New Records of Tardigrades from the Republic of South Africa with Integrative Description of a New Mesobiotus Species (Tardigrada: Eutardigrada: Macrobiotidae) ^†

Abstract

In this study, we provide a comprehensive description of a newly identified eutardigrade species, Mesobiotus longiconicus sp. nov., based on an integrative approach combining morphological and genetic data. The species was discovered at Lajuma Research Centre, Republic of South Africa, and is classified within the harmsworthi group. It can be differentiated from its closest relatives—Meb. barabanovi, Meb. ethiopicus, Meb. harmsworthi, Meb. reinhardti, and Meb. skorackii—by distinct morphological and morphometric characteristics of the bucco-pharyngeal apparatus, claws, and eggs. These morphological differences are further validated by genetic analyses using four molecular markers: 28S rRNA, 18S rRNA, COI, and ITS2. Additionally, two other tardigrade species were identified in the same locality, including Minibiotus pentannulatus, which is now recorded for the second time outside its type locality. With this discovery, the total number of tardigrade taxa reported from South Africa reaches 101, including Meb. longiconicus sp. nov.

1. Introduction

Tardigrades, also known as water bears, are a diverse phylum of microscopic multicellular invertebrates found in a wide range of terrestrial, freshwater, and marine environments worldwide [1]. Currently, ca. 1500 species and subspecies have been formally described across the world [2]. The tardigrade fauna of the Republic of South Africa (RSA) has been studied since the early 20th century, and, up to the present, 101 species have been reported from this area [3,4,5,6,7].

Currently, the genus Mesobiotus comprises 77 described species, making it the second most species-rich genus within the family Macrobiotidae [2]. According to Vecchi et al. [8], members of this genus are distinguished by several key traits, including a cuticle without pores, the presence of 10 peribuccal lamellae, a rigid buccal tube with three macroplacoids and a microplacoid positioned nearby (at a distance shorter than its length), Y-shaped double claws with a common tract featuring an internal septum that defines the distal portion, and eggs with conical or hemispherical processes. In this study, we describe a new Mesobiotus species from the Republic of South Africa using a combination of morphometric and genetic analyses.

2. Materials and Methods

2.1. Sampling and Specimen Extraction

Moss samples were collected in the vicinity of the Lajuma Research Centre, Limpopo Province, Republic of South Africa. Samples were then packed into paper envelopes, and each was labelled with a sample number, along with both geographical coordinates and altitude. They were later delivered to the laboratory at the Faculty of Biology, Adam Mickiewicz University in Poznań, Poland. Samples were examined following standard methods [9]. In summary, a piece of moss was submerged in a beaker filled with 200 mL of distilled water for six hours. Following this, the moss was vigorously agitated using a glass rod to dislodge tardigrades, their eggs, and other associated organisms. The resulting suspension, which also contained moss fragments and sediments, was transferred into a 250 mL cylinder and left undisturbed for 30 min to allow particulate matter to settle at the bottom. The upper 150 mL of liquid was carefully removed, while the remaining 50 mL was stirred and poured onto 10 cm Ø glass Petri dishes. Tardigrades and their eggs were then isolated using an Olympus SZ61 stereomicroscope. Adult specimens and eggs were subsequently mounted on microscope slides with Hoyer’s medium and dried in an incubator at 60 °C for two days.

A total of 54 individuals and 17 eggs of the new species were obtained from the sample. Among them, 37 specimens and 13 eggs were mounted on microscope slides using Hoyer’s medium for further analysis; 15 specimens and four eggs were prepared for scanning electron microscopy (SEM), following the protocol of Roszkowska et al. [10] and examined under high vacuum in Hitachi S3000N SEM; two specimens were used for DNA extraction and sequencing. One exoskeleton of a specimen used for genetic analyses was later included in the type series.

Additionally, specimens and eggs of two other species of tardigrades were extracted from the studied samples and were fixed on microscope slides in Hoyer’s medium.

2.2. Microscopy and Imaging

Tardigrades mounted on microscope slides, along with their eggs, were examined using a Phase Contrast Microscope (PCM) (Olympus BX41 equipped with an Olympus SC50 digital camera, Olympus Corporation, Shinjuku-ku, Japan). All figures were compiled using GIMP 2.10.36. For deeply positioned structures that could not be fully captured in a single image, a series of 2–8 photographs were taken at approximately 0.5 µm intervals and manually merged into a single deep-focus image using GIMP 2.10.36.

2.3. Morphometry and Morphological Nomenclature

All measurements are provided in micrometers (µm), and only structures positioned appropriately for accurate assessment were measured. The pt index, representing the ratio of a structure’s length to the length of the buccal tube as a percentage, follows the method described by Pilato [11]. The classification of the buccal apparatus and claws is based on the frameworks proposed by Pilato and Binda [12] and Vecchi et al. [8], while the terminology for the oral cavity armature (OCA) follows Pilato [13] with modifications described in Michalczyk and Kaczmarek [14]. Body length was measured from the anterior extremity to the posterior end of the body, excluding the IV pair of legs. Measurements of the buccal apparatus, claws, and eggs were conducted following the guidelines established by Kaczmarek and Michalczyk [15]. The macroplacoid length sequence is presented according to Kaczmarek et al. [16]. The eggshell morphology follows Kaczmarek et al. [17]. The nomenclature of the cuticular bars on the legs follows Kiosya et al. [18].

All morphometric data were processed with the usage of the template “Parachela” ver. 1.8, available from the Tardigrada Register [19] (The raw data are provided in SM.01.).

Tardigrade taxonomy follows Bertolani et al. [20] and Stec et al. [21], while genera abbreviations follow Perry et al. [22].

2.4. DNA Sequencing

Prior to genomic DNA extraction, two live specimens of the new species were examined under a light microscope for identification. Before proceeding with chemical extraction to obtain lysates for genetic analysis, photographic documentation of the specimens was created as reference vouchers. The DNA extraction was conducted using the Chelex^® 100 resin (Bio-Rad, Warsaw, Poland) extraction method established by Casquet et al. [23] and modified by Stec et al. [4]. To recover tardigrade exoskeletons following extraction, the remaining portion of the DNA extract containing Chelex^® 100 resin beads was diluted with ddH₂O and transferred to a glass staining block. The glass staining block was examined under stereomicroscope and the exoskeleton was mounted in a permanent slide in Hoyer’s medium. Sequencing of four DNA segments was attempted—one mitochondrial (COI) and three nuclear (18S rRNA, 28S rRNA, and ITS2)—using the following primers: HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) and LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) [24] for the COI gene fragment; SSU01_F (5′-AACCTGGTTGATCCTGCCAGT–3′) and SSU82_R (5′-TGATCCTT CTGCAGGTTCACCTAC-3′) (Sands et al. [25]) for the 18S rRNA gene fragment; 28SF0001 (5′-ACCCvCynAATTTAAGCATAT–3′) and 28SR 0990 (5′-CCTTGGTCCGTGTTTCAAGAC–3′) [26] for the 28S rRNA gene fragment; ITS-2 Eutar_Ff (5′-CGTAACGTGAATTGCAGGAC-3′) and ITS-2 Eutar_Rr (5′-TCCTCCGCTTATTGATATGC-3′) [27] for the ITS-2 gene fragment. All molecular markers underwent amplification using the protocol of Kaczmarek et al. [17]. The PCR products were purified with thermosensitive Exonuclease I and FastAP alkaline phosphatase (Thermo Scientific, Waltham, MA, USA) and sequenced using BigDye Terminator v3.1 on an ABI Prism 3130XL analyzer (Applied Biosystems, Foster City, CA, USA) according to manufacturer instructions. Sequence chromatograms were checked for accuracy using FinchTV 1.3.1 (Geospiza Inc., Seattle, WA, USA).

For comparative molecular analysis, sequence homology and identity were confirmed using the Basic Local Alignment Search Tool (BLAST; [28]). Sequence alignments were performed using the AUTO method for COI and ITS-2 markers, and the Q-INS-I method for ribosomal markers (18S rRNA and 28S rRNA) in MAFFT version 7 [29,30]. The alignments were then manually verified for non-conserved regions in BioEdit. After alignment, sequences were trimmed to a specific length for each marker, and uncorrected pairwise distances (p-distances) were calculated using MEGA 11 software [31]. The resulting distance matrices are included in the Supplementary Materials (SM.02.).

2.5. Species Identification

Identification of the new to science species and newly recorded species was carried out according to the recent keys to the species of the genus Mesobiotus [17] and original species descriptions and redescriptions [4,5,7,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47].

3. Results

3.1. New Records

1.

Echiniscus africanus Murray, 1907 [48]

Locality: 23°02′22″ S 29°27′15″ E, 1.272 m asl.: Republic of South Africa, Limpopo Province, Lajuma Research Centre, near Wilderness Camp, on a cliff above the waterfall, moss on stone, coll. Łukasz Kaczmarek, Marcelina Halicka, Adam Jęczkowski, Daria Rutkowska, 4 July 2024.

Remarks: Two collected specimens correspond perfectly to the redescription by Gąsiorek et al. [7]. Up to the present, this species has been known mainly from continental Africa (Angola, Lesotho, Republic of South Africa and Tanzania), Madagascar, and a very doubtful record from Vietnam [3,7,49,50,51].

2.

Minibiotus pentannulatus Londoño, Daza, Lisi & Quiroga, 2017 [34]

Localities: Lajuma Research Centre, Limpopo Province, Republic of South Africa: (1) 23°02′24″ S 29°27′07″ E, 1.202 m asl.: above the waterfall, moss on a stone, coll. Łukasz Kaczmarek, Aleksandra Baran, Gabriela Szczeblowska, Rafał Michalak, 2 July 2024; (2) 23°02′22″ S 29°27′15″ E, 1.193 m asl.: near the waterfall, moss on a tree, coll. Łukasz Kaczmarek, Aleksandra Baran, Gabriela Szczeblowska, Rafał Michalak, 2 July 2024; (3) 23°02′21″ S 29°27′08″ E, 1.283 m asl.: near Wilderness Camp, moss on a stone, coll. Łukasz Kaczmarek, Aleksandra Baran, Gabriela Szczeblowska, Rafał Michalak, 2 July 2024; (4) 23°01′20″ S 29°25′51″ E, 1.659 m asl.: Lajuma Mount, moss on a rock, coll. Łukasz Kaczmarek, 30 June 2024; (5) 23°01′20″ S 29°25′51″ E, 1659 m asl.: Lajuma Mount, moss on a tree, coll. Łukasz Kaczmarek 30 June 2024; (6) 23°01′23″ S 29°25′51″ E, 1686 m asl.: Lajuma Mount, moss on a rock, coll. Łukasz Kaczmarek, 30 June 2024; (7) 23°02′22″ S 29°27′15″ E, 1272 m asl.: on a cliff above the waterfall, near Wilderness Camp, moss on stone, coll. Łukasz Kaczmarek, Marcelina Halicka, Adam Jęczkowski, Daria Rutkowska, 4 July 2024.

Remarks: 355 specimens and 6 eggs found in seven samples correspond perfectly to the original description and the later amended description [4]. This is the second report (the first was from Tanzania) of this species outside of its type locality in Columbia [4,34].

3.2. Taxonomic Account of the New Species

Phylum: Tardigrada Spallanzani, 1777 [52]

Class: Eutardigrada Richters, 1926 [53]

Order: Parachela Schuster, Nelson, Grigarick, Christenberry, 1980 [54]

Superfamily: Macrobiotoidea Thulin, 1928 [55] (in Marley et al., 2011) [56]

Family: Macrobiotidae Thulin, 1928 [55]

Genus: Mesobiotus Vecchi, Cesari, Bertolani, Jönsoon, Guidetti, 2016 [8]

3.3. Description of the New Species

Mesobiotus longiconicus sp. nov. Dmuchowska, Nawrot, Warguła and Kaczmarek

(Figure 1, Figure 2, Figure 3 and Figure 4; Table 1 and Table 2)

LSID: urn:lsid:zoobank.org:act:38059656-9C2F-4E29-AC86-8E89E16CA876

3.3.1. Type Locality

23°02′19″ S, 29°27′14″ E, 1272 m asl: Republic of South Africa, Limpopo Province, Lajuma Research Centre, on Lion Trail near Wilderness Camp, on a cliff above the waterfall, moss on stone, coll. Łukasz Kaczmarek, Marcelina Halicka, Adam Jęczkowski, Daria Rutkowska, 4 July 2024.

3.3.2. Etymology

Species name refers to its long and conical egg processes. From Latin: longus = long, conicus = conical.

3.3.3. Type Depositories

The holotype (animal) (slide: RPA39/60) with 38 paratypes (27 animals and 11 eggs) (slides: RPA39/*, where the asterisk can be substituted by any of the following numbers: 1, 3, 8, 20, 22, 23, 32–35, 39, 41, 42, 57, 59–63, 65, 67) 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. Twelve paratypes (10 animals and two eggs) (slides: RPA39/*, where the asterisk can be substituted by any of the following numbers: 6, 7, 18, 19, 24, 28) are deposited in the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016, Kraków, Poland.

3.3.4. Description of Animals (Measurements and Statistics in Table 1)

Body white in living specimens and transparent after fixation in Hoyer’s medium (Figure 1A,B). Eyes present in specimens fixed in Hoyer’s medium. Body cuticle without pores, sculpturing or tubercles. A minute granulation on the entire dorsal and ventral cuticle, only visible in SEM (Figure 1C). Granulation is present on the external parts of leg pairs I–III, while on leg pair IV, it appears both on the sides and above the claws. Granulation is well visible on legs I–IV in SEM (Figure 1D) and weakly visible on PCM (Figure 1E,F).

Table 1. Measurements (in µm) of selected morphological structures of adults of Mesobiotus longiconicus sp. nov mounted in Hoyer’s medium. Abbreviations: N, number of specimens/structures measured; RANGE refers to the smallest and the largest structures 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
		µm			pt			µm	pt	µm	pt	µm	pt
Body length	19	301	–	591		–		448	–	71	–	591	–
Buccal tube
Buccal tube length	20	37.0	–	61.6		–		51.6	–	6.5	–	60.0	–
Stylet support insertion point	20	28.4	–	48.2	75.5	–	78.8	39.9	77.3	5.1	0.8	46.5	77.5
Buccal tube external width	20	5.0	–	10.2	13.5	–	18.3	8.0	15.4	1.3	1.0	9.1	15.1
Buccal tube internal width	20	3.9	–	7.8	10.3	–	14.8	6.1	11.7	1.1	1.1	7.2	12.0
Ventral lamina length	15	26.8	–	41.0	60.2	–	68.0	34.4	64.2	4.1	1.9	37.8	62.9
Placoid lengths
Macroplacoid 1	20	5.0	–	11.4	13.5	–	19.2	8.6	16.5	1.8	1.8	10.3	17.2
Macroplacoid 2	20	3.9	–	9.1	10.0	–	16.4	6.4	12.4	1.4	1.6	7.2	11.9
Macroplacoid 3	20	4.8	–	10.6	13.0	–	18.4	8.0	15.3	1.6	1.5	9.4	15.7
Microplacoid	20	2.6	–	7.8	7.0	–	12.6	5.3	10.1	1.2	1.6	4.7	7.9
Macroplacoid row	19	19.0	–	32.9	42.0	–	54.9	25.4	48.3	4.3	3.9	29.6	49.3
Placoid row	20	18.7	–	39.6	50.5	–	67.6	30.9	59.6	5.6	4.5	36.3	60.5
Claw I heights
External primary branch	12	9.2	–	13.6	19.9	–	26.5	12.3	23.8	1.3	2.3	13.6	22.7
External secondary branch	12	7.9	–	13.1	17.1	–	26.3	10.7	21.2	1.3	3.0	10.4	17.3
Internal primary branch	11	8.0	–	13.0	19.1	–	27.5	11.4	23.2	1.5	2.1	13.0	21.7
Internal secondary branch	9	6.8	–	10.8	17.1	–	22.4	9.6	19.6	1.2	1.6	10.3	17.1
Claw II heights
External primary branch	16	10.8	–	14.8	20.8	–	28.6	13.2	25.6	1.2	2.0	14.5	24.1
External secondary branch	15	8.7	–	13.1	17.2	–	26.3	11.0	21.3	1.3	2.2	11.3	18.8
Internal primary branch	15	10.3	–	15.0	21.8	–	27.0	12.7	24.3	1.1	1.6	13.8	22.9
Internal secondary branch	13	7.9	–	14.2	17.2	–	26.0	10.9	21.0	1.7	2.4	10.4	17.3
Claw III heights
External primary branch	12	11.5	–	15.9	22.8	–	29.7	13.5	25.8	1.4	1.9	13.7	22.8
External secondary branch	10	9.5	–	13.6	18.6	–	26.2	11.9	22.4	1.3	2.2	11.2	18.6
Internal primary branch	15	9.4	–	15.3	22.3	–	27.6	12.4	24.4	1.5	1.5	13.4	22.3
Internal secondary branch	10	8.0	–	13.5	17.6	–	25.2	11.2	21.6	2.0	2.1	11.7	19.5
Claw IV heights
Anterior primary branch	16	7.7	–	17.6	20.8	–	30.9	14.2	27.2	2.4	2.9	15.0	25.0
Anterior secondary branch	14	6.1	–	13.8	16.5	–	28.2	11.4	22.0	2.1	3.1	11.1	18.4
Posterior primary branch	16	7.5	–	18.2	20.3	–	32.1	14.9	28.5	2.5	3.1	14.8	24.7
Posterior secondary branch	15	5.8	–	13.8	15.7	–	26.6	11.7	22.4	2.0	2.7	12.4	20.7

Table 1. Measurements (in µm) of selected morphological structures of adults of Mesobiotus longiconicus sp. nov mounted in Hoyer’s medium. Abbreviations: N, number of specimens/structures measured; RANGE refers to the smallest and the largest structures 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
		µm			pt			µm	pt	µm	pt	µm	pt
Body length	19	301	–	591		–		448	–	71	–	591	–
Buccal tube
Buccal tube length	20	37.0	–	61.6		–		51.6	–	6.5	–	60.0	–
Stylet support insertion point	20	28.4	–	48.2	75.5	–	78.8	39.9	77.3	5.1	0.8	46.5	77.5
Buccal tube external width	20	5.0	–	10.2	13.5	–	18.3	8.0	15.4	1.3	1.0	9.1	15.1
Buccal tube internal width	20	3.9	–	7.8	10.3	–	14.8	6.1	11.7	1.1	1.1	7.2	12.0
Ventral lamina length	15	26.8	–	41.0	60.2	–	68.0	34.4	64.2	4.1	1.9	37.8	62.9
Placoid lengths
Macroplacoid 1	20	5.0	–	11.4	13.5	–	19.2	8.6	16.5	1.8	1.8	10.3	17.2
Macroplacoid 2	20	3.9	–	9.1	10.0	–	16.4	6.4	12.4	1.4	1.6	7.2	11.9
Macroplacoid 3	20	4.8	–	10.6	13.0	–	18.4	8.0	15.3	1.6	1.5	9.4	15.7
Microplacoid	20	2.6	–	7.8	7.0	–	12.6	5.3	10.1	1.2	1.6	4.7	7.9
Macroplacoid row	19	19.0	–	32.9	42.0	–	54.9	25.4	48.3	4.3	3.9	29.6	49.3
Placoid row	20	18.7	–	39.6	50.5	–	67.6	30.9	59.6	5.6	4.5	36.3	60.5
Claw I heights
External primary branch	12	9.2	–	13.6	19.9	–	26.5	12.3	23.8	1.3	2.3	13.6	22.7
External secondary branch	12	7.9	–	13.1	17.1	–	26.3	10.7	21.2	1.3	3.0	10.4	17.3
Internal primary branch	11	8.0	–	13.0	19.1	–	27.5	11.4	23.2	1.5	2.1	13.0	21.7
Internal secondary branch	9	6.8	–	10.8	17.1	–	22.4	9.6	19.6	1.2	1.6	10.3	17.1
Claw II heights
External primary branch	16	10.8	–	14.8	20.8	–	28.6	13.2	25.6	1.2	2.0	14.5	24.1
External secondary branch	15	8.7	–	13.1	17.2	–	26.3	11.0	21.3	1.3	2.2	11.3	18.8
Internal primary branch	15	10.3	–	15.0	21.8	–	27.0	12.7	24.3	1.1	1.6	13.8	22.9
Internal secondary branch	13	7.9	–	14.2	17.2	–	26.0	10.9	21.0	1.7	2.4	10.4	17.3
Claw III heights
External primary branch	12	11.5	–	15.9	22.8	–	29.7	13.5	25.8	1.4	1.9	13.7	22.8
External secondary branch	10	9.5	–	13.6	18.6	–	26.2	11.9	22.4	1.3	2.2	11.2	18.6
Internal primary branch	15	9.4	–	15.3	22.3	–	27.6	12.4	24.4	1.5	1.5	13.4	22.3
Internal secondary branch	10	8.0	–	13.5	17.6	–	25.2	11.2	21.6	2.0	2.1	11.7	19.5
Claw IV heights
Anterior primary branch	16	7.7	–	17.6	20.8	–	30.9	14.2	27.2	2.4	2.9	15.0	25.0
Anterior secondary branch	14	6.1	–	13.8	16.5	–	28.2	11.4	22.0	2.1	3.1	11.1	18.4
Posterior primary branch	16	7.5	–	18.2	20.3	–	32.1	14.9	28.5	2.5	3.1	14.8	24.7
Posterior secondary branch	15	5.8	–	13.8	15.7	–	26.6	11.7	22.4	2.0	2.7	12.4	20.7

Figure 1. Mesobiotus longiconicus sp. nov. (A) latero-ventral projection of entire animal (holotype, PCM); (B) lateral projection of entire animal (paratype, SEM); (C) dorsal cuticle with microgranulation (paratype, SEM); (D) granulation on leg IV (paratype, SEM); (E) granulation on leg I (paratype, PCM); (F) granulation on leg IV (paratype, PCM). Filled flat arrowhead represents granulation. All scale bars in μm.

Figure 1. Mesobiotus longiconicus sp. nov. (A) latero-ventral projection of entire animal (holotype, PCM); (B) lateral projection of entire animal (paratype, SEM); (C) dorsal cuticle with microgranulation (paratype, SEM); (D) granulation on leg IV (paratype, SEM); (E) granulation on leg I (paratype, PCM); (F) granulation on leg IV (paratype, PCM). Filled flat arrowhead represents granulation. All scale bars in μm.

The mouth is antero-ventral. The bucco-pharyngeal apparatus is of the Macrobiotus type with ventral lamina and ten small peribuccal lamellae (Figure 2A,B). The OCA is well developed and consists of three bands of teeth. The first band of teeth is composed of numerous small granules arranged in several discrete rows located anteriorly in the oral cavity (Figure 2B,C) just behind the bases of the peribuccal lamellae. The second band of teeth is located between the ring fold and the third band of teeth, and is composed of ridges parallel to the main axis of the buccal tube that are larger than those in the first band (Figure 2C–E). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the opening of the buccal tube. The third band of teeth is discontinuous and divided into a dorsal and ventral portion. Under PCM, the dorsal and ventral series of teeth are composed of two lateral and one median transverse ridges/crests (Figure 2C–E). The pharyngeal bulb is oval, with triangular apophyses, three rod-shaped macroplacoids, and a large, elongated microplacoid placed close to the third one (Figure 2A). The macroplacoid length sequence is 2 < 3 < 1. The first macroplacoid is anteriorly narrowed, and the third has a clearly defined subterminal constriction (Figure 2A).

Figure 2. Mesobiotus longiconicus sp. nov. (A) general view of the bucco-pharyngeal apparatus (paratype, PCM); (B) mouth opening with peribuccal lamellae and oral cavity armature (paratype, SEM); (C) oral cavity armature (paratype, PCM); (D) second and third bands of teeth in OCA on ventral side (paratype, PCM); (E) second and third bands of teeth in OCA on dorsal side (paratype, PCM). Filled flat arrowhead represents the first band of teeth, empty flat arrowhead represents second band of teeth, and filled indented arrowhead represents third band of teeth. Scale bars in μm.

Figure 2. Mesobiotus longiconicus sp. nov. (A) general view of the bucco-pharyngeal apparatus (paratype, PCM); (B) mouth opening with peribuccal lamellae and oral cavity armature (paratype, SEM); (C) oral cavity armature (paratype, PCM); (D) second and third bands of teeth in OCA on ventral side (paratype, PCM); (E) second and third bands of teeth in OCA on dorsal side (paratype, PCM). Filled flat arrowhead represents the first band of teeth, empty flat arrowhead represents second band of teeth, and filled indented arrowhead represents third band of teeth. Scale bars in μm.

Claws of the Mesobiotus type, with a peduncle connecting the claw to the lunula, a basal septum, and well-developed accessory points situated parallel to the primary branch (Figure 3A–C). Lunules under all claws are smooth (Figure 3A–D). A single continuous cuticular bar with shadowed extensions narrowing toward double muscle attachments is present below claws I–III (Figure 3A), while a horseshoe-shaped structure connects the anterior and posterior lunules on claws IV (Figure 3C).

Figure 3. Mesobiotus longiconicus sp. nov. (A) claws I (paratype, PCM); (B) claws II (paratype, SEM); (C) claws IV (paratype, PCM); (D) claws IV (paratype, SEM). Filled flat arrowhead represents cuticular bars, filled indented arrowhead represents horseshoe–shaped structure. Scale bars in μm.

Figure 3. Mesobiotus longiconicus sp. nov. (A) claws I (paratype, PCM); (B) claws II (paratype, SEM); (C) claws IV (paratype, PCM); (D) claws IV (paratype, SEM). Filled flat arrowhead represents cuticular bars, filled indented arrowhead represents horseshoe–shaped structure. Scale bars in μm.

3.3.5. Description of Eggs (Measurements and Statistics in Table 2)

The eggs are white, laid free, and spherical in shape, with sharp narrow cones (Figure 4A,B). Egg processes are terminated with one, or sometimes two, tips, i.e., bifurcated (Figure 4E,F). Egg surface between processes has visible pores and wrinkles, with semi-areolation (Figure 4C,D,G,H). The labyrinthine layer is visible under PCM as a reticulum in the process walls, with varying mesh size uniformly distributed within the process walls (Figure 4D–F). One or two large “bubbles” are present on the top of egg processes (Figure 4E,F). The flexible upper portions of egg processes are smooth and not covered with granules (Figure 4C).

Table 2. Measurements [in μm] of selected morphological structures of the eggs of Mesobiotus longiconicus sp. nov. mounted in Hoyer’s medium (N–number of eggs/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD–standard deviation).

Character	N	Range			Mean	SD
Egg bare diameter	10	60.2	–	78.6	68.9	6.0
Egg full diameter	10	127.4	–	155.0	139.0	8.9
Process height	31	27.8	–	44.2	35.6	4.1
Process base width	31	20.2	–	26.6	22.7	1.8
Process base/height ratio	31	52%	–	82%	64%	8%
Inter-process distance	22	2.3	–	6.0	4.3	1.1
Number of processes on the egg circumference	11	8	–	9	8.9	0.3

Table 2. Measurements [in μm] of selected morphological structures of the eggs of Mesobiotus longiconicus sp. nov. mounted in Hoyer’s medium (N–number of eggs/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD–standard deviation).

Character	N	Range			Mean	SD
Egg bare diameter	10	60.2	–	78.6	68.9	6.0
Egg full diameter	10	127.4	–	155.0	139.0	8.9
Process height	31	27.8	–	44.2	35.6	4.1
Process base width	31	20.2	–	26.6	22.7	1.8
Process base/height ratio	31	52%	–	82%	64%	8%
Inter-process distance	22	2.3	–	6.0	4.3	1.1
Number of processes on the egg circumference	11	8	–	9	8.9	0.3

Figure 4. Mesobiotus longiconicus sp. nov. (A,B) entire egg ((A): SEM; (B): PCM); (C) egg processes (SEM); (D) egg surface between processes (PCM); (E) bifurcated egg process (PCM); (F) pointed egg process (PCM); (G,H) semi-areolation on egg surface (SEM). Empty indented arrowhead represents semi-areolation, filled flat arrowhead represents bifurcation on egg process, and filled indented arrowhead represents “bubble” on the top of the egg process. Scale bars in μm.

Figure 4. Mesobiotus longiconicus sp. nov. (A,B) entire egg ((A): SEM; (B): PCM); (C) egg processes (SEM); (D) egg surface between processes (PCM); (E) bifurcated egg process (PCM); (F) pointed egg process (PCM); (G,H) semi-areolation on egg surface (SEM). Empty indented arrowhead represents semi-areolation, filled flat arrowhead represents bifurcation on egg process, and filled indented arrowhead represents “bubble” on the top of the egg process. Scale bars in μm.

3.3.6. DNA Sequences

We obtained good-quality sequences for the following molecular markers:

18S rRNA: GenBank: PV241371; 856 bp long (species voucher number: 39.1)

28S rRNA: GenBank: PV241372; 703 bp long (species voucher number: 39.1)

COI: GenBank: PV239604; 574 bp long (species voucher number: 39.2)

ITS-2: GenBank: PV241373; 418 bp long (species voucher number: 39.1)

3.3.7. Genetic Distances

The ranges of uncorrected genetic p-distances between the molecular markers of Meb. longiconicus sp. nov. obtained in our study and the sequences of all species of the genus Mesobiotus available in GenBank are as follows (SM.02):

28S rRNA: 0.7–14.8% (5.5% on average), with the most similar being Meb. diegoi Stec, 2022 [38] (GenBank: OP142520–21) [38] and the least similar being Meb. aradasi (Binda and Pilato, 2005) [57] (GenBank: OQ92999) [58].

18S rRNA: 0.6–5.6% (2.7% on average), with the most similar being Meb. diegoi Stec, 2022 [38] (GenBank: OP142526) [38] and the least similar being Meb. aff. furciger (GenBank: MH197148) [35].

COI: 17–25.1% (21.6% on average), with the most similar being Meb. aff. furciger (GenBank: MH195153) [35] and the least similar being Meb. hilariae Vecchi, Cesari, Bertolani, Jönsson, Rebecchi and Guidetti, 2016 [8] (GenBank: KT226108) [8] and Meb. cf. furciger (GenBank: MW727960) [59].

ITS2 rRNA: 8.5–38.2% (20.3% on average), with the most similar being Meb. diegoi Stec, 2022 [38] (GenBank: OP142514–15) [38] and the least similar being Meb. aradasi (Binda and Pilato, 2005) [57] (GenBank: OQ940183) [58].

4. Discussion

The new species possesses an egg surface between egg processes with not fully developed areolation (semi-areolation), making it similar to Meb. barabanovi (Tumanov, 2005) [33], Meb. ethiopicus Stec and Kristensen 2017 [60], Meb. harmsworthi (Murray, 1907) [61], Meb. reinhardti (Michalczyk and Kaczmarek, 2003) [14], and Meb. skorackii Kaczmarek, Zawierucha, Buda, Stec, Gawlak, Michalczyk and Roszkowska, 2018 [35]. However, it differs from the following:

1. Mesobiotus barabanovi, known only from Kirghizia [17,33] by the absence of ventral granulation between legs, the presence of granulation on legs I-III, the presence of typical Mesobiotus-type claws IV (elongated primary branches of claws IV in Meb. barabanovi), the absence of teeth on lunules IV, shorter claws (see Table 1 in the present paper for the new species vs. see Table 1 in Tumanov [33] for Meb. barabanovi), a different shape of egg processes (sharp narrow cones in the new species vs. cones with long slender endings in Meb. barabanovi), the lack of radial ridges on the egg surface between processes, a lower number of egg processes on the egg circumference (8–9 in the new species vs. ca. 20 in Meb. barabanovi), smaller egg bare diameter (60.2–78.6 µm in the new species vs. 81.2–91.3 µm in Meb. barabanovi), longer egg processes (27.8–44.2 µm in the new species vs. 11.0–17.2 µm in Meb. barabanovi), and wider egg processes bases (20.2–26.6 µm in the new species vs. 8.5–12.7 µm in Meb. barabanovi).

2. Mesobiotus ethiopicus, known from the type locality in Ethiopia [60] by the presence of eyes, a second band of teeth (several evidently larger teeth in the second band of teeth in Meb. ethiopicus), the presence of granulation on legs, smooth lunules under claws IV (serrated in Meb. ethiopicus), larger egg full diameter (127.4–155.0 µm in the new species vs. 89.5–117.5 µm in Meb. ethiopicus), longer egg processes (27.8–44.2 µm in the new species vs. 15.3–23.6 µm in Meb. ethiopicus), and a lower number of egg processes on the egg circumference (8–9 in the new species vs. 10–12 in Meb. ethiopicus).

3. Mesobiotus harmsworthi, known from its neotype locality in Norway [35], but also from Russia and the United Kingdom [35], by the lack of additional teeth in second row of the oral cavity armature, the absence of teeth on lunules IV, smaller accessory points on claws IV, a different shape of egg processes (sharp narrow cones in the new species vs. sharp wide cones in Meb. harmsworthi), the absence of areolation (although thin, they sometimes form complete areolae in M. harmsworthi), a lower number of egg processes on the egg circumference (8–9 in the new species vs. 11–12 in Meb. harmsworthi), a larger egg full diameter (127.4–155.0 µm in the new species vs. 101.0–120.0 µm in Meb. harmsworthi), and longer egg processes (27.8–44.2 µm in the new species vs. 14.3–19.7 µm in Meb. harmsworthi).

4. Mesobiotus reinhardti, known from the type locality in Cyprus [14] by the presence of granulation on legs I-III, better developed semi-areolation on the egg surface between egg processes, and the absence of pores on the processes’ surface.

5. Mesobiotus skorackii, known only from the type locality in the Kyrgyz Republic [35], by the absence of teeth on lunules IV, a longer external secondary branch on claw III (9.5–13.6 µm in the new species vs. 6.2–8.9 µm in Meb. skorackii), a different shape of egg processes (sharp narrow cones in the new species vs. sharp wide cones in Meb. skorackii), a lower number of egg processes on the egg circumference (8–9 in the new species vs. 10–12 in Meb. skorackii), a smaller egg full diameter (127.4–155.0 µm in the new species vs. 89.2–103.8 µm in Meb. skorackii), and longer egg processes (27.8–44.2 µm in the new species vs. 9.5–16.0 µm in Meb. skorackii).

Up to the present, 101 tardigrade taxa (including 25 unnamed species and one species listed as Pseudechiniscus cf. ehrenbergi) have been reported from the RSA [3,6,7]. The first studies on South African tardigrades were conducted by Murray [48,62] and later continued by other authors [5,63,64,65,66,67,68,69,70]. However, in most of the studies, a very limited number of samples had been analyzed, and the focus was on describing species new to science. The only comprehensive studies on larger numbers of samples were conducted by Morek et al. [6], who reported ca. 24 potential new species of the genus Milnesium, and Gąsiorek et al. [7], who reported 36 Heterotardigrada species. Tardigrades in the RSA are very unevenly studied. In some provinces, the number of reported tardigrades (not including unnamed species) is relatively high, like in KwaZulu-Natal (19 species), Cape (14), and Gauteng (10); however, in others, the tardigrade fauna is almost unknown, e.g., Eastern Cape (8), Limpopo (6), Western Cape (5), Mqumalanga (3), North West (2), and Northen Cape (2). It should be also mentioned that some of the species were reported a long time ago and require modern redescriptions, reconsiderations, or confirmations (e.g., Barbaria bigranulata (Richters, 1908) [71], Dianea sattleri (Richters, 1902) [72], Echiniscus duboisi (Richters, 1902) [73], Macrobiotus echinogenitus (Richters, 1903) [74], Pseudechiniscus (Pse.) suillus (Ehrenberg, 1853) [75], Pseudechiniscus (Pse.) and bispinosus (Murray, 1907) [61]), or are now considered as nomen dubium taxa, like Echiniscus crassispinosus fasciatus (Marcus, 1928) [76] and Pseudechiniscus jiroveci (Bartoš, 1963) [7,51,77,78].

Other taxa, like Macrobiotus hufelandi (C.A.S. Schultze, 1834) [79], Mesobiotus harmsworthi (Murray, 1907) [61], Minibiotus intermedius (Plate, 1888) [80], Milnesium tardigradum tardigradum (Doyère, 1840) [81], Paramacrobiotus richtersi (Murray, 1911) [82], and Ramazzottius oberhauseri (Doyère, 1840) [81], are now considered as nominal species for species complexes. In the past, these species were reported from numerous localities throughout the world, but most of them are now considered to have much more restricted distribution, and their old reports need to be confirmed [3,6,15,17,83,84,85].

To date, only eight tardigrade species have been reported from the entire Limpopo Province, including the new Mesobiotus species and Min. pentannulatus reported in the present study. In the analyzed samples, Ech. africanus and Min. pentannulatus have also been found. Both species had already been recorded in the same region, i.e., in the area surrounding the Lajuma Research Centre, Limpopo Province. [62,86].

To summarize, at present, 101 species (including 62 confirmed species, 14 problematic species, and 25 unnamed species) have been now reported in the RSA. However, the list of South African tardigrade species is still far from complete and further studies are necessary across the entire country.