spine D7 [8 um long] compare Figures 1, 2A, B and 3A, Qualitative traits ore) 3d Body appendage configuration A-C-D-D9-E A-C-D-E Cuticular sculpturing epicuticular ornamentation epicuticular ornamentation poor pronounced but see also Figures 2C and 3B for an atypically poor sculpturing inamale Quantitative traits 2o:x +SD,N=10 dd: xX +SD,N=10 t, p Body proportions: bs ratio 0.54-0.57 (= body larger and 0.48-0.49 (= body smaller and non-overlapping ranges; see also plump) slender) Fig. 2 Body length 175 = Wl) Lie Teo t.g= 3.27; p= 0.002 Scapular plate length 38.8 + 3.2 32.6+1.5 tg= 0.51; p< 0.001 Head appendages lengths Cephalic papilla 173 153 26.64 2.2 tg=-11.47; p< 0.001 Clava 14.2 + 1.6 20.8% 1.1 t.g= 10.44; p< 0.001 Body appendage lengths Spine C 43.0 + 8.3 FG, Oise 525 t.g= -8.90; p< 0.001 Spine D 41.3 + 8.3 70.4 + 8.5 tg=-7./5; p< 0.001 Spine E 48.3 + 8.2 7 eo Mave ste he t.g= -5.79; p< 0.001 Claw branch heights Claw | Poms peril Wad 2S OF 247 t.,= -3.99; p< 0.001 Claw II 24.44+1.4 28.44 2.5 t.,= -4.36; p< 0.001 Claw Ill 24,54+1.2 28.3: #:2.0 {= 9.01; p< 0.001 zse.pensoft.net 108 Figure 2. Morphology of males of £. masculinus sp. nov. (PCM). A. allotype (dorsolateral view, arrowheads indicate ar- eas 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 um. 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 haplo- types 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 FE. Jineatus + E. vir- zse.pensoft.net Piotr Gasiorek et al.: Dioecious Echiniscus masculinus sp. nov. from Borneo Figure 3. Close-up on the details of sculpturing of E. masculi- nus sp. nov. (PCM). A. evident epicuticular layer, endocuticu- lar 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 um. ginicus 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 (com- pare 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 In- stitute of Zoology and Biomedical Research, Jagiellonian University, Poland; slide MY.026.08 (499, 343, one juvenile) is deposited in the Natural History Museum of Denmark, University of Copenhagen, Denmark; slide MY.026.09 (499, 244, 2 juveniles) is deposited in the Catania University, Sicily, Italy. Found together with a new species of Echiniscus and a new species of Pseude- chiniscus (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 Rhodo- dendron ericoides bushes, moss on a stunted tree trunk. Etymology. From Latin masculinus = male (an adjec- tive in the nominative singular). The name underlines the presence of males in the new species, in contrast to close- ly related parthenogenetic E. /ineatus and E. virginicus. Zoosyst. Evol. 96 (1) 2020, 103-113 109 Table 5. Measurements [in um] of selected morphological structures of the juveniles of Echiniscus masculinus 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; sp — the proportion between the length of a given structure and the length of the scapular plate. Character N Range Mean SD ym sp ym sp ym sp Body length 5 115-148 431-477 129 454 ke 18 Scapular plate length 5 26.0-34.4 - 28.4 - 3.6 - Head appendages lengths Cirrus internus 5 7. 4-12.3 27.1-35.8 8.8 30:7 2.0 32D Cephalic papilla 5 3.8-6.4 13.1-19.6 4.9 eS 1.0 27 Cirrus externus 4 8.8-14.2 33.3-41.3 10.8 38.0 us 3.4 Clava 5 3.7-5.4 13.7-16.7 4.3 15.2 0.7 1.2 Cirrus A 5 19.5-28.6 74,1-83.5 22.8 80.1 3.5 37 Cirrus A/Body length ratio 5 16%-19% - 18% - 1% - Body appendages lengths Spine C 5 8.1-20.3 30.8-59.0 129: 44.6 4.6 1OZF Spine D 5 7.4-17.5 28.5-50.9 11.6 39.9 4.1 9.3 Spine D? 5 7.1-16.1 27.0-46.8 10.4 35.9 3.4 #23 Spine E 5 10.8-18.0 40.2-52.3 V2, 44,3 3.0 4.9 Spine on leg | length 4 1.9-2.7 7.2-9.1 22 7.9 0.4 0.9 Papilla on leg IV length 5 3.2-3.8 10.8-14.4 3.4 12.2 0.3 1.5 Number of teeth on the collar 5 7-8 - 7.6 - 0.5 - Claw | heights Branch 5 6.3-9.3 24.0-27.0 Fis 25.6 le? i? Spur 5 1.5-2.7 5.2-8.1 1.9 6.8 O:5 I. Spur/branch height ratio 5 21%-31% - 27% - 4% - Claw II heights Branch 4 6.1-6.8 23.2-25.0 6.5 24.0 0.3 0.9 Spur 4 1.4-1.9 5.4-7.2 1 6.2 Or 0.8 Spur/branch height ratio 4 22%-31% - 26% - 4% - Claw III heights Branch 4 6.3-8.9 23.0-25.9 Pai 24.3 LZ i e. Spur 4 1.7-2.5 5.8-7.3 2.0 6.7 0.4 0.6 Spur/branch height ratio 4 25%-29% - 28% - 1% - Claw IV heights Branch 4 6.7-9.1 25.4-27.7 7.6 PCRS 1:6 se | Spur 4 1.8-2.8 6.8-8.5 ae Ue], 0.4 0.8 Spur/branch height ratio 4 25%-33% - 29% - A% - 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 EF. virginicus Riggin, 1962 (Gasiorek et al. 2019a). Echiniscus masculinus sp. nov. can be differen- tiated from (body appendage configuration given collec- tively for both sexes): 1. E. clevelandi, recorded from China, the only oth- er dioecious representative of this group, by the body appendage configuration (A-C-D-(D*)-E in E. masculinus sp. nov. vs A-B-C-C?-D-D*-E in E. clevelandi) and dorsal sculpturing (faint and poor- ly 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). 2. E. hoonsooi, recorded from Korea, by the body ap- pendage configuration (A-C-D-(D‘)-E in E. mascu- linus sp. nov. vs A-(C)-(D)-E in E. hoonsooi), ho- momorphic spurs on all legs (heteromorphic spurs I-III and IV in E. hoonsooi; see Abe et al. 2000), and by the presence of males. 3. E. lineatus, distributed widely in the tropical and subtropical zone, by the body appendage configu- ration (A-C-D-(D‘)-E in E. masculinus sp. nov. vs A-(B)-C-C*-D-D*-E in E. lineatus), and by the pres- ence of males. 4. E. virginicus, native to the eastern Nearctic realm, by the body appendage configuration (4-C-D-(D- “)-E in E. masculinus sp. nov. vs A-(B)-C-C*-D- D*-E in E. virginicus), dorsal plate sculpturing (pseudopores in £. masculinus sp. nov. vs pores in E. virginicus), and by the presence of males. Discussion The Echiniscus virginicus complex contains species with well-defined geographical ranges: E. /ineatus is pantrop- ical, E. clevelandi and E. hoonsooi are known from Far East Asia, and E. virginicus has been recorded only from the Nearctic (Gasiorek et al. 2019a). Phylogenetic analy- ses inferred the new species as sister to the clade E. linea- tus + E. virginicus, with the latter two more closely related to each other than to E. masculinus sp. nov. (Fig. 4). This is surprising for two reasons: the same place of origin of E. masculinus sp. nov. and E. lineatus, the tropics, as both occur only there, and the morphological similarity of these two species, since they both have pseudopores. zse.pensoft.net 110 Piotr Gasiorek et al.: Dioecious Echiniscus masculinus sp. nov. from Borneo Echiniscus masculinus sp. nov. Borneo COl Echiniscus lineatus Pantropical Echiniscus virginicus Nearctic a ; 0.01 lf— Echiniscus succineus Madagascar — [TS-1 Echiniscus masculinus sp. nov. Borneo Echiniscus lineatus Pantropical Echiniscus virginicus Nearctic [fm————_Echiniscus succineus Madagascar ITS-2 Echiniscus masculinus sp. nov. Borneo Echiniscus lineatus Pantropical Echiniscus virginicus Nearctic f—— Echiniscus succineus Madagascar — 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. As it 1s generally assumed that dioecy is ancestral, and parthenogenetic thelytoky 1s an advanced character within Echiniscidae (e.g. Kristensen 1987), the presence of males within populations of £. masculinus sp. nov. 1s probably a retained plesiomorphy of the entire complex. Given that the new species 1s described from a very peculiar habitat, namely a prominent mountain peak with high levels of en- demism characterising many groups of animals (Merckx et al. 2015), the isolated locality suggests a contracted, relictual geographic range of EF. masculinus sp. nov. and its potentially restricted area of occurrence (only Gunung Kinabalu or maybe also other high mountains of Borneo). zse.pensoft.net In contrast to arthrotardigrades, usually ancestrally di- oecious (Fontoura et al. 2017), echiniscoidean taxa are more diversified in terms of reproductive modes and many groups embrace both parthenogenetic and dioecious spe- cies. Echiniscoididae and Oreellidae are bisexual (Kris- tensen and Hallas 1980; Dastych et al. 1998; Mobjerg et al. 2016), but sexual dimorphism is not well-marked in either of the two. The first observations on sexual dimor- phism within Echiniscidae were documented by Dastych (1987) and Kristensen (1987). At present, males have been reported for 14 echiniscid genera: Antechiniscus (Claxton 2001), Barbaria (Miller et al. 1999; Michalczyk and Kaczmarek 2007), Bryodelphax (Gasiorek and Deg- ma 2018), Claxtonia (Kaczmarek and Michalczyk 2002; Mitchell and Romano 2007), Cornechiniscus (Dastych 1979), Diploechiniscus (Vicente et al. 2013), Hypechinis- cus (Kristensen 1987), Mopsechiniscus (Dastych 2001), Novechiniscus (Rebecchi et al. 2008), Proechiniscus (Kristensen 1987), Pseudechiniscus (Cesari et al. 2020), Stellariscus (Gasiorek et al. 2018b), Testechiniscus (Ga- siorek et al. 2018a), and Echiniscus. Sexual dimorphism can be obvious, as in Mopsechiniscus, or restricted to different gonopore shapes (e.g. in Cornechiniscus). Un- til now, males have been reliably discovered only in 11 Echiniscus spp. (Degma et al. 2009-2019): EF. clevelandi (the virginicus complex), E. curiosus Claxton, 1996 and E. merokensis Richters, 1904 (the merokensis complex), E. duboisi Richters, 1902 and E. siticulosus Gasiorek & Michalczyk, 2020 (the spinulosus complex), E. ehrenber- gi Dastych & Kristensen, 1995 and E. rodnae Claxton, 1996 (the testudo complex), E. jamesi Claxton, 1996 (the granulatus complex), E. lentiferus Claxton & Dastych, 2017 (the gquadrispinosus complex), E. marleyi Li, 2007 (the b/umi—canadensis complex), E. nepalensis Dastych, 1975 (the /apponicus complex). The differences between the sexes are often minor (Dastych 1975; Dastych and Kristensen 1995; Miller et al. 1999), but some authors emphasised notable disparities in morphometric traits (Beasley 1999; Claxton 1996; Claxton and Dastych 2017; Gasiorek and Michalczyk 2020). These encompass main- ly differences in body proportions, and dimensions of claws, cephalic and trunk appendages (Claxton 1996; Ga- siorek and Michalczyk 2020). The sex ratio varies greatly even between populations of a single species (Miller et al. 1999), indicating that there may be seasonal variations in the presence of males within Echiniscus populations, as was observed for other micrometazoans (Gilbert and Williamson 1983). Originally, the “Gondwanan” hypothesis was postulat- ed to explain the distribution of dioecious Echiniscus spp. (Miller et al. 1999). In fact, except for the cosmopolitan E. merokensis and East Palaearctic E. marleyi, other di- oecious Echiniscus spp. inhabit exclusively post-Gond- wanan lands. Additionally, males are generally absent or present in almost negligible proportions in European and Central Asian populations of Echiniscus (Jorgensen et al. 2007; Guil and Giribet 2009). The evolutionary causes of this phenomenon are, however, still unknown. Zoosyst. Evol. 96 (1) 2020, 103-113 The sexual dimorphism of FE. masculinus sp. nov., evi- denced in both quantitative and qualitative traits (Table 4) is interesting in the context of usually poorly marked sex- ual differences in dioecious Echiniscus spp., and the fact that females of E. /ineatus, E. virginicus, and E. masculinus Sp. nov. are confusingly similar to each other. In fact, fe- males are a good example of profound evolutionary stasis in morphology, which led, for example, to a description of a synonymous species in the complex (E. dariae syn- onymised with FE. /ineatus by Gasiorek et al. 2019a). In contrast, males of E. masculinus sp. nov. and E. clevelan- di can be easily distinguished based on the differences in dorsal sculpturing and appendage configuration (compare Beasley 1999 and the present study). Consequently, a ques- tion arises: why do females of the virginicus complex tend to diverge morphologically at a slower rate than males? The acquisition of genetic data for E. clevelandi and E. hoon- sooi could help to resolve this conundrum, as the putative, basal, character of E. clevelandi and E. masculinus sp. nov. within the virginicus clade would support the hypothesis that asexually reproducing species are young and poorly phenotypically differentiated from each other and from the ancestral female phenotype. Finally, considering that the sexually reproducing E. masculinus sp. nov. is a sister taxon to the asexual EF. Jineatus + E. virginicus clade, we hypothe- sise that the males were originally present in the ancestor of the clade. Moreover, given the overall similarity of males of E. clevelandi and E. masculinus sp. nov., we also hypothe- sise that males in the ancestral lineage leading to E. /ineatus and FE. virginicus were phenotypically similar to males of E. masculinus sp. nov. Conclusions The description of sexually dimorphic E. masculinus sp. nov. elucidates the evolution of the virginicus complex and raises new questions about the phenotype evolution in tardigrades. Females of three species (E. /ineatus, E. virginicus and E. masculinus sp. nov.) represent an exemplary case of delusively similar taxa (1.e. almost identical under PCM but easily identifiable with SEM analysis). The tardigrade fauna of the Indomalayan re- gion requires more sampling effort to uncover its diver- sity and uniqueness. Acknowledgements We are most grateful to Maciej Barczyk (Senckenberg Bio- diversity and Climate Research Centre, Goethe University Frankfurt, Germany) for the collection of the sample. We would also like to thank Diane Nelson, Reinhardt M. Kris- tensen, and an anonymous reviewer, who contributed to the improvement of this manuscript. The study was supported by the Polish Ministry of Science and Higher Education via the Diamond Grant (DI2015 014945 to PG, supervised by LM) and by the Sonata Bis programme of the Polish 1 IA National Science Centre (grant no. 2016/22/E/NZ8/00417 to LM). We owe our sincere thanks to the Museum fiir Naturkunde, Berlin, for covering the publication charge. References Abe W, Ito M, Takeda M (2000) First record of Echiniscus hoonsooi (Tardigrada: Echiniscidae) from Japan. Species Diversity 5: 103— 110. https://doi.org/10.12782/specdiv.5.103 Bartels PJ, Apodaca JJ, Mora C, Nelson DR (2016) A global biodi- versity estimate of a poorly known taxon: phylum Tardigrada. Zo- ological Journal of the Linnean Society 178: 730-736. https://doi. org/10.1111/zoj.12441 Beasley CW (1999) A new species of Echiniscus (Tardigrada, Echinisci- dae) from Northern Yunnan Province, China. Zoologischer Anzeiger 238: 135-138. Casquet J, Thebaud C, Gillespie RG (2012) Chelex without boiling, a rapid and easy technique to obtain stable amplifiable DNA from small amounts of ethanol-stored spiders. Molecular Ecology Resources 12: 136-141. https://doi.org/10.1111/j.1755-0998.2011.03073.x Cesari M, Montanari M, Kristensen RM, Bertolani R, Guidetti R, Rebecchi L (2020) An integrated study of the biodiversity within the Pseudechiniscus suillus—facettalis group (Heterotardigrada: Echiniscidae). Zoological Journal of the Linnean Society 188: 717— 732. https://doi.org/10.1093/zoolinnean/z1z045 Claxton SK (1996) Sexual dimorphism in Australian Echiniscus (Tar- digrada, Echiniscidae) with descriptions of three new species. Zo- ological Journal of the Linnean Society 116: 13-33. https://doi. org/10.1111/j.1096-3642.1996.tb02330.x Claxton SK (2001) Antechiniscus in Australia: Description of Antechiniscus moscali sp. n. and redescription of Antechiniscus parvisentus (Horning & Schuster, 1983) (Heterotardigrada: Echiniscidae). Zoologischer An- zeiger 240: 281—289. https://doi.org/10.1078/0044-523 1-00035 Claxton SK, Dastych H (2017) A new bisexual species of Echiniscus C.A.S. Schultze, 1840 (Heterotardigrada: Echiniscidae) from Tas- mania, Australia. Entomologie Heute 29: 105-119. Dastych H (1975) Some Tardigrada from the Himalayas (Nepal) with a description of Echiniscus (E.) nepalensis n. sp. Memorie dell’ Istitu- to Italiano di Idrobiologia 32 (Supplement): 61-68. Dastych H (1979) Tardigrada from Afghanistan with a description of Pseudechiniscus schrammi sp. nov. Bulletin de la Société des Amis des Sciences et des Lettres de Poznan, Série D, sciences biologiques 19: 99-108. Dastych H (1987) Two new species of Tardigrada from the Canadi- an Subarctic with some notes on sexual dimorphism in the family Echiniscidae. Entomologische Mitteilungen aus dem Zoologischen Museum Hamburg 8: 319-334. Dastych H (2001) Notes on the revision of the genus Mopsechiniscus (Tardigrada). Zoologischer Anzeiger 240: 299-308. https://doi. org/10.1078/0044-523 1 -00037 Dastych H, Kristensen RM (1995) Echiniscus ehrenbergi sp. n., a new water bear from the Himalayas (Tardigrada). Entomologische Mit- teilungen aus dem Zoologischen Museum Hamburg 11: 221—230. Dastych H, McInnes SJ, Claxton SK (1998) Oreella mollis Murray, 1910 (Tardigrada): a redescription and revision of Oreella. Mittei- lungen aus dem Hamburgischen Zoologischen Museum und Institut 95: 89-113. zse.pensoft.net Ne Piotr Gasiorek et al.: Dioecious Echiniscus masculinus sp. nov. from Borneo de Bruyn M, Stelbrink B, Morley RJ, Hall R, Carvalho GR, Cannon CH, van den Bergh G, Meijaard E, Metcalfe I, Boitani L, Maiorano L, Shoup R, von Rintelen T (2014) Borneo and Indochina are major evolutionary hotspots for Southeast Asian biodiversity. Systematic Biology 63: 879-901. https://doi.org/10.1093/sysbio/syu047 Degma P, Bertolani R, Guidetti R (2009-2019)Actual checklist of Tardigrada species. Ver. 36: 01-09-2019. https://doi.org/10.25431/11380_ 1178608 Degma P, Guidetti R (2007) Notes to the current checklist of Tardigrada. Zootaxa 1579: 41-53. https://doi.org/10.11646/zootaxa.1579.1.2 Fontoura P, Bartels PJ, Jogensen A, Kristensen RM, Hansen JG (2017) A dichotomous key to the genera of the marine heterotardigrades (Tardigrada). Zootaxa 4294: 1-45. https://doi.org/10.11646/zoot- axa.4294.1.1 Gasiorek P (2018) New Bryodelphax species (Heterotardigrada: Echiniscidae) from Western Borneo (Sarawak), with new molecular data for the genus. Raffles Bulletin of Zoology 66: 371-381. Gasiorek P, Degma P (2018) Three Echiniscidae species (Tardigrada: Heterotardigrada) new to the Polish fauna, with the description of a new gonochoristic Bryodelphax Thulin, 1928. Zootaxa 4410: 77-96. https://doi.org/10.11646/zootaxa.4410.1.4 Gasiorek P, Jackson KJ, Meyer HA, Zajac K, Nelson DR, Kristensen RM, Michalezyk L (2019a) Echiniscus virginicus complex: the first case of pseudocryptic allopatry and pantropical distribution in tar- digrades. Biological Journal of the Linnean Society 128: 789-805. https://doi.org/10.1093/biolinnean/blz147 Gasiorek P, Michalezyk L (2020) Echiniscus siticulosus (Echiniscidae: spinulosus group), a new tardigrade from Western Australian scrub. New Zealand Journal of Zoology 47: 87-105. https://doi.org/10.108 0/03014223.2019.1603166 Gasiorek P, Morek W, Stec D, Michalczyk L (2019b) Untangling the Echiniscus Gordian knot: paraphyly of the “arctomys group” (Het- erotardigrada: Echiniscidae). Cladistics 35: 633-653. https://doi. org/10.1111/cla.12377 Gasiorek P, Stec D, Zawierucha K, Kristensen RM, Michalczyk L (2018a) Revision of 7estechiniscus Kristensen, 1987 (Heterotardigra- da: Echiniscidae) refutes the polar-temperate distribution of the genus. Zootaxa 4472: 261-297. https://doi.org/10.11646/zootaxa.4472.2.3 Gasiorek P, Suzuki AC, Kristensen RM, Lachowska-Cierlik D, Michal- czyk L (2018b) Untangling the Echiniscus Gordian knot: Ste/lariscus gen. nov. (Heterotardigrada: Echiniscidae) from Far East Asia. Inver- tebrate Systematics 32: 1234-1247. https://doi.org/10.1071/IS18023 Gilbert JJ, Williamson CE (1983) Sexual dimorphism in zooplankton (Copepoda, Cladocera, and Rotifera). Annual Review of Ecol- ogy and Systematics 14: 1-33. https://doi.org/10.1146/annurev. es.14.110183.000245 Guidetti R, Cesari M, Bertolani R, Altiero T, Rebecchi L (2019) High diversity in species, reproductive modes and distribution within the Paramacrobiotus richtersi complex (Eutardigrada, Macrobiotidae). Zoological Letters 5: 1. https://doi.org/10.1186/s40851-018-0113-z Guidetti R, Bertolani R (2005) Tardigrade taxonomy: an updated check list of the taxa and a list of characters for their identification Zootaxa 845: 1-46. https://do1.org/10.11646/zootaxa.845.1.1 Guil N, Giribet G (2009) Fine scale population structure in the Echinis- cus blumi—canadensis series (Heterotardigrada, Tardigrada) in an Iberian mountain range — When morphology fails to explain genet- ic structure. Molecular Phylogenetics and Evolution 51: 606-613. https://doi.org/10.1016/j.ympev.2009.02.019 Jorgensen A, Kristensen RM, Mobjerg N (2018) Phylogeny and inte- grative taxonomy of Tardigrada. In: Schill O (Ed.) Water Bears: The zse.pensoft.net Biology of Tardigrades. Zoological Monographs, Springer, 95—114. https://doi.org/10.1007/978-3-319-95702-9 3 Jorgensen A, Mobjerg N, Kristensen RM (2007) A molecular study of the tardigrade Echiniscus testudo (Echiniscidae) reveals low DNA sequence diversity over a large geographical area. Journal of Lim- nology 66(S1): 77-83. https://doi.org/10.4081/jlimnol.2007.s1.77 Jorgensen A, Mobjerg N, Kristensen RM (2011) Phylogeny and evolu- tion of the Echiniscidae (Echiniscoidea, Tardigrada) — an investiga- tion of the congruence between molecules and morphology. Journal of Zoological Systematics and Evolutionary Research 49(S1): 6-16. https://doi.org/10.1111/j.1439-0469.2010.00592.x Kaczmarek L, Michalczyk L (2002) Echiniscus barbarae, a new spe- cies of tardigrade from Cuba Island (Tardigrada: Heterotardigra- da, Echiniscidae, ‘arctomys group’). Zootaxa 53: 1-4. https://doi. org/10.11646/zootaxa.53.1.1 Kaczmarek L, Michalczyk L (2010) The genus Echiniscus Schultze 1840 (Tardigrada) in Costa Rican (Central America) rain forests with descriptions of two new species. Tropical Zoology 23: 91-106. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fou- rier transform. Nucleic Acids Research 30: 3059-3066. https://doi. org/10.1093/nar/gkf436 Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program. Briefings in Bioinformatics 9: 286— 298. https://doi.org/10.1093/bib/bbn013 Kitayama K (1992) An altitudinal transect study of the vegetation on Mount Kinabalu, Borneo. Vegetatio 102: 149-171. https://doi. org/10.1007/BF00044731 Kristensen RM (1987) Generic revision of the Echiniscidae (Heterotar- digrada), with a discussion of the origin of the family. In: Bertolani R (Ed.) Biology of Tardigrades. Selected Symposia and Monographs U.Z.1., Modena, 261-335. Kristensen RM, Hallas TE (1980) The tidal genus Echiniscoides and its variability, with erection of Echiniscoididae fam. n. (Tardigrada). Zoologica Scripta 9: 113-127. https://doi. org/10.1111/j.1463-6409.1980.tb00657.x Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874. https://doi.org/10.1093/molbev/msw054 Li X (2007) Tardigrades from the Tsinling Mountains, central Chi- na with descriptions of two new species of Echiniscidae (Tardi- grada). Journal of Natural History 41: 2719-2739. https://do1. org/10.1080/00222930701711046 Lohman DJ, de Bruyn M, Page T, von Rintelen K, Hall R, Ng PKL, Shih H-T, Carvalho GR, von Rintelen T (2011) Biogeography of the In- do-Australian Archipelago. Annual Review of Ecology, Evolution, and Systematics 42: 205—226. https://doi.org/10.1146/annurev-ecol- sys-102710-145001 Merckx VSFT, Hendriks KP, Beentjes KK, Mennes CB, Becking LE, Peijnenburg KTCA, Afendy A, de Boer ANH, Biun A, Buang MM, Chen P, Chung AYC, Dow R, Feijen FAA, Feijen H, Feijen-van Soest C, Geml J, Geurts R, Gravendeel B, Hovenkamp P, Imbun P, Ipor I, Janssens SB, Jocqué M, Kappes H, Khoo E, Koomen P, Lens F, Majapun RJ, Morgado LN, Neupane S, Nieser N, Pereira JT, Rahman H, Sabran S, Sawang A, Schwallier RM, Shim P, Smit H, Sol N, Spait M, Stech M, Stokvis F, Sugau JB, Suleiman M, Sumail S, Thom- as DC, van Tol J, Tuh FYY, Yahya BE, Nais J, Repin R, Lakim M, Schilthhuizen M (2015) Evolution of endemism on a young tropical mountain. Nature 524: 347-350. https://doi.org/10.1038/nature14949 Zoosyst. Evol. 96 (1) 2020, 103-113 Michalczyk L, Kaczmarek L (2007) Echiniscus ganczareki, a new species of Tardigrada (Heterotardigrada: Echiniscidae, bigran- ulatus group) from Costa Rica. Zootaxa 1471: 15-25. https://doi. org/10.11646/zootaxa.1471.1.2 Miller WR, Claxton SK, Heatwole HF (1999) Tardigrades of the Austra- lian Antarctic Territories: Males in the genus Echiniscus (Tardigra- da: Heterotardigrada). Zoologischer Anzeiger 238: 303-309. Mitchell CR, Romano FA (2007) Sexual dimorphism, population dy- namics and some aspects of life history of Echiniscus mauccii (Tar- digrada; Heterotardigrada). Journal of Limnology 66 (Supplement 1): 126-131. https://doi.org/10.4081/jlimnol.2007.s1.126 Moon SN, Kim HS (1990) A new species of Echiniscus (Tardigrada: Echiniscidae) from Korea. Korean Journal of Systematic Zoology 6: 231-234. Mora C, Tittensor DP, Adl S, Simpson AGB, Worm B (2011) How many species are there on Earth and in the ocean? PLoS Biology 9: e1001127. https://doi.org/10.1371/journal.pbio. 1001127 Mobjerg N, Kristensen RM, Jorgensen A (2016) Data from new taxa infer [soechiniscoides gen. nov. and increase the phylogenetic and evolutionary understanding of echiniscoidid tardigrades (Echinis- coidea: Tardigrada). Zoological Journal of the Linnean Society 178: 804-818. https://do1.org/10.1111/zoj.12500 Ohsawa M, Nainggolan PHJ, Tanaka N, Anwar C (1985) Altitudinal zo- nation of forest vegetation on Mount Kerinci, Sumatra: with compari- sons to zonation in the temperate region of east Asia. Journal of Tropi- cal Ecology 1: 193-216. https://doi.org/10.1017/S0266467400000286 Pilato G, Binda MG, Lisi O (2004) Famelobiotus scalicii, n. gen. n. sp., a new eutardigrade from Borneo. New Zealand Journal of Zoology 31: 57-60. https://doi.org/10.1080/03014223.2004.9518359 Pilato G, Fontoura P, Lisi O, Beasley C (2008) New description of Echiniscus scabrospinosus Fontoura, 1982, and description of a new species of Echiniscus (Heterotardigrada) from China. Zootaxa 1856: 41-54. https://doi.org/10.11646/zootaxa.1856.1.4 Pleijel F, Jondelius U, Norlinder E, Nygren A, Oxelman B, Schander C, Sundberg P, Thollesson M (2008) Phylogenies without roots? A plea for the use of vouchers in molecular studies. Molecular Phylogenetics and Evolution 48: 369-371. https://doi.org/10.1016/j.ympev.2008.03.024 Rambaut A, Suchard MA, Xie D, Drummond AJ (2014) Tracer v1.6. https://beast.bio.ed.ac.uk/Tracer Rebecchi L, Altiero T, Eibye-Jacobsen J, Bertolani R, Kristensen RM (2008) A new discovery of Novechiniscus armadilloides (Schuster, 1975) (Tardigrada, Echiniscidae) from Utah, USA with consider- ations on non-marine Heterotardigrada phylogeny and biogeog- raphy. Organisms, Diversity & Evolution 8: 58-65. https://doi. org/10.1016/j.ode.2006.11.002 Richters F (1902) Beitrage zur Kenntnis der Fauna der Umgebung von Frankfurt a. M. Bericht tiber die Senckenbergische Naturforschende Gesellschaft in Frankfurt am Main, 23—26. Richters F (1904) Arktische tardigraden. Fauna Arctica 3: 495-511. Riggin GT (1962) Tardigrada of the Southwest Virginia: with the addi- tion of a description of a new marine species from Florida. Virginia Agricultural Experiment Station Technical Bulletin 152: 1-147. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenet- ic inference under mixed models. Bioinformatics 19: 1572-1574. https://do1.org/10.1093/bioinformatics/btg 180 Stec D, Morek W, Gasiorek P, Michalczyk L (2018) Unmasking hid- den species diversity within the Ramazzottius oberhaeuseri com- plex, with an integrative redescription of the nominal species for the family Ramazzottiidae (Tardigrada: Eutardigrada: Parachela). Igns Systematics and Biodiversity 16: 357-376. https://doi.org/10.1080 /14772000.2018.1424267 Stec D, Smolak R, Kaczmarek L, Michalczyk L (2015) An integrative description of Macrobiotus paulinae sp. nov. (Tardigrada: Eutardi- grada: Macrobiotidae: hufelandi group) from Kenya. Zootaxa 4052: 501-526. https://doi.org/10.11646/zootaxa.4052.5.1 van Steenis CGGJ (1984) Floristic altitudinal zones in Malesia. Bo- tanical Journal of the Linnean Society 89: 289-292. https://doi. org/10.1111/j.1095-8339. 1984 .tb02560.x Vecchi M, Cesari M, Bertolani R, Jonsson KI, Rebecchi L, Guidetti R (2016) Integrative systematic studies on tardigrades from Antarcti- ca identify new genera and new species within Macrobiotoidea and Echiniscoidea. Invertebrate Systematics 30: 303-322. https://doi. org/10.1071/1S15033 Vicente F, Fontoura P, Cesari M, Rebecchi L, Guidetti R, Serrano A, Bertolani R (2013) Integrative taxonomy allows the identification of synonymous species and the erection of a new genus of Echiniscidae (Tardigrada, Heterotardigrada). Zootaxa 3613: 557-572. https://doi. org/10.11646/zootaxa.3613.6.3 Supplementary material | Raw morphometric data for the type population Authors: Piotr Gasiorek, Katarzyna Von¢ina, Lukasz Michalczyk Data type: morphometric data Explanation note: The dataset comprises individual mea- surements provided separately for all life stages. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.96.49989 suppl 1 Supplementary material 2 Uncorrected pairwise distances Authors: Piotr Gasiorek, Katarzyna Von¢ina, Lukasz Michalczyk Data type: genetic data Explanation note: p-distances between haplotypes of fastly evolving DNA fragments (ITS-1, ITS-2, COT) provided for the members of the virginicus complex. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.96.49989 suppl2 zse.pensoft.net