Macrobiotus paulinae Stec, Smolak, Kaczmarek & Michalczyk, 2015, sp. nov.
Creators: name: Stec, Daniel
name: Smolak, Radoslav
name: Kaczmarek, Łukasz
name: Michalczyk, Łukasz
Publisher: name: Zenodo
Year: 2015
Subjects: Biodiversity
Taxonomy
Animalia
Tardigrada
Eutardigrada
Parachela
Macrobiotidae
Macrobiotus
Macrobiotus paulinae

description: Macrobiotus paulinae sp. nov.
(Tables 4–5, Figs 1–45)
Material examined. 173 animals and 2 eggs isolated directly from the moss sample and 14 animals and 12 eggs obtained from the in vitro culture. Specimens mounted on microscope slides in Hoyer’s medium, fixed on SEM stubs and processed for DNA sequencing.
Description of the new species.
Animals (measurements and statistics in Table 4): Body from white in juveniles to light yellow in adults, transparent after fixation in Hoyer’s medium (Fig. 1). In older, live animals the portion of the body posterior to legs III is swollen (Fig. 2). Eyes present (also in mounted specimens). Cuticle covered with pores and faint granulation (Figs 3–12). Small oval and round pores, 0.3–0.5 Μm in diameter (Figs 4–6), are scattered randomly on the entire cuticle, although they are larger on dorso-lateral parts of the body, including the outer surface of legs (Fig. 9). A ring of pores is present around the mouth opening, below the peribuccal sensory lobes (Fig. 12, empty arrow). Due to the small size of pores, they are difficult to observe under light microscopy (LM) (see Fig. 6, asterisks). Granulation is arranged in patches on the dorso-lateral and caudo-dorsal cuticle (body granulation) as well as on the outer surface of all legs (leg granulation) (Figs 3, 6–11). There are seven dorso-lateral patches of sparse granulation arranged symmetrically on both sides of the body. The patches are spaced regularly, with patches I, III and V being placed slightly anteriorly to legs I, II and III, respectively and with patches II, IV and VI being placed slightly posteriorly to legs I, II and III, respectively. Patch VII is placed between legs III and IV, at the level of the cloaca (Fig. 3). All body patches are arranged around cribriform areas (circled in Fig. 7). Rarely, and only in some of larger animals, the dorso-lateral patches may extend to the dorsum and join to form seven transverse stripes of granulation, narrow dorsally and wide laterally. The granulation on all legs is arranged into two distinct patches: a small area of fine and dense granulation just above claws (distal patch) and a larger area of more robust and sparse granulation located in the middle of each leg (proximal patch) (Figs 8–11, in all figures the distal patch is outlined with a dashed curve whereas the proximal patch is outlined with a solid curve). Compared to granulation on legs I– III (Figs 8–9), the granulation on legs IV is more distinct and forms larger patches, with the proximal patches merged into a single transverse band of granulation (Figs 10–11). Both pores and granulation are poorly visible, with the dorso-lateral patches being the least visible, thus the cuticle has to be observed under a good quality contrast microscope and at the highest magnification in order to ensure correct interpretation.
Mouth antero-ventral (Fig. 12). Bucco-pharyngeal apparatus of the Macrobiotus type, with the ventral lamina and ten small peribuccal lamellae. The lamellae are only clearly visible under SEM (Figs 13–14) and very difficult to identify under LM (Fig. 15). The oral cavity armature composed of three bands of teeth, but under LM only the third band is visible (Fig. 15) and SEM is required to reveal the first and the second bands of teeth (see Figs 13–14). The first band of teeth comprises extremely small cones arranged in a single row situated at the anterior portion of the oral cavity, on the bases of peribuccal lamellae (Fig. 13, indented arrowhead). The second band of teeth is composed of ca. five rows of slightly larger cones, positioned at the rear of the oral cavity, between the ring fold and the third band of teeth (Figs 13–14, flat arrowheads). The teeth of the third band are positioned at the rear of the oral cavity, between the second band of teeth and the buccal tube opening (Figs 13–15). Under LM, the teeth of the third band appear as a single ventral and a single dorsal thin transversal ridge (Fig. 15 and the lower insert). However, SEM reveals that both ventral and dorsal teeth indeed form continuous ridges, but with evident median and lateral peaks corresponding to median and lateral teeth in species with stronger oral cavity armatures (Fig. 14). Median teeth are smaller than the lateral teeth, which appears under LM as a thinning in the central portion of the ridges (Fig. 15 and the lower insert). In addition, there are a number of smaller accessory teeth placed laterally to the lateral teeth. These accessory teeth are more likely to develop on the ventral side than on the dorsal. Buccal tube walls are slightly thickened posterior to the stylet support insertion point (Fig. 15, empty indented arrowhead). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a triangular microplacoid (Fig. 15 and the upper insert). Macroplacoid length sequence 2<1. First macroplacoid with a central constriction (Fig. 15, upper insert).
Claws small and slender, of the hufelandi type (Figs 16–19). Primary branches with distinct accessory points. Lunules on legs I–III smooth (Figs 16–17), but those on legs IV from crenulate in young animal to dentate in larger specimens (Figs 18–19). Bars under claws absent, but extremely faint (barely visible under LM, only clearly visible in SEM) paired muscle attachments below claws I–III present.
Eggs (measurements and statistics in Table 5): Laid freely, light yellow, spherical and with a hufelandi type chorion ornamentation (Figs 20–45). The surface between processes is covered with a very dense regular reticulum (mesh diameter 0.05–0.20 Μm, reticulum thickness 0.05–0.30 Μm) (Figs 32–34, 38, 40–42). As these diameters are on the margin or below the resolution of LM, the mesh is only clearly visible with SEM. Under LM the mesh outline can merely be detected only on some eggs (usually only around the processes, where meshes are slightly larger), but on the majority of eggs the surface appears to be covered with faint dots when observed with PCM (Figs 24 and 26). Thus, eggs have to be observed under a good quality contrast microscope and at the highest magnification in order to ensure correct identification. Importantly, however, SEM also does not guarantee the correct identification regarding the chorion sculpture. In some of the eggs we observed with SEM, the reticulum was partially covered by a layer of an unknown substance that fills the mesh and makes the egg surface appear smooth (Figs 31, 33, 35, 39, 41 and 43). Figures 33 and 41 show a transition from a clearly visible reticulum to an obscured smooth surface. As eggs with an obscured reticulum appear smooth, they could be erroneously classified as the persimilis type (i.e. with a smooth chorion). Thus, the examination of a larger number of eggs and at magnifications>30,000× is necessary in order to avoid false interpretations.
Processes are in the shape of inverted goblets with slightly concave conical trunks and well-defined terminal discs (Figs 20–45). When observed under SEM, all trunks have 5–7 distinct ring undulations (Figs 36–41), whereas in LM the undulations are lightly outlined only in some processes (Fig. 28, indented arrowheads), with the majority of processes appearing to have smooth trunks. Processes exhibit a considerable variation in height and, therefore, also in the base/height ratio (e.g. see Figs 22–23). Terminal discs are cog-shaped, with a concave central area and with 8–10 small irregular teeth (Figs 28–29, 34–45). In SEM, small granules 0.05–0.08 Μm in diameter, are visible on the teeth (Figs 42–45). Approximately half the processes have one to a few teeth on the disc that are elongated into thin flexible filaments, less than 0.5 Μm in diameter and up to 20 Μm in length (Figs 34–41). The filaments are hair-like under LM, but under SEM they are covered with the same type of granulation as the disc teeth (Fig. 41), so they probably enhance the adhesive function of egg processes. Eggs found in the sample were devoid of filaments and we were able to observe them only on eggs obtained from cultured animals. The most probable explanation for this is that the filaments are very fragile and easily broken, thus are not present on some eggs. Also, the filaments are very thin so could be overlooked and/or misinterpreted as dirt attached to eggs. Therefore, extreme care must be taken when examining the eggs in order to avoid incorrect conclusions.
DNA sequences. First, we sequenced all four DNA fragments for four individuals. As we found no polymorphism in any of the sequences, for the remaining ten individuals we have amplified and sequenced only ITS-2, potentially the most variable fragment. Again, there were no differences between all fourteen sequences. Since the type population exhibited only one haplotype, only a single sequence for each of the four DNA fragments was uploaded to the GenBank. The typical DNA sequences for Ma. paulinae sp. nov. are as follows:
The 18S rRNA sequence (GenBank: KT935502), 1731 bp long:
AGATTAGCCATGCATGTCTCAGTACTTGCTTTTACAAGGCGAAACCGCGAATGGCTCATTAAATCAGTTATGGTTCACTA GATCGTAAATTTTACACGGATAACTGTGGTAATTCTAGAGCTAATACGTGCAAGCAGCTCGTTTCCTTGTGGAGCGAGCG CAGTTATTAGAACAAGACCAATCCGGCCTTCGGGTCGGTACAATTGGTGACTCTGAATAACCGAAGCGGAGCGCATGGTC TCGTACCGGCGCCAGATCTTTCAAGTGTCTGACTTATCAGCTTGTTGTTAGGTTACGTTCCTAACAAGGCTTCGACGGGT AACGGGGTATTAGGGTCCGATACCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAA TTACCCACTCCTAGCACAGGGAGGTAGTGACGAAAAATAACGATGCGAGGGCTAATAGCTTCTCGTAATCGGAATGGGTA CACTTTAAATCCTTTAACGAGGATCTATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGT ATATTAAAGTTGCTGCGGTTAAAAAGCTCGTAGTTGGATCTGGGCTTCTGAATGGACGGTTCACTTTACGGTGTAACTGC TCGTTTGGTGCCACAAGCCGGCCATGTCTTGCATGCCCTTTGCTGGGTGTGCTTGGCGACCGGAACGTTTACTTTGAAAA AATTAGAGTGCTCAAAGCAGGCGTATGGCCTTGCATAATGGTGCATGGAATAATGGAATAGGACCTCGGTTCTATTTTGT TGGTTTTCGGAACTCGAGGTAATGATTAAGAGGAACAGACGGGGGCATTCGTATTGCGGCGTTAGAGGTGAAATTCTTGG ATCGTCGCAAGACGAACTACTGCGAAAGCATTTGCCAAGAATGTTTTCATTAATCAAGAACGAAAGTTAGAGGTTCGAAG GCGATCAGATACCGCCCTAGTTCTAACCATAAACGATGCCAACCAGCGATCCGTCGGTGTTTTTTTTATGACTCGACGGG CAGCTTTCCGGGAAACCAAAGTGCTTAGGTTCCGGGGGAAGTATGGTTGCAAAGCTGAAACTTAAAGGAATTGACGGAAG GGCACCACCAGGCGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTTACCCGGCCCGGACACTGTAAGGATTG ACAGATTGAGAGCTCTTTCTTGATTCGGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGCGATTTGTCTGGTTAA TTCCGATAACGAACGAGACTCTAGCCTGCTAAATAGCCGACCGATCCGCAGCGTCGGTTGCTACAAAAGCTTCTTAGAGG GACAGGCGGCGTTTAGTCGCACGAGATTGAGCAATAACAGGTCTGTGATGCCCTTAGATGTCCGGGGCCGCACGCGCGCT ACACTGAAGGGACCAGAGTGCTTAACTACCTTGGCCGGAAGGCCTGGGGAATCCGGTTAAACCCCTTCGTGATTGGGATT GAGCTTTGTAATTATCGCTCATGAACGAGGAATGCCCAGTACTCGCGAGTCATAAGCTCGCGATGATTACGTCCCTGCCC TTTGTACACACCGCCCGTCGCTACTACCGATTGAATGATTTAGTGAGGTCTTCGGACTGGCCGTCGATGCTGACTCTGTT GGCGTCGGTTGGATCGGAAAGACGACCAAACTGGCTCATTAGAGGAAGTAA
The 28S rRNA sequence (GenBank: KT935501), 788 bp long:
TACTAAGCGGAGGAAAAGAAACCAACGGGGATGCCGATAGTACTGCGAGTGAAATCGGCCAAGCCCAGCGCCGAATCCTG TTGCTGGCGACGGTGACAGGAACTGTGGCGTGAAGAACGTCCTTACCGGTACGGTTTGCGTGCGTAAGTTCTCCTGAGTG AGGCTCCATTCCAAGGAGGGTGCAAGACCCGTATCGCGTGCAACCGGTGTCGGTGTAAGATGTTCGGAGAGTCGCCTTGT TTGTGAGTACAAGGTGAAGTCGGTGGTAAACTCCATCGAAGGCTAAATATGACCACGAGTCCGATAGCGAACAAGTACCG TGAGGGAAAATTGAAAAGCACTTTGAAGAGAGAGCGAAACAGTGCGTGAAACCGCTCAGAGGCAAGCAAATGGGGCCTCG AAGGCAAGGCAGCGAATTCAGCTGGTGGTCTGCGCGGCTGGTTGGTTTGGAGATCTTACGACTCTGGCCGGCTGGGCTCT GAGCGTAGGTGCCAGTGCACTTTTGTTGCTTGTACGCCACCGCCGTTGAGTGGGCATCCGTCGAGTTGGCAATGCGAAGC CTTGAGCCTTTACGGGCCTAGGTGCTTGCAGCCGGCTTTTGTACGCGTTTGCACTTCAACCGGTCATGTTTGCATGTGCC AGCAATTTGGCGTTGGATCGGCTTGCTCTGCCGTTTGTCGTGAGATGACGAGCTTGCTCGGCTCTTCGGCATCTATGGTA GAATCGGGTCGGTTTCAACGTGGGCACATTGTAATTCGGTGGCGAGTAGATGGCTGCCCATTTAACCC
The COI sequence (GenBank: KT951668), 707 bp long:
TTGACAGCTTGTGTAGGAACATCACTAAGCTTTTTAATCCGAACAGAACTCAGCCWACCAGGACTTCTTTTAGCTGATGA ACAAATATATAATGTCATTGTCACAAGCCACGCCTTTATTATAATTTTTTTTTTTGTAATGCCAATTCTTATTGGAGGAT TTGGTAATTGATTAATCCCTTTAATAATCAGAGCCCCCGACATAGCTTTCCCTCGTATAAATAATTTAAGATTTTGAATA CTACCTCCCTCATTTCTTCTAATTACATTAAGAACTATGGCTGAACAGGGAGCCGGTACGGGATGAACTGTGTACCCCCC TCTCTCGCATTTTTTTGCTCATAGGGGGCCTAGGGTAGACTTGACTATTTTTTCTCTTCATGTAGCCGGTATCTCCTCCA TTTTAGGGGCTATTAATTTTATTTCTACAATTATTAATATGCGAGCCCCGTTTATAAGGTTAGAAAAAATACCTCTTTTT GTTTGGTCAGTACTCCTAACTGCCATTTTACTTTTATTAGCTCTGCCTGTGCTTGCAGGGGGCATCACTATATTATTACT AGACCGAAATTTTAACACTTCTTTTTTTGACCCTGCGGGCGGGGGAGACCCTATTTTATACCAACATTTATTTTGATTTT TTGGGCATCCCGAAGTGTATATTTTAATTTTGCCTGGTTTTGGGATTATCTCTCAAATTGTAATTCA
The ITS-2 sequence (GenBank: KT935500), 440 bp long:
AAAATGCGAGACGTAACGTGAATTGCAGGACTTTGTGAACGTTAATTCTTCGAACGCACATTGCGGCTTCGGGTTAACTG AAGCCATGCCTGGTTGAGGGTCAGTTGAAATAAAAAATCGTAATCGYGCATTGATTACGGATTGTCTGGTTTTAACGGCC TTGTGTGCCGTTTCCGGATAAAGTTGAGACCAGATGTGTGCGCTCATTTGACCGGTGCAAGCAACGCTTTGCCGAGTTGG AGCATCCGACTTGTTTAGTCGTGCGCCGCAGCTGCACAATGGCTAAGCATGGTCAACCAACGGCGTTTGATGGCAAAGAA AGACTGGTACAAAAGTGCGCAAGCGCATAGACACGTCTGTGGCCGAAAAAGAACGCACCCAAGTGTGTTTTTGCTCATTC TTTTGACCTCAGCTCAGACAAGATTACCCGCTGAACTTAA
Type locality. 02°39'15.75''N, 36°56'9.99''E; 1824 m asl: Kenya, Eastern Province, Marsabit County, Mount Kulal Biosphere Reserve, Kulal Mt., near Gatab. Habitat: compact high, dense and shady forest (with approximately 30 min of sunshine a day reaching the forest floor). Sample: moss from a dead fallen tree (Chionanthus sp.). Coll. Radoslav Smolak.
Etymology. The species is named after a former member of the Michalczyk lab and the first author’s friend, Miss Paulina Kosztyła, who wears braids that resemble fine filaments on the egg processes of the new species.
Type depositories. Holotype: slide KE.001.01 (with 17 paratypes), 108 paratypes (slides: KE.001/*, where the asterisk can be substituted by any of the following numbers: 1, 2, 3, 4, 5, 6, 8, 9, 11, 13, 14, 16, 17, 18, 19, 22, 26, 27, 28) and 7 eggs (slides: KE.001/*: 21, 23, 24) are deposited at the Department of Entomology, Institute of Zoology, Jagiellonian University, Gronostajowa 9, 30-387, Kraków, Poland; 25 paratypes (slides: KE.001/*: 10, 12, 15, 20) and 4 eggs (slide KE.001/25) are deposited at the Department of Animal Taxonomy and Ecology, Institute of Environmental Biology, Adam Mickiewicz University in Poznań, Umultowska 89, 61-614 Poznań, Poland.
description: Published as part of Stec, Daniel, Smolak, Radoslav, Kaczmarek, Łukasz & Michalczyk, Łukasz, 2015, An integrative description of Macrobiotus paulinae sp. nov. (Tardigrada: Eutardigrada: Macrobiotidae: hufelandi group) from Kenya, pp. 501-526 in Zootaxa 4052 (2) on pages 506-517, DOI: 10.11646/zootaxa.4052.5.1, http://zenodo.org/record/238460