Continued Exploration of Tanzanian Rainforests Reveals a New Echiniscid Species (Heterotardigrada)

© 2020 Academia Sinica, Taiwan Open Access Continued Exploration of Tanzanian Rainforests Reveals a New Echiniscid Species (Heterotardigrada) Marcin Bochnak1, Katarzyna Vončina1, Reinhardt M. Kristensen2,§, and Piotr Gąsiorek1,§* 1Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland. *Correspondence: E-mail: piotr[email protected] (Gąsiorek) E-mail: [email protected] (Bochnak); [email protected] (Vončina) 2Section for Biosystematics, Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, Copenhagen Ø DK-2100, Denmark. E-mail: [email protected] (Kristensen) §RMK and PG share joint senior authorship. Received 13 January 2020 / Accepted 28 April 2020 / Published 15 June 2020 Communicated by Benny K.K. Chan The Afrotropical tardigrade fauna is insufficiently studied, and consequently its diversity in this region is severely underestimated. Ongoing sampling in the Udzungwa Mountains, Morogoro Region of Tanzania has revealed a new representative of the genus Echiniscus C.A.S. Schultze, 1840 (Echiniscidae). Echiniscus tantulus sp. nov. belongs to the spinulosus group, but it stands out from other members of this speciose Echiniscus clade by having a heteromorphic sculpture of the dorsal plates and an uncommonly stable body appendage configuration A-C-Cd-Dd-E. The new species is characteristic by being equipped with long dorsal spines and very short lateral spicules, which so far have been found only in one other species of the group, Echiniscus spinulosus (Doyère, 1840). An updated checklist of Tanzanian Echiniscidae is provided, incorporating recent advances in their classification. Key words: Biodiversity, Chaetotaxy, Cuticular sculpturing, The spinulosus group, Udzungwa Mountains. Citation: Bochnak M, Vončina K, Kristensen RM, and Gąsiorek P. 2020. Continued exploration of Tanzanian rainforests reveals a new echiniscid species (Heterotardigrada). Zool Stud 59:18. doi:10.6620/ZS.2020.59-18. BACKGROUND The Eastern Arc Mountains of Kenya and Tanzania consist of numerous ranges covered with mostly isolated and fragmented Afromontane rainforest, which possesses a staggering species richness and remarkable endemism (Rodgers and Homewood 1982; Bjørndalen 1992; Newmark 2002; Burgess et al. 2007). Most of the system lies in Tanzania, and only Taita Hills are situated in Kenya. The boundaries of the Eastern Arc are delineated by volcanic mountains, e.g., Kilimanjaro and Meru, to the north, and the Southern Highlands at its southern extremity, close to the border of Zambia (Bjørndalen 1992). The Udzungwa Mountains, located to the south of the Tanzanian capital Dodoma, are among the largest in the Eastern Arc ranges, and constitute a primeval habitat for the numerous endemic species (Scharff et al. 2015). Tardigrades are generally poorly studied in Africa (McInnes et al. 2017), which, considering the diversity of specific habitats that they usually dominate, like cryofauna of glaciers (Zawierucha and Shain 2019), renders real tardigrade abundance obscure. The heterotardigrade family Echiniscidae has been the subject of several studies on animals inhabiting mosses and lichens from Tanzania (Van Rompu et al. 1991; Binda and Pilato 1993 1995; McInnes et al. 2017; Gąsiorek and Kristensen 2018). Jørgensen (2001) pointed out an overall under-sampling in Africa, and Gąsiorek and Kristensen (2018) conjectured that more undescribed species await discovery and formal description in Tanzania. Here, by applying classic light and scanning microscopy and DNA barcoding (e.g., Wang et al. 2018), we provide an integrative description Zoological Studies 59:18 (2020) doi:10.6620/ZS.2020.59-18 1 © 2020 Academia Sinica, Taiwan of Echiniscus tantulus sp. nov. from the Udzungwa Mountains (Fig. 1), and place it in the Echiniscus phylogeny. The composition of the Tanzanian echiniscid fauna is discussed in relation to the progress made in unravelling its taxonomy and biogeography. MATERIALS AND METHODS Sample collection, morphometry and terminology Twenty-five animals representing the new species were extracted from fruticose lichen samples collected in the Udzungwa Mountains by Thomas Pape on 18th August 2018. The air-dried samples stored in unbleached paper envelopes were rehydrated in water for several hours before being agitated and squeezed, and the obtained sediment was poured into Petri dishes to search for microfauna under a stereomicroscope with darkfield illumination. Specimens isolated from samples TZ.072 and TZ.073 were used for a number of analyses: (I) imaging with light microscopy (morphology and morphometry; nineteen specimens), (II) imaging with scanning electron microscopy (SEM – ultrastructure; three specimens), and (III) DNA sequencing + phylogenetics (three specimens). Specimens for light microscopy and morphometry were mounted in a small drop of Hoyer’s medium and examined under a Nikon Eclipse 50i phase contrast microscope (PCM) associated with a Nikon Digital Sight DS-L2 digital camera. Fig. 1. The type locality of the new species. page 2 of 13Zoological Studies 59:18 (2020) © 2020 Academia Sinica, Taiwan Specimens were prepared for SEM in accordance with the protocol provided by Stec et al. (2015). Images 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 2–4 images were taken every ca. 0.2 μm and then assembled into a single deep-focus image. All measurements are given in micrometres (μm) and were performed under PCM. Structures were measured only if they were undamaged and their orientations suitable. Body length was measured from the anterior to the posterior end of the body, excluding the hind legs. The sp ratio is the ratio of the length of a given structure to the length of the scapular plate (Dastych 1999). Morphometric data were handled using the Echiniscoidea ver. 1.3 template, available from the Tardigrada Register, www.tardigrada. net/register (Michalczyk and Kaczmarek 2013). Raw morphometric data are included as supplementary material S1 and deposited in the Tardigrada Register under www.tardigrada.net/register/0067.htm. Morphological terminology follows Kristensen (1987) and subsequent changes proposed in Gąsiorek et al. (2019a). Genotyping and phylogenetics DNA was extracted following the Chelex® 100 resin (Bio-Rad) extraction method by Casquet et al. (2012) with modifications as detailed in Stec et al. (2015). All specimens were mounted on temporary water slides and examined under PCM before DNA extraction to ensure correct taxonomic identification. Two hologenophore cuticles (Pleijel et al. 2008) were retrieved from Eppendorf tubes, mounted on permanent slides, and deposited in the Institute of Zoology and Biomedical Research in Kraków. Five markers of variable mutation rates were sequenced: 18S rRNA, 28S rRNA, ITS-1, ITS-2, and COI. All fragments were amplified and sequenced according to the protocols described in Stec et al. (2015). Primers and original references for specific PCR programmes are listed in table S1. Sequences were aligned with default settings of MAFFT ver. 7 (Katoh et al. 2002; Katoh and Toh 2008) under G-INS-i strategy. COI sequences were examined in MEGA7 (Kumar et al. 2016) to exclude possible pseudogenes. Uncorrected pairwise distances were calculated using MEGA7 and are enclosed in the table S2. ITS-1 and ITS-2 sequences were used to reconstruct separate Maximum Likelihood (ML) phylogenies; GenBank accession numbers for the newly sequenced taxa and for sequences retrieved from GenBank are presented in table 1. Alignments were 724 bp (ITS-1) and 540 bp (ITS-2) long. ModelFinder (Kalyaanamoorthy et al. 2017) was used to choose the best-fit models—TIM2+F+G4 (ITS-1) and TPM2u+F+G4 (ITS-2)—according to the Bayesian information criterion. W-IQ-TREE was used for ML reconstruction (Nguyen et al. 2015; Trifinopoulos et al. 2016). One thousand ultrafast bootstrap (UFBoot) replicates were applied to provide support values for branches (Hoang et al. 2018). Trees were rooted on Diploechiniscus oihonnae (Richters, 1903). The final consensus trees were visualized using FigTree ver. 1.4.3 (available at: http://tree.bio.ed.ac.uk/software/figtree/). Table 1. GenBank accession numbers for the sequences used in the present study. New sequences are in boldface Species COI ITS-1 ITS-2 Origin of new sequences Echiniscus belloporus Gąsiorek & Kristensen, 2018 MT374157 MT374193 MT374181 type population (TZ.030) Echiniscus blumi Richters, 1903 EU046198 - - Echiniscus canadensis Murray, 1910 MF459630 - - Echiniscus granulatus (Doyère, 1840) EU244600 - - Echiniscus lineatus Pilato et al., 2008 MN548147 MN545741 MN545749 Echiniscus manuelae da Cunha & do Nascimento Ribeiro, 1962 MT374158 MT374194 MT374182 population UG.004 Echiniscus merokensis Richters, 1904 FJ435813 - - Echiniscus ornamentatus Gąsiorek & Kristensen, 2018 MT374159 MT374195 MT374183 type population (TZ.022) Echiniscus quadrispinosus Richters, 1902 JX683821 - - Echiniscus siticulosus Gąsiorek & Michalczyk, 2020 MT374160 MT374196–7 MK726308–9 type population (AU.080) Echiniscus succineus Gąsiorek & Vončina, 2019 MK649675 MT374198 MK675925 type population (MG.005) Echiniscus tantulus sp. nov. MT107427 MT108138 MT108137 type population (TZ.072) Echiniscus testudo (Doyère, 1840) MG025605 MT374199 MG016456 neotype population (FR.057) Echiniscus trisetosus Cuénot, 1932 MF459627 - - Echiniscus tristis Gąsiorek & Kristensen, 2018 MT374161 MT374200 MT374184 type population (TZ.030) Echiniscus virginicus Riggin, 1962 MN548176 MN545748 MN545756 Diploechiniscus oihonnae (outgroup) MG063724 MT374201 MT374185 population GB.098 page 3 of 13Zoological Studies 59:18 (2020) © 2020 Academia Sinica, Taiwan RESULTS TAXONOMY Phylum Tardigrada Doyère, 1840 Class Heterotardigrada Marcus, 1927 Order Echiniscoidea Richters, 1926 Family Echiniscidae Thulin, 1928 Genus Echiniscus C.A.S. Schultze, 1840 Echiniscus tantulus sp. nov. Gąsiorek, Bochnak, Vončina & Kristensen (Figs. 2–5, Tables 2–3) urn:lsid:zoobank.org:act:4CB6B6FC-A376-4464-8338B434C82FD8D5 Description: Females (i.e., from the third instar onwards; measurements and statistics in table 2): Body orange with minute red eyes present in live specimens; colours disappearing soon after mounting. Cylindrical, Echiniscus-type cephalic papillae (secondary clavae) and (primary) clavae; cirri growing out from bulbous cirrophores (Figs. 2A–B, 3B, 4A, 5A). The body appendage configuration is A-C-Cd-Dd-E, with trunk appendages formed as: spines (Cd, Dd) or spicules (C, E). Spicules formed precisely at the posterior edges of segmental plates, usually as a prolongation of the cuticular margin. All trunk appendages smooth (Figs. 2A–B, 3, 4A). Single cases of asymmetry in all positions (e.g., Fig. 3A). Dorsal plates with the spinulosus type of sculpturing (see Gąsiorek et al. 2019a), that can be easily subdivided into (I) typical pores present in the majority of plates, (II) more densely arranged pores at the anterior portions of the segmental plates and median plate 2, and the entirety of median plate 3 that are clearly seen under SEM and as darker areas under PCM (Figs. 2A–B, 3, 4), (III) very fine epicuticular wrinkling present on bands dividing the segmental plates and median plate 2, and the posteriormost part of median plate 3, which are visible only in SEM (Fig. 3B). Pores are smaller and more sparsely distributed in the lateral portions of all plates. Pores without endocuticular rings (Figs. 3, 4B–C). The cephalic plate with typical pores, divided into halves (Figs. 2A, 5A). The cervical (neck) plate poorly delineated from the scapular plate, formed as thin grey belt without pores (Figs. 2A, 3A, 4A). The scapular plate large, with poorly marked lateral sutures separating narrow rectangular lateral portions without pores (Figs. 2A–B, 3A, 4). Paired segmental plates divided into a smaller, much narrower anterior and a prominent posterior part by a light nonporous, transverse band (wrinkled in SEM, Figs. 2A–B, 3, 4A). Posterior parts heterogenic, with the anteriormost margins adjacent to the belts being identical to the anterior parts of the plate (Fig. 3). The caudal (terminal) plate with short incisions and horizontal and vertical epicuticular ridges, forming a cross, i.e., dividing the plate into four facets (Figs. 2A–B, 3, 4C). Median plates 1 and 3 unipartite, whereas median plate II divided into a very narrow anterior and wide posterior portion (Fig. 3). Ventral cuticle with minute endocuticular pillars distributed evenly throughout the entire venter (Fig. 5B), with rudimentary subcephalic plates in the form of convex swellings (Figs. 4A, 5A). Sexpartite gonopore between genital plates, and a trilobed anus between legs IV. Pedal plates seen as dark areas on the central leg portions under PCM, without pores; plate IV with a typical dentate collar composed of short teeth (Figs. 2A– B, 4A, 5D). Distinct pulvini on all legs (Figs. 2A, 4A). A small spine on leg I and a papilla on leg IV present (Figs. 2A–B, 4A). External claws on all legs smooth. Internal claws with large, acute spurs positioned at ca. 1/4–1/3 of the claw height and bent downwards (Fig. 5B–D). Buccal apparatus short, with a rigid, stout tube and a roundish pharynx containing serrated, chitinous placoids. Stylet supports absent. Males: Unknown (likely a parthenogenetic species). Juveniles (i.e., the second instar, measurements and statistics in Table 3): Clearly smaller than adult females, but with the trunk appendage configuration as in sexually mature individuals. In one individual, spines Cd displaced in the direction of the dorsolateral position (Fig. 2C). Pores fainter than in adult females; the differences in dorsal sculpturing between anterior and posterior portions of segmental plates not apparent. Lacking gonopore. No other significant disparities were found between the juvenile and mature life stages. Larvae: Unknown. Eggs: Up to five round, orange eggs per exuvia were found in two exuviae. Molecular markers and phylogenetic position: All five genetic markers were represented by single haplotypes (GenBank accession numbers: 18S rRNA – MT126785, 28S rRNA – MT126765, COI – MT107427, ITS-1 – MT108138, and ITS-2 – MT108137). Both ITS-1 and ITS-2-based phylogenies reflected the topology of the Echiniscus clades from Gąsiorek et al. (2019a), with the virginicus complex as basal and E. testudo (Doyère, 1840) as sister to the spinulosus complex, which included E. tantulus sp. nov. According to the ITS-1 tree, E. tantulus sp. nov. is a sister species to the clade E. succineus Gąsiorek & Vončina, 2019 + E. ornamentatus Gąsiorek & Kristensen, 2018 (Fig. 6). In page 4 of 13Zoological Studies 59:18 (2020) © 2020 Academia Sinica, Taiwan the ITS-2 tree, E. tantulus sp. nov. is also most closely related to these two taxa, but their relationships are unclear as the tree is inconclusive. The p-distances were calculated for both ITS and COI markers on the dataset of selected sequences (see Table S2). Type material: Holotype (slide TZ.072.01), and 16 paratypes on slides TZ.072.01–06. Three paratypes mounted on SEM stub no. 19.15. Moreover, two voucher specimens (hologenophores) mounted on the slides TZ.073.01–02. The slides TZ.072.02–03 (5♀♀) deposited in the Natural History Museum of Denmark, University of Copenhagen, Denmark; the slide TZ.072.04 (5♀♀) deposited in the Catania University, Sicily, Italy. All remaining slides deposited in the Institute of Zoology and Biomedical Research, Jagiellonian University, Poland. The present species was found together with abundant populations of E. tristis Gąsiorek & Kristensen, 2018. Additional material: Two females mixed with a population of E. tristis in an additional sample collected in the locus typicus (sample reference TZ.069). Type locality: 7°49'04"S, 36°50'39"E, ca. 2100 m asl; Mwanihana Peak, Udzungwa Mountains, Tanzania; Afromontane rainforest, lichens growing on exposed bedrock. Etymology: From Latin tantulus = “so small”. The Table 2. Measurements [in µm] of selected morphological structures of the adult females of E. tantulus 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 Holotype µm sp µm sp µm sp µm sp Body length 16 182–237 476–534 204 503 17 21 232 523 Scapular plate length 16 35.1–44.9 -40.5 - 3.0 - 44.4 - Head appendages lengths Cirrus internus 16 7.6–14.4 17.0–36.6 10.8 26.8 1.9 4.9 14.2 32.0 Cephalic papilla 15 4.6–8.0 11.0–20.1 6.6 16.2 0.8 2.0 7.4 16.7 Cirrus externus 16 11.4–19.0 29.7–47.0 15.5 38.3 2.1 4.5 19.0 42.8 (Primary) C lava 16 4.2–6.8 10.7–15.8 5.4 13.4 0.7 1.6 6.8 15.3 Cirrus A16 27.2–40.3 68.7–99.0 34.1 84.2 3.8 7.4 39.9 89.9 Cirrus A/Body length ratio 16 14%–20% -17% -1% -17% - Body appendages lengths Spine C15 2.5–6.1 6.4–14.3 4.3 10.6 1.0 2.2 6.1 13.7 Spine Cd16 12.4–18.9 29.1–46.7 15.9 39.3 2.0 5.1 16.6 37.4 Spine Dd16 15.2–29.9 38.7–67.3 21.8 54.0 3.7 8.7 29.9 67.3 Spine E15 2.5–4.1 6.1–10.3 3.3 8.1 0.5 1.3 2.9 6.5 Spine on leg I length 16 2.3–3.4 5.8–9.4 2.9 7.0 0.3 0.8 3.4 7.7 Papilla on leg IV length 16 2.5–3.9 5.8–9.9 3.1 7.6 0.4 1.0 3.2 7.2 Number of teeth on the collar 16 9–16 - 11.3 - 1.9 - 13 - Claw I heights Branch 16 10.3–13.3 26.5–31.4 11.6 28.6 1.0 1.5 13.3 30.0 Spur 14 1.8–3.2 4.3–7.8 2.7 6.6 0.4 0.9 3.2 7.2 Spur/branch length ratio 14 16%–26% -23% -3% -24% - Claw II heights Branch 16 9.9–12.9 26.0–29.9 11.2 27.7 0.8 1.2 12.9 29.1 Spur 15 2.1–3.2 4.7–7.6 2.6 6.5 0.4 0.8 2.8 6.3 Spur/branch length ratio 15 17%–28% -23% -3% -22% - Claw III heights Branch 16 9.8–13.7 25.4–30.9 11.4 28.0 1.1 1.7 13.7 30.9 Spur 14 2.1–3.5 5.6–7.9 2.6 6.4 0.4 0.7 2.5 5.6 Spur/branch length ratio 14 18%–28% -23% -3% -18% - Claw IV heights Branch 16 10.5–15.0 27.3–35.9 12.8 31.6 1.4 2.4 15.0 33.8 Spur 13 2.5–3.9 5.6–8.8 3.1 7.5 0.4 0.9 3.6 8.1 Spur/branch length ratio 13 19%–31% -24% -3% -24% - page 5 of 13Zoological Studies 59:18 (2020) © 2020 Academia Sinica, Taiwan name refers to the microscopic size of the animal. An adjective in the nominative singular. Differential diagnosis: The stable body appendage configuration A-C-Cd-Dd-E, with the dorsal spines several times longer than short lateral spicules, makes E. tantulus sp. nov. unique among the members of the spinulosus group (Gąsiorek et al. 2019a), which are typically highly variable in terms of the development of trunk spines and frequent asymmetries (e.g., see Pilato et al. 2008; Meyer 2016). There is only one other species showing a combination of long dorsal spines and short lateral spicules: E. spinulosus (Doyère, 1840). Echiniscus canedoi da Cunha & do Nascimento Ribeiro, 1962 also bears some resemblance to E. tantulus sp. nov. due to the dorsal spines Dd two times longer than the longest lateral spines C, yet the new species can be differentiated from: E. canedoi, so far found only in Madeira, based on the trunk appendage configuration (C-Cd-Dd-E in E. tantulus sp. nov. vs C-D-Dd in E. canedoi), and dissimilarities in the dorsal sculpturing (markedly darker anterior portions of segmental plates, with densely arranged pores in E. tantulus sp. nov. vs anterior portions with sparsely arranged pores in E. canedoi); E. spinulosus, reliably reported only from numerous locales in Western Palaearctic (McInnes 1994), by Table 3. Measurements [in µm] of selected morphological structures of the juveniles of E. tantulus 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 µm sp µm sp µm sp Body length 3 127–157 465–503 142 488 15 20 Scapular plate length 3 27.2–31.2 - 29.1 - 2.0 - Head appendages lengths Cirrus internus 35.9–10.5 20.4–33.7 8.4 28.8 2.3 7.3 Cephalic papilla 33.7–5.5 12.8–17.6 4.4 14.9 1.0 2.5 Cirrus externus 3 8.9–12.3 32.7–40.8 11.0 37.7 1.8 4.3 (Primary) clava 3 3.7–4.0 12.8–14.0 3.8 13.2 0.2 0.7 Cirrus A323.7–25.8 82.7–87.9 25.0 85.9 1.1 2.8 Cirrus A/Body length ratio 3 16%–19% -18% -1% - Body appendages lengths Spine C32.6–3.2 8.3–11.8 2.8 9.7 0.3 1.8 Spine Cd36.1–10.2 21.1–37.5 8.8 30.3 2.3 8.4 Spine Dd313.2–16.6 46.4–53.2 14.4 49.4 1.9 3.5 Spine E3 2.1–2.7 7.7–8.7 2.4 8.1 0.3 0.5 Spine on leg I length 3 1.7–2.3 5.9–7.4 1.9 6.5 0.3 0.8 Papilla on leg IV length 3 1.9–2.6 7.0–8.3 2.3 7.8 0.4 0.7 Number of teeth on the collar 3 9–11 - 9.7 - 1.2 - Claw I heights Branch 3 7.9–9.0 28.8–29.8 8.5 29.2 0.6 0.5 Spur 3 1.9–2.1 6.7–7.3 2.0 7.0 0.1 0.3 Spur/branch length ratio 3 23%–24% -24% -1% - Claw II heights Branch 3 7.9–9.0 27.7–29.0 8.3 28.5 0.6 0.7 Spur 3 1.3–2.3 4.5–7.4 1.8 6.0 0.5 1.4 Spur/branch length ratio 3 16%–26% -21% -5% - Claw III heights Branch 2 7.4–7.9 27.2–27.3 7.7 27.3 0.4 0.1 Spur 2 1.6–1.6 5.5–5.9 1.6 5.7 0.0 0.2 Spur/branch length ratio 2 20%–22% -21% -1% - Claw IV heights Branch 3 8.6–10.3 29.8–33.0 9.2 31.7 0.9 1.7 Spur 2 1.9–2.0 6.9–7.0 2.0 7.0 0.1 0.0 Spur/branch length ratio 2 22%–23% -22% -1% - page 6 of 13Zoological Studies 59:18 (2020) © 2020 Academia Sinica, Taiwan Fig. 2. Habitus of Echiniscus tantulus sp. nov. (PCM): A, adult female (holotype, dorsolateral view); B, adult female (paratype, lateral view); C, juvenile (paratype, dorsolateral view). White arrowheads indicate spicules in lateral positions C and E, black arrowheads indicate pulvini, and empty arrowheads point out pedal plates. All scale bars in µm. page 7 of 13Zoological Studies 59:18 (2020) © 2020 Academia Sinica, Taiwan the distinct trunk appendage configuration (C-CdDd-E in E. tantulus sp. nov. vs (B)-C-Cd-D-Dd-E in E. spinulosus), dissimilarities in the dorsal sculpturing (markedly darker anterior portions of segmental plates, with densely arranged pores in E. tantulus sp. nov. vs uniform sculpturing in E. spinulosus, see Pilato et al. 2008), the presence of epicuticular ridges on the caudal plate (forming a cross in E. tantulus sp. nov. vs absent in E. spinulosus, see Pilato et al. 2008), and the level of development of pedal plates (poorly developed and lacking pores in E. tantulus sp. nov. vs well-developed, with large pores identical to the pores present on the dorsal plates in E. spinulosus, see Gąsiorek and Degma 2018). Fig. 3. Dorsal sculpturing of E. tantulus sp. nov.: A, in PCM (note one spine Cd asymmetrically lacking); B, in SEM (note wrinkled belts of cuticle between anterior and posterior portions of segmental plates and median plate 2, and in the posterior portion of median plate 3. All scale bars in µm. page 8 of 13 Zoological Studies 59:18 (2020) © 2020 Academia Sinica, Taiwan DISCUSSION To date, twelve echiniscid species have been reported from Mount Kilimanjaro and the Udzungwa Mountains (Binda and Pilato 1995; Gąsiorek and Kristensen 2018). Ten are representatives of the genus Echiniscus, and can be divided into three phyletic lineages: the spinulosus group (7 spp.: E. angolensis da Cunha & do Nascimento Ribeiro, 1964, E. baius Marcus, 1928, E. belloporus Gąsiorek & Kristensen, 2018, E. ornamentatus, E. scabrospinosus Fontoura, 1982, E. tantulus sp. nov., E. tristis), the africanus group (2 spp.: E. africanus Murray, 1907 and E. pusae Marcus, 1928), and the virginicus group (E. lineatus Pilato et al., 2008). In Gąsiorek and Kristensen (2018), E. pusae was misidentified and listed as E. africanus (first reported from Tanzania by Binda and Pilato 1995); however, a comparison of specimens from Tanzania, the Malay Archipelago and Australia (data in preparation) under PCM revealed a consistent low morphological variation within this species. Consequently, E. pusae is likely to be another pantropical echiniscid species (or at least one with a very wide range that extends from Africa to Australasia), along with E. baius (McInnes 1994) and E. lineatus (E. lineatus was listed as E. dariae Kaczmarek & Michalczyk, 2010 in Gąsiorek and Kristensen 2018, and later synonymised in Gąsiorek et al. 2019b). Fig. 4. Close-up on the details of sculpturing of E. tantulus sp. nov. (SEM): A, female in lateral view (note sparsely distributed pores on the lateral portions of all dorsal plates); B, scapular plate; C, caudal (terminal) plate. Arrowheads indicate spicules in lateral positions C and E. All scale bars in µm. page 9 of 13Zoological Studies 59:18 (2020)