Abstract

African tropical forests are highly underexplored regarding microinvertebrate diversity, including tardigrades. Here, we describe two new tardigrade species from the genera Parahypsibius Gąsiorek et al., 2024 and Echiniscus C.A.S. Schultze, 1840, collected in the tropical rainforest of the Kibale National Park in southwestern Uganda. Species delimitation was based on an integrative approach combining morphological, morphometric, and molecular data. Morphological analyses employed light and scanning electron microscopy, while molecular characterization used mitochondrial COI and nuclear 18S rRNA, 28S rRNA, ITS1, and ITS2 markers. Additionally, we report Echiniscus lineatus Pilato, Fontoura, Lisi and Beasley, 2008 from Uganda for the first time, providing new sequences and reconstructing haplotype networks using previously published genetic data of this species. Phylogenetic analyses of subfamily Hypsibiinae Pilato, 1969 and genus Echiniscus clarify the relationships of the new taxa and place them within the broader phylogenetic context. Our study highlights the underestimated diversity of African tardigrades and demonstrates the importance of integrative taxonomy for species delineation. The new records from Kibale National Park provide also baseline data for understanding the distribution and phylogeny of tardigrades of the Central Africa.

Keywords

Africa, DNA barcoding, Haplotype network, Taxonomy, Water bears

1. Introduction

Tropical forest ecosystems cover ca. 10% of the Earth’s land surface and occur on almost every continent except for Antarctica (Malhi et al. 2014). In Africa, this ecosystem covers approximately 216 million ha and its degradation is continuously progressing (FAO 2020). From both an ecological and taxonomical perspective, tropical forests in Africa are classified as biodiversity hotspots, understood as areas with high levels of species diversity (Myers et al. 2000). These types of forests are present in 11 African countries (FAO 2020). Among them is Uganda, with its Kibale National Park (Kibale NP) located in the southwestern part of the country, forming part of the Guinean Forests of West Africa within the Eastern Afromontane biodiversity hotspot (Mittermeier et al. 2011).

Kibale NP is one of the main areas where biological studies are conducted by numerous of national and international organizations, initially it was established as a nature reserve in 1932, but its rank was elevated to the National Park in 1993 (https://www.kibaleforestnationalparkuganda.com/history-of-kibale-forest). About the animal kingdom, studies in Kibale NP have focused primarily on vertebrates, while the best-studied group of invertebrates are butterflies (Nyafwono et al. 2014). For most other groups, knowledge is limited or outdated, and this includes also tardigrades.

Tardigrades are ubiquitous invertebrates found in a wide range of environments, from the highest mountain ranges to the deep sea. They occur in soil, lichens, mosses, in freshwater and marine sediments, as well as, in algae, and leaf litter (Nelson et al. 2015). Tardigrade diversity is best known in Europe (ca. 800 species), North America (ca. 380 species), and the Arctic (ca. 100 species: Meier 2017; Kaczmarek et al. 2016; Roszkowska et al. 2019; Coulson et al. 2024). Compared to these regions, the knowledge of African tardigrades remains very limited, especially considering the size of the continent, with only ca. 170 species reported across Africa (McInnes et al. 2017). In Uganda, only 20 tardigrade species have been recorded so far (four freshwater and 16 limno-terrestrial species), representing ca. 9% of all known African species (Kaczmarek et al. 2008; Gąsiorek and Kristensen 2018; Zawierucha et al. 2018; Warguła et al. 2025). Among these tardigrades are genera representing the families Hypsibiidae Pilato, 1969 and Echiniscidae Thulin, 1928, including Echiniscus C.A.S. Schultze, 1840 Pseudechiniscus Thulin, 1911, Notahypsibius Tumanov, 2020, and Diphascon Plate, 1888.

The genus Parahypsibius, belonging to the family Hypsibiidae, was recently established by Gąsiorek et al. (2024a) based on morphological and molecular characters of the widespread species Parahypsibius scabropygus (Cuénot 1929). Currently, the genus comprises nine species, all characterized by dorsal sculpturing, the presence of two rounded macroplacoids, and Ramazzottius-like claws (sensu Tumanov 2020: Gąsiorek et al. 2024a). Importantly, most of these species were described in the second half of the 20^th century and all of them (excluding Pap. scabropygus) require integrative redescriptions (Abe 2004; Bartoš 1935, 1941, 1960; Beasley 1988; Binda and Pilato 1985; Cuénot 1929; Nelson and McGlothin 1993; Ramazzotti and Maucci, 1983).

The genus Echiniscus, belonging to the family Echiniscidae includes over 130 species and one of the biggest, morphologically coherent group recognized within the genus is the spinulosus morpho-group, comprising ca. 30 species (Gąsiorek and Sørensen 2025). Members of this group are characterized by trunk spines of varying lengths, often coarse and/or serrated, and by dorsal plate sculpturing with irregular and circular pores or granules, rarely connected by striae (Bartylak et al. 2019; Gąsiorek et al. 2021; Gąsiorek et al. 2022; Dey et al. 2024). Species from the spinulosus morpho-group occur mainly in tropical and subtropical regions, with their vast intra-group morphological diversity demonstrated by recently described species such as Echiniscus brunus Dey, Gąsiorek and Michalczyk 2024 and Echiniscus minutus Gąsiorek and Michalczyk 2024 (Dey et al. 2024; Gąsiorek and Michalczyk 2024). Despite being well defined in terms of morphology, the spinulosus morpho-group has so far lacked phylogenetic evidence supporting its monophyly (Dey et al. 2024, 2025; Gąsiorek et al. 2022, Gąsiorek and Sørensen 2025).

The genus Echiniscus is among the best-studied tardigrade genera, and the geographic distribution of its species has been widely discussed in the literature (Gąsiorek et al. 2019a; Gąsiorek and Michalczyk 2024; Dey et al. 2024; Surmacz et al. 2025). A notable example is Echiniscus lineatus Pilato, Fontoura, Lisi and Beasley, 2008, a member of the Ech. virginicus species complex, which is characterized by a pantropical distribution (Gąsiorek et al. 2019b). The steadily increasing number of DNA sequences available for different populations of this species has facilitated studies on relationships among its haplotypes, making it a recurrent subject of recent research (Gąsiorek et al. 2019b; Tumanov and Khabibulina 2024).

In the present study, we analysed three tardigrade species. We describe two of them as new to science, representing the genera Parahypsibius Gąsiorek et al. 2024a and Echiniscus, discovered in the rainforest of Kibale NP. Additionally, we analysed the distribution of Ech. lineatus, supplementing existing data with new sequences obtained from Ugandan samples. We used light and scanning electron microscopy to collect detailed phenotypic data from the prepared specimens. Additionally, we obtained DNA sequences of five molecular markers that are commonly used in tardigrade taxonomy (18S rRNA, 28S rRNA, ITS-1, ITS-2, COI). We constructed phylogenetic trees for the subfamily Hypsibiinea and of the genus Echiniscus, to establish phyletic positions of the studied species. Finally, we calculated haplotype networks in order to examine distribution of Ech. lineatus.

2. Material and methods

2.1. Samples and sample processing

One moss and one lichen samples examined for this study were collected in Kibale NP, Uganda in July 2022 and packed in paper envelopes, dried at the temperature of ca. 25°C and transported to the laboratory at the Faculty of Biology, Adam Mickiewicz University in Poznań, Poland. Moss sample (No UG77) was collected from tree trunk: 00°34'1.25"N 30°22'34.642"E; ca. 1535 m asl: Uganda, Kabarole District, Kibale NP. Lichen sample (No UG79) was collected from a tree branch: 00°34'2,600"N 30°21'12,373"E; ca. 1535 m asl: Uganda, Kabarole District, Kibale NP. Tardigrades were extracted from the samples and studied following the protocol by Stec et al. (2015). In short, a piece of moss or lichen was placed in a beaker filled with 250 ml of H₂O for six hours. Afterwards, the moss/lichen was vigorously stirred within the beaker with the use of a glass rod. The supernatant (containing tardigrades, their eggs and other animals inhabiting the substratum alongside substratum particles) was transferred into a 250 ml cylinder for 30 minutes, to allow all the particles to fall to the bottom of the cylinder. Then, the top 200 ml of water was discarded, and the remaining 50 ml was stirred, and poured onto 10 cm Ø glass Petri dishes. Tardigrades and their eggs were extracted using an Olympus SZ61 stereomicroscope. The isolated tardigrades were allocated to morphological and genetic analyses; the exact numbers are detailed below in the “Material examined” section for each of the three species.

2.2. Microscopy and imaging

Animals for light microscopy were prepared by mounting them on microscope slides using Hoyer’s medium and secured with cover slips. These slides were subsequently analysed using an Olympus BX41 Phase Contrast Light Microscope (PCM) equipped with an Olympus SC50 digital camera (Olympus Corporation, Shinjuku-ku, Japan). For scanning electron microscopy, seven specimens from sample UG77 were prepared following the method described in Roszkowska et al. (2018). These specimens were examined under a high vacuum using a Hitachi S3000N Scanning Electron Microscope (SEM).

Photomicrographs and drawings were compiled using GIMP 2.10.36. For thick structures that could not be adequately shown in a single image, a series of 2–10 photomicrographs were taken at approximately 0.5 μm intervals. These images were then manually merged into a single deep-focus image using GIMP 2.10.36.

2.3. Morphometrics and morphological nomenclature

All measurements are given in micrometres [μm]. Structures were measured only if their orientation was suitable. The body length was measured from the anterior extremity to the end of the body, excluding hind legs. Measurements of the buccal tube length and the position of the stylet support insertion point were conducted in accordance with the methodologies described by Pilato (1981). The pt index was calculated as the ratio of the length of a specific structure to the length of the buccal tube, expressed as a percentage, following the guidelines proposed by Pilato (1981). The dorsal protuberances in the genus Parahypsibius were measured by determining the diameter of five protuberances on each of the seven bands. Due to subtle differences in their shape, the protuberances selected for measurement were chosen at random. The claws of Parahypsibius were measured following Beasley et al. (2008). The new species were identified using the key in Ramazzotti and Maucci (1983; English translation by Beasley 1995) and original descriptions and re-descriptions by Pilato et al. (1989), Binda and Pilato (1994, 1995), Kaczmarek and Michalczyk (2010), Gąsiorek and Kristensen (2018), Bartylak et al. (2019), Bochnak et al. (2020), Gąsiorek and Michalczyk (2020), Kiosya et al. (2021), Gąsiorek et al. (2022), Gąsiorek and Michalczyk (2024), Gąsiorek and Vončina (2023), and Gąsiorek et al. (2024a). Measurements were recorded using the “Parachela” ver. 1.8 template and “Echiniscoidea” ver. 1.3 template available from the Tardigrada Register (Michalczyk and Kaczmarek 2013). Raw morphometric data for both new species are given in Files S1, S2. Generic abbreviations follow Perry et al. (2019).

2.4. DNA extraction and genotyping

Before genomic DNA extraction, specimens were identified in vivo using light microscopy. The DNA extraction was performed using the Chelex®100 resin (Bio-Rad) protocol (Casquet et al. 2012) modified by Stec et al. (2020). To obtain tardigrade exoskeleton the remaining portion of the DNA extract containing Chelex®100 resin was diluted with ddH₂O and transferred to a glass cube. The glass cube was examined under the stereomicroscope and when exoskeleton was present it was mounted in a permanent slide in Hoyer’s medium. Four molecular markers with different effective mutation rates were sequenced: three nuclear fragments (28S rRNA, 18S rRNA, and ITS2) and one mitochondrial fragment (COI). All fragments were amplified and sequenced using the primers and protocols of Stec et al. (2020), except for the forward 28S rRNA primer 28SF0002 (Stec 2022). Sequencing products were read with the ABI 3130xl sequencer by the Genomed company (Warsaw, Poland). Sequences were processed in BioEdit ver. 7.2.5 (Hall 1999) and submitted to GenBank. Prior to submission, all obtained COI sequences were translated into protein sequences in MEGA11 (Tamura et al. 2021) to check against pseudogenes.

2.5. Phylogenetic analysis and genetic comparisons

To determine the phyletic position of the new species of the genus Parahypsibius and Echiniscus, a phylogenetic tree was constructed based on the sequence dataset used in Gąsiorek et al. (2024a) in case of Parahypsibius and from Gąsiorek et al. (2023) in case of Echiniscus. These datasets were supplemented with sequences newly obtained in this study and sequences published in the meanwhile (Gąsiorek and Michalczyk 2024; Dey et al. 2024, 2025; Li et al. 2025). The list of all sequences used to construct trees for both groups is provided in File S3. Sequence alignments were performed using the AUTO setting for COI, ITS-1, ITS-2 markers, and the Q-INS-I method for ribosomal markers 18S rRNA and 28S rRNA in MAFFT version 7 (Katoh et al. 2002; Katoh and Toh 2008). Alignments were then manually verified for non-conserved regions in BioEdit. After alignment, sequences were trimmed to lengths of 878 bp (18S rRNA), 805 bp (28S rRNA), 641 bp (COI), 518 bp (ITS-2) in case of Parahypsibius, 900 bp (18S rRNA), 802 bp (28S rRNA), 538 bp (COI), 423 bp (ITS-1), 356 bp (ITS-2) in case of Echiniscus, and subsequently concatenated with SequenceMatrix (Vaidya et al. 2011). Uncorrected pairwise distances (p-distances) were calculated using MEGA 11 (Tamura et al. 2021) in Echiniscus alignment. For Parahypsibius, the concatenated alignment was divided into six blocks: three blocks for the ribosomal markers and three blocks for the different codon positions of the COI dataset. For Echiniscus, the concatenated alignment was divided into seven blocks: four blocks for the ribosomal markers and three blocks for the different codon positions of the COI dataset. PartitionFinder (Lanfear et al. 2017), using the Akaike Information Criterion (AIC), was employed to select the optimal partitioning scheme and substitution models for the phylogenetic analysis. Bayesian inference (BI) was performed on the concatenated datasets, 18S rRNA + COI and 18S rRNA + 28S rRNA + COI + ITS-1 + ITS-2, using MrBayes v3.2 (Ronquist et al. 2003), with random starting trees. Both analyses were run for 10 million generations, sampling every 1000 generations, and convergence was confirmed by an average standard deviation of split frequencies below 0.01. Tracer v1.6 (Rambaut and Drummond 2014) was used to ensure the Markov chains had reached stationarity and to determine the appropriate ‘burn-in’ period, which was the first 10% of generations, Effective Sample Size (ESS) values exceeded 200, and the consensus tree was obtained by summarizing the topologies after discarding the burn-in phase. The final tree was visualized using FigTree v1.4.3, available at http://tree.bio.ed.ac.uk/software/figtree (accessed on 3 November of 2025). The raw trees and the best models and partitions suggested by the Partition Finder are included in Files S4, S5. The raw results of the p-distance analysis are provided in File S6.

To investigate the distribution of Ech. lineatus, haplotype networks were constructed for all available sequences of COI, ITS-1 and ITS-2 markers of that species. Sequence alignments were performed using the AUTO setting for COI, ITS-1, ITS-2 markers in MAFFT version 7 (Katoh et al. 2002; Katoh and Toh 2008). After alignment, sequences were trimmed to lengths of 572 bp (COI), 625 bp (ITS-1) and 450 bp (ITS-2). Separate single-gene Median-Joining haplotype networks (Bandelt et al. 1999) were generated in PopART ver.1.7 (Leigh and Bryant 2015).

3. Results

3.1. Taxonomic account of Parahypsibius

Phylum: Tardigrada Doyère, 1840

Class: Eutardigrada Richters, 1926

Order: Parachela Schuster, Nelson, Grigarick and Christenberry, 1980

Superfamily: Hypsibioidea Pilato, 1969 (in Marley et al. 2011)

Family: Hypsibiidae Pilato, 1969

Genus: Parahypsibius Gąsiorek, 2024 (in Gąsiorek et al. 2024a)

3.1.1. Abbreviation of genus Parahypsibius

The genus Parahypsibius has not been assigned an official abbreviation. Herein, we designate this abbreviation following the rules proposed by Perry et al. (2019). The official abbreviation of the genus Parahypsibius is Pap.

3.1.2. Parahypsibius arletae sp. nov. Warguła, Dmuchowska, Stec, Kaczmarek

https://zoobank.org/947A5344-BA7C-4436-B6BD-2AD01F71E820

Figures 1, 2, 3, 4; Table 1

Material examined.

Seven specimens; UGANDA; Kibale NP, Kabarole District; 00°34'2.600"N 30° 21'12.373"E; ca. 1535 m asl; 5 Jul. 2022; coll. Zuzanna Kudelska, Bogna Malinowska, Marta Janecka; lichen on fallen tree branch; barcodes (GenBank: PZ129105–6, PZ129999–30000); sample code (UG79).

Figure 1.

Parahypsibius arletae sp. nov.: A Habitus, dorso-ventral view (holotype, PCM). B A drawing illustrating a sculpture divided into seven bands. Scale bar in μm.

Figure 2.

Parahypsibius arletae (holotype) sp. nov.: A Claws I; B Claws II; C Claws III; D Claws IV. White indented arrowheads indicate pseudolunulae. All in PCM. Scale bars in μm.

Figure 3.

Parahypsibius arletae sp. nov.: A Buccal apparatus, dorsal view (holotype); B buccal apparatus, dorsal view; C buccal apparatus, dorsal view; D buccal apparatus, ventral view. Black blunt arrowhead indicates first macroplacoid. White sharp and blunt arrowheads indicate dorsal and ventral apophyses, respectively. Scale bars in μm.

Figure 4.

Parahypsibius arletae sp. nov.: A Leg IV with protuberances on dorsal surface (white indented arrowhead); B pair of elliptical organs (white blunt arrowheads) (paratype, PCM). Scale bars in μm.

Table 1.

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Measurements [in μm] and pt values of selected morphological structures of Parahypsibius arletae sp. nov. N – number of specimens/structures measured; range – measurements taken for the smallest and the largest structure among all measured specimens; SD – standard deviation; pt – ratio of the length of a given structure to the length of the buccal tube expressed as a percentage.

CHARACTER	N	RANGE						MEAN		SD		Holotype
CHARACTER	N	µm			pt			µm	pt	µm	pt	µm	pt
Body length	7	117	–	254		–		195	–	42	–	207	–
Buccal tube
Buccal tube length	7	16,7	–	25.8		–		23.0	–	3.1	–	24.9	–
Stylet support insertion point	7	7.8	–	13.6	46.4	–	55.0	11.8	51.0	2.0	3.1	13.2	52.8
Buccal tube external width	7	1.0	–	1.8	6.0	–	7.3	1.5	6.3	0.2	0.5	1.5	6.1
Buccal tube internal width	7	0.4	–	0.9	2.3	–	3.8	0.7	2.9	0.2	0.6	0.7	2.8
Placoid lengths
Macroplacoid 1	7	1.5	–	2.3	7.6	–	9.7	2.0	8.8	0.3	0.6	2.3	9.1
Macroplacoid 2	7	1.3	–	2.3	6.9	–	9.3	1.8	8.0	0.3	0.9	1.8	7.3
Macroplacoid row	7	3.0	–	5.0	18.0	–	20.7	4.4	19.0	0.6	1.0	4.7	18.7
Claw I heights
External base	6	1.8	–	3.4	8.4	–	14.0	2.4	10.8	0.6	2.3	2.1	8.4
External primary branch	6	4.5	–	6.9	22.1	–	28.3	5.7	25.3	1.0	2.3	6.4	25.7
External secondary branch	6	2.4	–	4.4	14.6	–	18.1	3.8	16.6	0.7	1.1	4.2	16.8
External base/primary branch (cct)	6	32.6	–	54.8		–		43.1	–	9.3	–	32.6	–
Internal base	5	1.7	–	3.2	7.8	–	13.4	2.3	10.0	0.6	2.3	2.0	7.8
Internal primary branch	6	3.4	–	5.5	17.0	–	22.5	4.2	18.9	0.7	2.1	4.5	17.8
Internal secondary branch	6	2.1	–	3.3	11.9	–	14.9	3.0	13.2	0.5	1.1	3.3	13.3
Internal base/primary branch (cct)	5	43.8	–	63.5		–		52.3	–	9.0	–	43.8	–
Claw II heights
External base	7	1.8	–	3.4	10.4	–	14.1	2.7	11.6	0.5	1.3	2.6	10.4
External primary branch	7	3.9	–	7.4	17.4	–	30.7	6.1	26.7	1.3	4.7	7.1	28.6
External secondary branch	7	2.5	–	4.4	13.8	–	18.1	3.7	16.0	0.7	1.4	4.1	16.3
External base/primary branch (cct)	7	36.1	–	67.1		–		45.0	–	11.3	–	36.4	–
Internal base	6	1.6	–	3.5	9.7	–	14.3	2.7	11.5	0.6	1.6	2.7	10.7
Internal primary branch	6	2.2	–	5.3	12.9	–	22.0	4.3	18.2	1.3	3.6	5.2	20.9
Internal secondary branch	6	2.7	–	3.5	12.2	–	19.3	3.2	14.1	0.3	2.6	3.3	13.0
Internal base/primary branch (cct)	6	51.3	–	77.6		–		64.9	–	11.4	–	51.3	–
Claw III heights
External base	6	1.8	–	3.5	10.8	–	15.0	3.0	13.0	0.6	1.5	3.0	12.1
External primary branch	6	4.6	–	7.6	23.5	–	30.4	6.4	27.8	1.2	2.8	7.6	30.4
External secondary branch	6	2.5	–	5.2	15.1	–	21.3	3.9	17.0	0.9	2.3	3.9	15.5
External base/primary branch (cct)	6	39.0	–	58.8		–		47.3	–	9.0	–	40.0	–
Internal base	6	2.0	–	3.2	11.1	–	13.2	2.7	11.8	0.4	0.8	2.8	11.2
Internal primary branch	6	3.5	–	5.4	16.8	–	22.3	4.5	19.3	0.7	2.1	4.8	19.4
Internal secondary branch	6	2.3	–	4.4	13.3	–	18.1	3.4	14.7	0.7	1.9	3.5	13.8
Internal base/primary branch (cct)	6	57.8	–	71.0		–		61.4	–	5.4	–	57.8	–
Claw IV heights
Anterior base	7	2.0	–	4.5	11.8	–	18.6	3.2	13.9	0.7	2.3	33	13.3
Anterior primary branch	7	5.2	–	10.0	31.0	–	41.4	8.0	34.5	1.5	3.6	8.6	34.5
Anterior secondary branch	7	3.1	–	5.3	15.7	–	21.2	4.5	19.4	0.8	2.3	5.3	21.1
Anterior base/primary branch (cct)	7	36.9	–	45.3		–		40.2	–	3.4	–	38.6	–
Posterior base	6	2.0	–	3.3	9.6	–	14.3	2.8	12.3	0.5	1.6	3.3	13.1
Posterior primary branch	6	3.8	–	5.6	17.1	–	23.5	4.7	20.9	0.8	2.4	5.6	2.3
Posterior secondary branch	6	2.5	–	4.0	12.8	–	16.3	3.4	14.9	0.6	1.6	4.0	16.0
Posterior base/primary branch (cct)	6	50.3	–	68.3		–		59.1	–	7.4	–	58.7	–

Type repository.

The holotype and two paratypes (slide: UG79/1) and one paratype (slide: UG79/2) and one exoskeleton after DNA extraction (slide: UG79/5) are deposited in the Department of Animal Taxonomy and Ecology, Institute of Environmental Biology, Adam Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, 61-614 Poznań, Poland. Two paratypes (slide: UG79/1-2) are deposited in Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016, Kraków, Poland.

Etymology.

We dedicate this species to our friend, Professor Małgorzata Arlet from the Institute of Human Biology and Evolution, Faculty of Biology, Adam Mickiewicz University in Poznań, who initiated the Tropical Biology course series at the Faculty of Biology.

Description.

Body cylindrical, measurements in Table 1. Eyespots absent after fixing in Hoyer’s medium. The cuticle exhibits a distinct sculpture composed of irregular, roughly polygonal protuberances (2.5–3.7 μm in diameter) arranged in seven transverse bands (Fig. 1A, B). The first two bands are situated at the level of the first pair of legs and are considerably less distinct than the remaining ones, whereas the subsequent bands are arranged progressively along the body. The seventh and widest band is situated in the caudal region and extends then uniformly as far as the caudal end, which itself is entirely covered by an irregular cuticular pattern of sculpturing composed of irregular protuberances (Figs 1A, 1B, 2A). Areas between bands are smooth. Protuberances gradually increase in size along the rostro-caudal axis, reaching their largest dimensions in the caudal region. Delicate sculpture is present also on the external surface of leg pair IV (Fig. 2A).

A pair of elliptical organs (Kihm et al. 2023) present (Fig. 2B). Peribuccal structures not visible under PCM. Bucco-pharyngeal apparatus of the Ramazzottius type (Fig. 3A–C). The oral cavity armature absent or not visible under PCM. Apophyses for the insertion of stylet muscles (AISMs) asymmetrical with respect to the frontal plane: dorsal apophysis is shorter, higher, and stumpy, with a prominent caudal apex (Fig. 3A, C). Ventral apophysis slenderer, with more developed caudal processes (Fig. 3D). Furcae unmodified, of the Hypsibius type, with well-developed condyles (Fig. 3A). Buccal tube straight and narrow (Fig. 3A–C). Pharynx circular, with only two roundish granular macroplacoids, similar in size (Fig. 3A). Constrictions in macroplacoids absent or not visible under PCM (Fig. 3B). Microplacoid absent (Fig. 3A).

Claws of Ramazzottius-type (sensu Tumanov 2020), with elongated primary branches of external/posterior claws and thinned connective portions in the form of light-refracting units (Fig. 4A–D). Posterior claws IV always longer than external claws in legs I–III. Accessory points on all primary branches evident and clearly divergent (Fig. 4A–D). Pseudolunulae at all claws bases present (Fig. 4B), but often poorly visible, and most prominent under claws IV. Pulvini and cuticular bars on legs absent. Eggs not found.

DNA sequences.

The sequences obtained from one specimen for two out of four molecular markers analysed in this study were of good quality. The amplification of 28S rRNA and ITS-2 was not successful. The 18S rRNA sequences (GenBank: PZ129105–6) 878 bp long; COI sequences (GenBank: PZ129999–30000) 641 bp long.

Differential diagnosis.

Currently there are nine species included in the genus Parahypsibius: Pap. biscuitiformis (Bartoš, 1960); Pap. calcaratus (Bartoš, 1935); Pap. camelopardalis (Ramazzotti and Maucci, 1983); Pap. macrocalcaratus (Beasley, 1988); Pap. ragonesei (Binda and Pilato, 1985); Pap. roanensis (Nelson and McGlothin, 1993); Pap. runae (Bartoš, 1941); Pap. scabropygus (Cuénot, 1929), and Pap. stiliferus (Abe, 2004). The new species differs specifically from:

(1) Pap. biscuitiformis, known only from the type locality in the Czech Republic (Bartoš 1960), by: the absence of sculpture on the head region (the entire body covered with uniform granular sculpture in Pap. biscuitiformis), the absence of the subcentral constriction on macroplacoid I, shorter macroplacoid I (1.5–2.3 μm in Pap. arletae sp. nov. vs. ca. 4.05 μm in Pap. biscuitiformis) and reticulated cuticle on legs IV.

(2) Pap. calcaratus, known only from its type locality in Slovakia (Bartoš 1935), by: the absence of sculpture on the head region (the entire body is covered with an irregular sculpturing in Pap. calcaratus).

(3) Pap. camelopardalis, known only from type locality in Portugal and Spain (Ramazzotti and Maucci 1983), by: the absence of sculpture on the head region (the entire body covered with granular sculpture in Pap. camelopardalis), lack of the sculpture on the legs I–III, wider buccal tube (1.0–1.8 μm in Pap. arletae sp. nov. vs. 4.0 μm in Pap. camelopardalis) and smaller protuberances on the dorsal side of the body (2.5–3.7 μm in Pap. arletae sp. nov. vs. ca. 6.0 μm in Pap. camelopardalis).

(4) Pap. macrocalcaratus, known only from USA (Beasley 1988, 1990), by: the absence of sculpture on the head region (the entire body covered with an irregular sculpturing in Pap. macrocalcaratus) and larger protuberances on the dorsal side of the body (2.5–3.7 μm in Pap. arletae sp. nov. vs. ca. 2.0 μm in Pap. macrocalcaratus).

(5) Pap. ragonesei, known only from the type locality in Italy (Binda and Pilato 1985), by: the absence of sculpture on the head region (the entire body covered with an irregular sculpturing in Pap. ragonesei) and the absence of cuticular bars between claws on all the legs.

(6) Pap. roanensis, known only from the type locality in USA (Nelson and McGlothin 1993), by: the absence of sculpture on the head region (the entire body covered with an irregular sculpturing in Pap. roanensis).

(7) Pap. runae, known only from the type locality in Ukraine (Bartoš 1941), by: the absence of sculpture on the head region (the entire body covered with an irregular sculpturing in Pap. runae).

(8) Pap. scabropygus, known from Denmark, France (type locality), Poland and Scotland (Cuénot 1929; Gąsiorek et al. 2024a), by: the different dorsal sculpture (sculpture composed of seven bands extending from the first pair of legs to the caudal region in Pap. arletae sp. nov. vs. sculpture present only on caudal region in Pap. scabropygus).

(9) Pap. stiliferus, known only from the type locality in Russia (Abe 2004), by: the absence of sculpture on the head region (the entire body covered with sculpturing in Pap. stiliferus).

Remarks.

Due to the very limited number of sequences currently available in public databases for the genus Parahypsibius, p-distances were not calculated. At present, sequences for only three taxa have been published, which is insufficient to perform a meaningful comparison.

3.2. Taxonomic account of Echiniscus

Class: Heterotardigrada Marcus 1927

Order: Echiniscoidea Richters 1926

Family: Echiniscidae Thulin 1928

Genus: Echiniscus C.A.S. Schultze 1840

3.2.1. Echiniscus osiejuki sp. nov. Warguła, Dmuchowska, Polishchuk, Stec, Kaczmarek

https://zoobank.org/45E1E910-4554-4695-916A-2EA5A4315C6C

Figures 5, 6, 7, 8, 9; Tables 2, 3

Material examined.

Twelve specimens; UGANDA; Kibale NP, Kabarole District; 00°34'1.253"N 30°22'34.642"E; ca. 1535 m asl; 4 Jul. 2022; coll. Barbara Wiśniewska; moss on tree trunk; barcodes at GenBank: PZ127154-5, PZ129998, PZ127159, PZ127161; specimen code UG77.

Figure 5.

Echiniscus osiejuki sp. nov.: A Habitus, dorso-ventral view(holotype); B habitus, dorsal view (paratype). All in PCM. Scale bars in μm.

Figure 6.

Echiniscus osiejuki sp. nov.: A Habitus, dorsal view (paratype); B habitus, ventrolateral view (paratype). All in SEM. Scale bars in μm.

Figure 7.

Echiniscus osiejuki sp. nov.: A Dorsal view of cephalic appendages with cephalic and scapular plates (paratype, PCM); B lateral projection of cephalic appendages (paratype, SEM), white arrowhead indicates secondary clava. Scale bars in μm.

Figure 8.

Echiniscus osiejuki sp. nov.: Drawing of the plates in dorsal view along with all the spines.

Figure 9.

Echiniscus osiejuki sp. nov.: A A spine on leg I (paratype) indicated by white blunt arrowhead; B claws on leg II (paratype), spur on internal claws indicated by dark blunt arrowhead; C dentate collar with papilla on leg IV (paratype), papilla indicated by white indented arrowhead; D gonophore (paratype) indicated by black indented arrowhead. All in SEM. Scale bars in μm.

Table 2.

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Measurements [in μm] and sp values of selected morphological structures of females Echiniscus osiejuki sp. nov. N – number of specimens/structures measured; range – measurements taken for the smallest and the largest structure among all measured specimens; SD – standard deviation; pt – ratio of the length of a given structure to the length of the scapular plate.

CHARACTER	N	RANGE						MEAN		SD		Holotype
CHARACTER	N	µm			sp			µm	sp	µm	sp	µm	sp
Body length	8	144	–	202	512	–	564	174	534	16	19	166	523
Scapular plate length	8	25.6	–	36.5		–		32.6	–	3.3	–	31.8	–
Head appendages lengths	0
Cirrus internus	8	9.5	–	14.5	37.2	–	42.9	12.9	39.5	1.6	2.4	12.0	37.8
Cephalic papilla	8	4.6	–	6.2	16.4	–	19.1	5.7	17.6	0.5	1.0	6.1	19.1
Cirrus externus	7	11.8	–	15.9	41.7	–	47.7	14.5	45.0	1.3	2.0	14.2	44.5
Clava	8	4.2	–	6.7	13.3	–	18.3	5.1	15.6	0.9	1.7	4.2	13.3
Cirrus A	8	15.6	–	23.5	59.0	–	65.4	20.4	62.5	2.4	2.5	20.1	63.2
Cirrus A / Body length ratio	8	11%	–	13%		–		12%	–	1%	–	12%	–
Body appendages lengths	0
Cirrus B	7	3.8	–	12.7	15.0	–	34.8	9.2	27.8	2.9	6.8	7.9	24.8
Cirrus C	8	6.6	–	16.4	25.7	–	46.7	13.6	41.1	3.1	6.6	13.3	41.9
Cirrus C^d	8	3.8	–	15.2	11.2	–	46.3	11.7	35.6	4.2	11.8	14.7	46.3
Cirrus D	5	5.9	–	15.3	18.9	–	43.8	11.0	33.9	4.5	12.0	13.8	43.3
Cirrus D^d	8	12.9	–	22.0	50.3	–	63.8	18.4	56.3	2.7	4.7	17.9	56.2
Cirrus E	8	5.2	–	15.5	20.3	–	46.9	12.8	38.6	3.5	9.2	14.8	46.5
Spine on leg I length	8	1.1	–	2.7	4.4	–	8.0	2.0	6.0	0.4	1.0	1.8	5.5
Papilla on leg IV length	8	2.4	–	4.2	9.5	–	11.5	3.5	10.6	0.5	0.7	3.4	10.8
Number of teeth on the collar	8	7	–	12		–		9.4	–	2.1	–	7	–
Claw I heights	0
Branch	8	7.6	–	10.8	27.9	–	31.5	9.6	29.6	0.9	1.2	9.3	29.3
Spur	8	1.3	–	2.2	4.1	–	6.6	1.7	5.3	0.3	0.7	1.7	5.3
Spur/branch height ratio	8	15%	–	21%		–		18%	–	2%	–	0	–
Claw II heights	0
Branch	7	7.1	–	10.5	27.8	–	30.4	9.2	28.5	1.0	0.9	8.9	27.8
Spur	7	1.4	–	1.9	4.2	–	5.6	1.6	4.9	0.2	0.4	1.5	4.7
Spur/branch height ratio	7	15%	–	20%		–		17%	–	2%	–	0	–
Claw III heights	0
Branch	8	7.1	–	10.2	27.4	–	30.6	9.2	28.2	0.9	1.0	8.8	27.7
Spur	7	1.5	–	1.8	4.7	–	5.9	1.6	5.1	0.1	0.4	1.5	4.7
Spur/branch height ratio	7	17%	–	21%		–		18%	–	2%	–	0	–
Claw IV heights	0
Branch	8	8.3	–	12.4	31.1	–	36.3	10.8	33.1	1.2	1.5	10.4	32.8
Spur	8	1.6	–	2.4	5.0	–	6.6	2.0	6.0	0.3	0.5	1.9	5.9
Spur/branch height ratio	8	16%	–	20%		–		18%	–	1%	–	0	–

Table 3.

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Measurements [in μm] and sp values of selected morphological structures of males and one juvenile of Echiniscus osiejuki sp. nov. (N – number of specimens/structures measured; range – measurements taken for the smallest and the largest structure among all measured specimens; SD – standard deviation; sp – ratio of the length of a given structure to the length of the scapular plate.

CHARACTER	N	MALES										JUVENILE
		RANGE						MEAN		SD		RANGE
		µm			sp			µm	sp	µm	sp	µm	sp
Body length	2	118	–	136	473	–	506	127	489	13	24	115	686
Scapular plate length	2	23.2	–	28.8		–		26.0	–	3.9	–	16.8	–
Head appendages lengths	0
Cirrus internus	2	7.0	–	7.9	27.4	–	30.1	7.4	28.8	0.6	1.9	6.8	38.5
Cephalic papilla	2	4.3	–	4.3	15.0	–	18.6	4.3	16.8	0.0	2.5	3.0	17.8
Cirrus externus	2	8.6	–	9.5	32.8	–	36.8	9.0	34.8	0.6	2.8	8.6	50.9
Clava	2	3.8	–	4.4	15.3	–	16.4	4.1	15.8	0.4	0.7	3.5	20.7
Cirrus A	2	13.5	–	15.9	55.2	–	58.2	14.7	56.7	1.7	2.1	13.1	78.1
Cirrus A / Body length ratio	2	11%	–	12%		–		12%	–	0%	–	11%	–
Body appendages lengths	0
Cirrus C^d	1	3.4	–	3.4	11.9	–	11.9	3.4	11.9
Cirrus D^d	2	10.7	–	16.0	45.8	–	55.7	13.3	50.8	3.8	7.0
Cirrus E	2	6.6	–	9.3	28.3	–	32.2	7.9	30.3	1.9	2.8
Spine on leg I length	2	1.4	–	2.3	6.1	–	7.8	1.8	7.0	0.6	1.2
Papilla on leg IV length	1	2.6	–	2.6	9.0	–	9.0	2.6	9.0			1.8	10.9
Number of teeth on the collar	1	5	–	5		–		5.0	–		–	4.0	–
Claw I heights	0
Branch	2	6.3	–	6.9	24.0	–	26.9	6.6	25.4	0.5	2.1	5.6	33.1
Spur	1	1.5	–	1.5	5.1	–	5.1	1.5	5.1			1.0	5.9
Spur/branch height ratio	1	21%	–	21%		–		21%	–		–	18%	–
Claw II heights	0
Branch	2	6.1	–	6.8	23.5	–	26.3	6.4	24.9	0.5	2.0	5.2	30.8
Spur	0											0.9	5.5
Spur/branch height ratio	0					–			–		–	18%	–
Claw III heights	0
Branch	2	5.7	–	6.4	22.3	–	24.5	6.1	23.4	0.5	1.5	5.2	30.6
Spur	1	0.9	–	0.9	3.8	–	3.8	0.9	3.8			0.9	5.5
Spur/branch height ratio	1	15%	–	15%		–		15%	–		–	18%	–
Claw IV heights	0
Branch	2	7.0	–	7.5	25.9	–	29.9	7.2	27.9	0.3	2.9	5.4	32.4
Spur	1	1.1	–	1.1	4.7	–	4.7	1.1	4.7
Spur/branch height ratio	1	16%	–	16%		–		16%	–		–		–

Type repository.

The holotype (slide: UG77/13 and 8 paratypes (slides: UG77/*, where the asterisk can be substituted by any of the following numbers: 11, 13, 14, 15, 16, 17) are deposited in the Department of Animal Taxonomy and Ecology, Institute of Environmental Biology, Adam Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, 61-614 Poznań, Poland. Two paratypes (slide: UG79/12) are deposited in Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016, Kraków, Poland.

Etymology.

We dedicate this species to our friend, Professor Tomasz Osiejuk from the Department of Behavioural Ecology, Faculty of Biology, Adam Mickiewicz University in Poznań, who initiated the Tropical Biology course series at the Faculty of Biology.

Description.

Adults (measurements and statistics in Tables 2, 3). Before mounting, the body orange and plump (Figs 5, 6A, B), with red eyespots. Bulbous cirrophores are present at the base of all cirri (Fig. 7A, B). Dactyloid/ovoid cephalic papillae (secondary clavae) and minute (primary) clavae present (Fig. 7A, B). Cirrus A short. The spines configuration is A-(B)-C-C^d-(D)-D^d-E (Figs 5A, 5B, 6A, 6B, 8). Spines D and E may have serrated margins (Fig. 6A). Asymmetry in the development of appendages is frequently observed, with one of the spines B or D, which are sometimes absent. Specifically, the absence of spine B was noted in one of the eight specimens examined, while spine D was absent in three of the eight specimens. Considerable variation in the length of all spines is observed across all examined individuals. The female gonopore is rosette-shaped (Fig. 9D).

The dorsal plates with spinulosus type of sculpture, characterized by irregularly distributed pores, across all plates (Figs 5A, 5B, 6A). However, pores on anterior portion of the first paired plate often gradually become smaller in the direction from the head to the caudal region. Most of pores on the dorsal plate have a diameter ranging from 1.1–2.0 μm in all measured females, 0.9–2.2 μm in all measured males. Dark central rings in pores absent (Figs 5, 7A). Intracuticular pillars not visible under PCM. The ventral sculpture is visible as tiny granulation, but only in PCM. The cephalic plate is narrow and with an anterior incision (Fig. 7). The cervical (neck) plate is also narrow and formed as a thin dark belt without pores (Figs 5B, 7A). A smooth, thin transverse stripe divides the first and second paired plates into smaller, narrower anterior and larger posterior parts (Figs 5B, 6A). Median plates I and III are unipartite, whereas median plate II is divided into a narrower anterior and a wider posterior part (Figs 5B, 6A). Median plate III is tiny, but densely covered by pores, often partially hidden under the paired plate II and the caudal plate (Figs 5B, 6A). The caudal plate with well visible incisions (Fig. 5B). Pedal plates absent but pulvini present (Fig. 6B). The first pair of legs with small spine, the fourth pair of legs with papilla (Fig. 9A). Dentate collar with 7–12 large teeth, present on leg IV (Fig. 9C). All claws of similar length. External claws smooth. Internal claws with small, slender spurs located close to the claw bases (Fig. 9B).

One juvenile (identified by the absence of reproductive organs) was found in the sample and its morphology is similar to that of adult females, including appendage configuration and sculpturing (measurements and statistics in Table 3). Pores on dorsal plates are well visible. Gonopore absent.

No eggs and larvae were found.

DNA sequences.

The sequences obtained from one specimen for all five molecular markers analysed in this study were of good quality. The 28S rRNA sequences (GenBank: PZ127155) 711 bp long; 18S rRNA sequences (GenBank: PZ127154) 758 bp long; COI sequences (GenBank: PZ129998) 650 bp long; ITS-1 sequences (GenBank: PZ127159) 607 bp long; ITS-2 sequences (GenBank: PZ127161) 380 bp long.

Genetic distances.

The ranges of uncorrected genetic p-distances between the molecular markers of Ech. osiejuki sp. nov. obtained in our study and the sequences of all species of the genus Echiniscus available in GenBank are as follows (File S3):

28S rRNA: 0.01–0.6% (0.3% on average), with the most similar being Ech. manuelae Gąsiorek et al., 2019 (GenBank: MK529708) and the least similar being Ech. perarmatus Gąsiorek et al., 2022 (GenBank: OM517009).

18S rRNA: 0.0–3.9% (1.8% on average), with the most similar being Ech. succineus Gąsiorek & Voncina, 2019 (GenBank: MK675903) and the least similar being Ech. evelinae Gąsorek et al., 2021 (GenBank: MZ467757).

COI: 16.3–25.3% (19.2% on average), with the most similar being Ech. tristis Bochnak et al., 2020 (Genbank: MT374161) and the least similar being Ech. tantulus Bochnak et al., 2020 (GenBank: MT107427).

ITS1 rRNA: 1.9–13.8% (10.64% on avarage), with the most similar being Ech. cavagnaroi Gąsiorek et al., 2022 (Genbank: OM516846) and the least similar being Ech. spinulosus Gąsiorek & Voncina, 2023 (GenBank: PQ283268).

ITS2 rRNA: 1.4–14.9% (14.2% on average), with the most similar being Ech. tristis Bochnak et al., 2020 (GenBank: MT374184) and the least similar being Ech. aonikenk Gąsiorek et. al., 2021 (GenBank: MZ467833).

Differential diagnosis.

Echiniscus osiejuki sp. nov. is diagnosed by the combination of the following morphological characters: a spines configuration [A–(B)–C–C^d–(D)–D^d–E], lacking internal dark rings inside pores and pores not connected by striae, and the presence of spurs on internal claws of all legs. Taking these diagnostic features the new species is similar to nine Echiniscus species, but it differs specifically from:

(1) Echiniscus dikenli Maucci, 1972, known only from the type locality in Turkey (Maucci 1972), by: the presence of median plate III, shorter cirrus A (15.6–23.5 μm in Ech. osiejuki sp. nov. vs. 60.0–115.0 μm in Ech. dikenli), shorter spine C^d (3.5–15.2 μm in Ech. osiejuki sp. nov. vs. 22.0–34.0 μm in Ech. dikenli) and shorter spine E (5.2–15.5 μm in Ech. osiejuki sp. nov. vs. 42.0–90.0 μm in Ech. dikenli).

(2) Echiniscus kosickii Kaczmarek and Michalczyk, 2010, known only from the type locality in Costa Rica (Kaczmarek and Michalczyk 2010), by: a longer cirrus internus (9.5–14.5 μm in Ech. osiejuki sp. nov. vs. 8.2–9.1 μm in Ech. kosickii), shorter cephalic papilla (4.6–6.2 μm in Ech. osiejuki sp. nov. vs. 6.3–6.8 μm in Ech. kosickii), a cirrus A / body length ratio (11–13% in Ech. osiejuki sp. nov. vs. 14–17% in Ech. kosickii) and a longer spurs on claws I and IV (I: 1.3–2.2; IV: 1.6–2.4, respectively in Ech. osiejuki sp. nov. vs. I and IV: ca. 1.0 in Ech. kosickii).

(3) Echiniscus marcusi Pilato, Claxton and Binda, 1989, known only from the type locality in Australia (Pilato et al. 1989; Claxton 2004), by: the absence of granulation on legs I–III, shorter cephalic papilla (4.6–6.2 μm in Ech. osiejuki sp. nov. vs. ca. 10.2 μm in Ech. marcusi), a shorter cirrus A (15.6–23.5 μm in Ech. osiejuki sp. nov. vs. ca. 37.8 μm in Ech. marcusi), shorter spine C^d (3.8–15.2 μm in Ech. osiejuki sp. nov. vs. ca. 22.5 μm in Ech. marcusi) and longer spine D (12.9–22.0 μm in Ech. osiejuki sp. nov. vs. ca. 8.6 μm in Ech. marcusi).

(4) Echiniscus minutus Gąsiorek and Michalczyk, 2024, known only from the type locality in Indonesia (Gąsiorek and Michalczyk 2024), by the absence of pedal plates on legs I–III, longer spine D^d (12.9–22.0 μm in Ech. osiejuki sp. nov. vs. 3.3–9.8 in Ech. minutus) and a longer spurs on claws III (1.5–1.8 μm in Ech. osiejuki sp. nov. vs. 1.0–1.4 μm in Ech. minutus).

(5) Echiniscus pooensis Rodriguez-Roda, 1947 known only from the type locality in Ecquatorial Guinea (Rodriguez-Roda 1947), by the orange body color, the presence of papilla on leg IV, a shorter cirrus A (15.6–23.5 μm in Ech. osiejuki sp. nov. vs. ca. 30.0 μm in Ech. pooensis), shorter spine B (3.8–12.7 μm in Ech. osiejuki sp. nov. vs. ca. 22.0 μm in Ech. pooensis), shorter spine C (6.6–16.4 μm in Ech. osiejuki sp. nov. vs. ca. 28.0 μm in Ech. pooensis), shorter spine C^d (3.8–15.2 μm in Ech. osiejuki sp. nov. vs. ca. 20.0 μm in Ech. pooensis), shorter spine D (5.9–15.3 μm in Ech. osiejuki sp. nov. vs. ca. 24.0 μm in Ech. pooensis) and shorter spine E (5.2–15.5 μm in Ech. osiejuki sp. nov. vs. ca. 22.0 μm in Ech. pooensis).

(6) Echiniscus rugospinosus Marcus, 1927 known from Uganda and Kenya (McInnes et al. 2017), by the presence of median plate III. However, considering that Ech. rugospinosus is regarded in the literature as a nomen inquirendum (Gąsiorek and Michalczyk 2024), it is impossible to carry out a detailed morphological comparison between these two species.

(7) Echiniscus siticulosus Gąsiorek and Michalczyk, 2020, known only from the type locality in Australia, by: the lack of pedal plates, the lack of caudal plate faceting, a larger body size (144–202 μm in Ech. osiejuki sp. nov. vs. 110–132 μm in Ech. siticulosus), longer cirrus internus (9.5–14.5 μm in Ech. osiejuki sp. nov. vs. 4.9–5.1 μm in Ech. siticulosus), longer clava (4.2–6.7 μm in Ech. osiejuki sp. nov. vs. 2.2–3.6 μm in Ech. siticulosus) and longer papilla on leg IV (2.4–4.2 μm in Ech. osiejuki sp. nov. vs. 1.9–2.3 μm in Ech. siticulosus).

(8) Echiniscus spinulosus (Doyère, 1840), known from Asia, Europe and Hawaiian Islands (McInnes 1994; Gąsiorek and Vončina 2023), by: the presence of pedal plates on legs I–III, the lack of granulation on legs, a smaller body size (144–202 μm in Ech. osiejuki sp. nov. vs. 236–314 μm in Ech. spinulosus), narrower scapular plate (25.6–36.5 μm in Ech. osiejuki sp. nov. vs. 54.3–64.8 μm in Ech. spinulosus), shorter cephalic papilla (4.6–6.2 μm in Ech. osiejuki sp. nov. vs. 8.6–10.5 μm in Ech. spinulosus), a shorter cirrus externus (11.8–15.9 μm in Ech. osiejuki sp. nov. vs. 20.2–29.1 μm in Ech. spinulosus), shorter cirrus A (15.6–23.5 μm in Ech. osiejuki sp. nov. vs. 117.1–166.6 μm in Ech. spinulosus), lower cirrus A / body length ratio (11–13% in Ech. osiejuki sp. nov. vs. 44–56% in Ech. spinulosus), longer spine C (6.6–16.4 μm in Ech. osiejuki sp. nov. vs. 2.2–5.2 μm in Ech. spinulosus), shorter spine C^d (3.8–15.2 μm in Ech. osiejuki sp. nov. vs. 48.7–62.2 μm in Ech. spinulosus), shorter spine D^d (12.9–22.0 μm in Ech. osiejuki sp. nov. vs. 43.6–56.1 μm in Ech. spinulosus), shorter spine on leg I (1.1–2.7 μm in Ech. osiejuki sp. nov. vs. 4.2–6.1 μm in Ech. spinulosus), smaller papilla on leg IV (2.4–4.2 μm in Ech. osiejuki sp. nov. vs. 4.5–5.5 μm in Ech. spinulosus), shorter claws on legs I–IV (I: 7.6–10.8 μm; II: 7.1–10.5 μm; III: 7.1–10.2 μm; IV: 8.3–12.4 μm, respectively in Ech. osiejuki sp. nov. vs. I: 19.1–22.4 μm; II: 16.2–21.5 μm; III: 15.6–22.6 μm; IV: 19.5–28.5 μm, respectively in Ech. spinulosus) and shorter spurs on legs I–IV (I: 1.3–2.2 μm; II: 1.4–1.9 μm; III: 1.5–1.8 μm; IV: 1.6–2.4 μm, respectively in Ech. osiejuki sp. nov. vs. I: 2.4–3.3 μm; II: 2.3–3.4 μm; III: 2.6–3.4 μm; IV: 3.0–4.5 μm, respectively in Ech. spinulosus).

(9) Ech. tropicalis Binda and Pilato, 1995, known from (Indonesia, Malaysia and Seychelles (type locality) Singapore (Binda and Pilato 1995; Kiosya et al. 2021), by: the presence of pores on the entire median plates I and II (lack of pores on anterior part of median plates I and II in Ech. tropicalis) and a shorter spine D^d (12.9–22.0 μm in Ech. osiejuki sp. nov. vs. 5.6–6.6 μm in Ech. tropicalis).

3.2.2. Taxonomic account of Echiniscus lineatus (Ugandan population)

Echiniscus lineatus Pilato, Fontoura, Lisi and Beasley, 2008

Material examined.

One specimen; UGANDA; Kibale NP, Kabarole District; 00°34'1.253"N, 30°22'34.642"E; ca. 1535 m asl; 4 Jul. 2022; coll. Barbara Wiśniewska; moss on tree trunk; barcodes (GenBank: PZ120891, GenBank: PZ120889, GenBank: PZ120886, GenBank: PZ124266-7); sample code (UG77L). — One specimen was examined for DNA isolation. The exoskeleton after DNA extraction was mounted in Hoyer’s medium.

Specimen depository.

The slide UG77/19 with one individual is deposited in the Department of Animal Taxonomy and Ecology, Institute of Environmental Biology, Adam Mickiewicz University, Poznań, Uniwersytetu Poznańskiego 6, 61-614 Poznań, Poland.

Morphological identification.

The morphological characteristics fully correspond to the original species description.

DNA sequences.

The sequences obtained from one specimen for all five molecular markers analysed in this study were of good quality. 28S rRNA sequence (GenBank: PZ120891) 710 bp long; 18S rRNA sequence (GenBank: PZ120889) 714 bp long; COI sequence (GenBank: PZ120886) 619 bp long; ITS-1 sequence (GenBank: PZ124266) 613 bp long; ITS-2 sequence (GenBank: PZ124267) 387 bp long.

Haplotype distribution.

The analysis included the following numbers of sequences: 30 sequences for COI, nine sequences for ITS-1, and ten sequences for ITS-2. The total number of countries from which the material originated was nine for COI (Brazil, the United States, Indonesia, Uganda, Seychelles, Taiwan, Vietnam, Madagascar and Tanzania; Fig. 12A), five for ITS-1 (Indonesia, Uganda, Seychelles, Taiwan and Brazil; Fig. 12B), and five for ITS-2 (Uganda, Seychelles, Indonesia, Brazil and USA; Fig. 12C). Results of the analysis revealed no discernible geographic patterns, as haplotype clustering was random and individuals from similar geographic regions were often distantly related in the presented networks.

Figure 10.

Bayesian phylogeny of the subfamily Hypsibiinae constructed from concatenated sequences (18S rRNA + 28S rRNA + ITS-2 + COI). Numbers at nodes indicate Bayesian posterior probabilities (pp), black dots represent pp = 1. Taxa belonging to genus Parahypsibius highlighted in green. Clade comprising the remaining representatives of Hypsibiinae highlighted in blue. Outgroup highlighted in grey. Scale bar represents substitutions per position.

Figure 11.

Bayesian phylogeny of the genus Echiniscus constructed from concatenated sequences (18S rRNA + 28S rRNA + ITS-1 + ITS-2 + COI). Numbers at nodes indicate Bayesian posterior probabilities (pp), black dots represent pp = 1. Nodes with pp < 0.75 were collapsed. New species is highlighted in orange. Species belonging to the spinulosus morphogroup and south-african endemic clade highlighted in green and yellow, respectively. Species Ech. aonikenk and Ech. meridionalis highlighted in red. Outgroup highlighted in grey. Scale bar represents substitutions per position.

Figure 12.

Median-joining haplotype network for Echiniscus lineatus. A COI marker (N = 30); B ITS-1 marker (N = 9); C ITS-2 marker (N = 10). The sequences included in the analysis are distributed among countries as follows: for COI, Brazil (1), USA (1), Indonesia (16), Uganda (1), Seychelles (1), Taiwan (4), Vietnam (2), Madagascar (1), and Tanzania (3); for ITS-1, Brazil (1), USA (1), Indonesia (5), Uganda (1), Seychelles (1), Taiwan (1); for ITS-2, Brazil (1), USA (1), Indonesia (6), Uganda (1), Seychelles (1). The size of circles correlates with the number of individuals representing a single haplotype. The colours of the circles indicate country of origin. Black small circles indicate hypothetical intermediate haplotypes that were not found, but are necessary to link the observed haplotypes. Hatch marks in the network represent single mutations.

3.4. Phylogenetic results

3.4.1. Parahypsibius phylogeny

The phylogenetic analysis was performed using Bayesian inference on nine taxa from three genera belonging to the subfamily Hypsibiinae Pilato, 1969: Cryobiotus Dastych, 2019, Hypsibius Ehrenberg, 1848, and Parahypsibius, with Mesocrista revelata Gąsiorek, Stec, Morek, Zawierucha, Kaczmarek, Lachowska-Cierlik and Michalczyk, 2016 (Gąsiorek et al. 2016) and Platicrista angustata (Murray, 1905) (Gąsiorek et al. 2024b) used as outgroup (Fig. 10). Results demonstrate the paraphyly of the genus Hypsibius, as the genus Cryobiotus is positioned within Hypsibius. The new species of Parahypsibius clusters together with the Pap. cf. scabropygus and Pap. scabropygus (Gąsiorek et al. 2024a) forming a monophyletic clade. Parahypsibius clade was recovered as being in a sister relationship with a clade comprising the remaining representatives of the subfamily Hypsibiinae (Fig. 10).

3.4.2. Echiniscus phylogeny

The phylogenetic analysis was performed using Bayesian inference for 54 taxa belonging to the genus Echiniscus (Fig. 11). The largest clade in the phylogenetic tree which stays in sister relationship with Echiniscus testudo comprises endemic species from the Republic of South Africa (Fig. 11; yellow area), species belonging to the spinulosus morpho-group (Fig. 11; green area), and two other species, Ech. aonikenk Gąsiorek, Bochnak, Vončina and Michalczyk, 2021 and Ech. meridionalis Murray, 1906 (Fig. 11; red area). This large clade was recovered with high number of internal polytomies and a lack of clear patterns related to morphology or zoogeography, except for the South African endemic species, which cluster into a single clade. The new species Ech. osiejuki sp. nov. clusters with species of the spinulosus morpho-group, with the closest relative being with Ech. cavagnaroi Schuster and Grigarick, 1966. The phylogenetic results also demonstrate the paraphyly of the spinulosus morpho-group. The remaining part of the Echiniscus phylogeny also suffers from several polytomies. Importantly, it includes populations of Ech. lineatus from four different countries (Fig. 11 orange vertical bar), which stays in sister relationship with Ech. virginicus. The sequences of the species Diploechinsicus oihonnae (Richters, 1904) (Guil et al. 2013) and Testechiniscus spitzbergensis (Scourfield, 1897) (Gąsiorek et al. 2018) were used to represent the outgroup.

4. Discussion

Although the rate of tardigrade species discoveries and descriptions in Africa has increased over the past decade, the continent remains poorly explored in terms of tardigrade diversity (e.g., McInnes et al. 2017; Stec and Kristensen 2017; Stec 2022; Stec et al. 2018; Zawierucha et al. 2018; Gąsiorek and Kristensen 2018, Gąsiorek et al. 2022; Morek et al. 2021; Kaczmarek et al. 2023; Dmuchowska et al. 2025; Warguła et al. 2025). The best-known tardigrade species diversity is in the Republic of South Africa (ca. 100 species; Dmuchowska et al. 2025), which is in large contrast to countries such as Gabon, Guinea, Malawi, or Somalia with only single species reported up to present (McInnes et al. 2017). Even when considering other African countries that are better studied, such as Algeria (ca. 36 species), Morocco (ca. 35 species), or Kenya (ca. 27 species), the majority of records originate from studies published in the 20^th Century, without support from molecular data or based on redescriptions of nominal taxa (McInnes et al. 2017). Consequently, a large proportion of species listed in checklists of African countries consists of taxa such as Macrobiotus hufelandi C.A.S. Schultze 1834, Mesobiotus harmsworthi (Murray, 1907a), Milnesium tardigradum Doyère, 1840, Minibiotus intermedius (Plate, 1888), Murrayon pullari (Murray, 1907b), Paramacrobiotus areolatus (Murray, 1907b), and Pam. richtersi (Murray, 1911). As is now known, many earlier records of the above-mentioned species, following detailed morphological and molecular analyses, have proven to represent distinct species (e.g., Kaczmarek et al. 2018; Guidetti et al. 2019; Surmacz et al. 2019; Stec et al. 2021). Therefore, it can be suggested that current data on tardigrade diversity are strongly underestimated and that extensive studies employing integrative taxonomy are required.

The genus Parahypsibius was recently established by Gąsiorek et al. (2024a) based on morphological and genetic evidence from the nominal species Pap. scabropygus, which is characterised by dorso-lateral sculpturing restricted to the caudal part of the body. This trait is unique to this species, whereas other members of the genus display sculpture over a much larger portion of the dorsal body surface. There is a substantial gap in genetic data for the genus Parahypsibius (Gąsiorek et al. 2024a). One reason for this is that species with extensive dorsal sculpturing were described more than 20 years ago, prior to the widespread adoption of integrative taxonomy. Thus, the data presented here not only include the description of a new species of Parahypsibius, but also provide additional molecular evidence confirming the genus as a distinct phylogenetic lineage, based on sequences from a species exhibiting sculpture that covers most of the dorsal body surface (Fig. 10). For deeper phylogenetic and taxonomic analyses within the genus, it will be essential to redescribe the remaining Parahypsibius taxa using detailed morphological data combined with modern molecular approaches.

The newly described species Ech. osiejuki sp. nov. provides additional phylogenetic data within the genus Echiniscus, particularly when considering the spinulosus morpho-group, which contains the largest number of taxa within the genus. Both morphological and molecular data confirmed the inclusion of the new species within the aforementioned group (Fig. 11). The tree topology reveals numerous polytomies within the clade containing species traditionally assigned to the spinulosus morpho-group. Three main groups can be distinguished. The first includes two lineages of closely related species that share the diagnostic morphological characters of the spinulosus morpho-group. The second consists of endemic clade of South African species, which does not exhibit the morphological traits defining this group. The third comprises two species, Ech. aonikenk and Ech. meridionalis, which form an unresolved polytomy together with the spinulosus group and the South African endemics, but differ markedly in morphology (e.g., the presence of ventral plates in Ech. meridionalis and an elongated cirrus A in Ech. aonikenk; Gąsiorek et al. 2022). In addition, the nominal species Ech. spinulosus is recovered on a separate branch, outside the large clade containing most species assigned to the spinulosus morphogroup. Taken together, these patterns are inconsistent with the monophyly of the spinulosus morphogroup. However, the generally low support values across the tree and multiple polytomies prevent a definitive interpretation of the relationships. Improved taxonomic and phylogenetic sampling will be necessary to clarify the phylogenetic relationships within the genus Echiniscus.

In our study, we also provided a new haplotype sequence for Ech. lineatus from Uganda, which constitute a second record of this species in continental Africa, alongside with individual previously described from Tanzania. The analysis of the mitochondrial COI fragment and the nuclear ITS-1 and ITS-2 regions, based on a haplotype network, shows no clear geographical pattern in the distribution of haplotypes across different regions in the world (Fig. 12). This is evidenced by the small nucleotide differences between the Ugandan specimen and the Brazilian specimen, compared to the African individuals from Madagascar or Tanzania, which form a distinct group within the network. The distribution of haplotypes from Indonesia is similarly erratic, with individuals located in random positions, clustering closely with specimens from both Africa and Vietnam, as well as from South America. Similar results were reported by Tumanov and Khabibulina (2024), who observed closer similarity between haplotypes from Seychelles Islands and those from Asia than with those from Africa.

Based on our phylogenetic analysis, the absence of a geographic pattern in distribution was also observed in the spinulosus morpho-group (Fig. 11). Within this group, taxa from widely separated regions occupy phylogenetic positions that appear randomly distributed, showing no clear signal of geographic relatedness. As a result, species from the Nearctic, Palaearctic, and Ethiopian regions may cluster together within a single clade [e.g., Ech. manuelae (Argentina) + Ech. tristis (Tanzania) + Ech. scabrospinosus (Portugal)]. A plausible explanation for this pattern is that Echiniscus species are capable of efficient long-distance passive dispersal, allowing species to colonize distant regions and maintain low levels of genetic divergence even across continents (Jørgensen et al. 2007; Gasiorek et al. 2019a, 2019b). If such dispersal events occurred relatively recently, common mitochondrial and nuclear markers may not yet show regional differentiation, resulting in the intermixed phylogenetic and haplotype patterns observed. Based on the current data, no clear pattern associated with climate or zoogeographic regions can be identified. The absence of a geographic signal may instead reflect human-mediated dispersal of tardigrades, which may be particularly plausible in areas that function as research or tourist centers, as recent studies have demonstrated that human activity can influence tardigrade distributions (Surmacz et al. 2025). However, the apparent lack of clear biogeographic structure may also reflect substantial gaps and biases in available sequence data, which remain unevenly distributed across both geographic regions and tardigrade taxonomic groups (Ugarte and Garraffoni 2024; Surmacz et al. 2026).

5. Summary

In this study, we described two new species belonging to the genera Parahypsibius and Echiniscus discovered in the tropical forests of Kibale NP, Uganda. The species were delineated based on both morphological and molecular data, utilizing the mitochondrial marker COI and ribosomal markers 18S rRNA, 28S rRNA, ITS1, and ITS2. Additionally, we provide sequences for these markers for Ech. lineatus, which is reported from Uganda for the first time. For all mentioned genera, we conducted phylogenetic analyses and haplotype network reconstruction for all known populations of Ech. lineatus.

6. Acknowledgements

Samples were collected during the Tropical Biology course in Uganda organized by Adam Mickiewicz University in Poznań. The study has been partially conducted in the framework of activities of BARg (Biodiversity and Astrobiology Research group). The study was supported by the Institute of Systematics and Evolution of Animals of the Polish Academy of Sciences and the Department of Animal Taxonomy and Ecology at Adam Mickiewicz University in Poznań.

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Supplementary material

Supplementary material 1

Files S1–S6

Warguła J, Stec D, Dmuchowska W, Krakowiak M, Polishchuk A. Gawlak M, Kaczmarek Ł (2026)

Data type: .zip

Explanation notes: File S1. Raw measurements of Parahypsibius arletae sp. nov. [.xlsx file] — File S2. Raw measurements of Echiniscus osiejuki sp. nov. [.xlsx file] — File S3. List of accession numbers for all sequences used in the study [.pdf file]. — File S4. Raw tree with best evolutionary model for Parahypsibius arletae sp. nov. [.txt file; .nwk file]. — File S5. Raw tree with best evolutionary model for Echiniscus osiejuki sp. nov. [.txt file; .nwk file]. — File S6. Result of p-distance analysis for genus Echiniscus [.xlsx file].

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 users 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.

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