Research Article |
Corresponding author: Łukasz Kaczmarek ( kaczmar@amu.edu.pl ) Academic editor: Sandra McInnes
© 2019 Łukasz Kaczmarek, Daria Grobys, Adam Kulpa, Tomasz Bartylak, Hanna Kmita, Marta Kepel, Andrzej Kepel, Milena Roszkowska.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Kaczmarek Ł, Grobys D, Kulpa A, Bartylak T, Kmita H, Kepel M, Kepel A, Roszkowska M (2019) Two new species of the genus Milnesium Doyère, 1840 (Tardigrada, Apochela, Milnesiidae) from Madagascar. ZooKeys 884: 1-22. https://doi.org/10.3897/zookeys.884.29469
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The knowledge of the diversity and distribution of tardigrades on Madagascar is rather poor. To date, only 13 tardigrade taxa have been reported from this region (including one Milnesium species). We examined 46 specimens belonging to two new-to-science species of the genus Milnesium described herein using an integrative approach, including classical morphology and molecular marker (COI, ITS-2 and 28S rRNA) analysis. The species were found in two moss and lichen samples collected in the Ivohibory forest in Fianarantsoa Province. Milnesium matheusi sp. nov., with claw configuration [3-3]–[3-3] and rather wide buccal tube, morphologically is most similar to: Mil. beatae Roszkowska, Ostrowska & Kaczmarek, 2015, Mil. bohleberi Bartels, Nelson, Kaczmarek & Michalczyk, 2014, Mil. eurystomum Maucci, 1991, Mil. shilohae Meyer, 2015 and Mil. tumanovi Pilato, Sabella & Lisi, 2016; however, it differs from these by morphometric characteristics. Milnesium wrightae sp. nov., by the presence of four points on secondary branches of claws IV, is most similar to Mil. quadrifidum Nederström, 1919. However, Mil. wrightae sp. nov. differs from Mil. quadrifidum by claw configuration ([4-4]–[4-4] in Mil. quadrifidum vs. [3-3]–[4-4] in Mil. wrightae sp. nov.), but also by the position of the fourth points on secondary branches of claws IV, which are located near the base of the claw in the new species and near the top of the claw in Mil. quadrifidum. Genotypic analysis showed that Mil. matheusi sp. nov. is most similar to Milnesium sp. (28S rRNA), Mil. variefidum (COI) and Mil. t. tardigradum (ITS-2) while Mil. wrightae sp. nov. is most similar to Milnesium sp. (28S rRNA), Mil. variefidum (COI) and Mil. matheusi (ITS-2). Five Milnesium taxa are recorded from the African region, including the two new species from Madagascar reported in this study.
integrative taxonomy, Milnesiidae, Milnesium matheusi sp. nov., Milnesium wrightae sp. nov., tropical region
Madagascar stretches from ~12° to ~26°S latitude on the Indian Ocean, more than 400 km east of Africa. With an area of ca. 590,000 km2, Madagascar is the world’s fourth largest island; however, it is sometimes considered a microcontinent due to its geological and biological history. First, it separated from Gondwana as part of East Gondwana, comprising the Antarctic, Madagascar, Indian, and Australian plates. After several subsequent breakups, it finally separated from the Seychelles and India ca. 66–90 My ago (
The area studied is located in south-central Madagascar (approximately 22.598830S, 46.720841E Ivohibe District, Fianarantsoa Province) on the eastern slopes of a hill located on the dry side of the main mountain range. The Ivohibory forest – which is a humid rainforest with some patches of grassy clearings – covers an area of approximately 1400 ha with an elevation gradient stretching from 900 to 1500 m asl, surrounded by human-created savannah, with a few lasting micro-patches of dry forest. It is situated on quartzite deposits, which is unique for this region (
The phylum Tardigrada currently consists of ca. 1200 species (
Species of the genus Milnesium Doyère, 1840 are large and carnivorous, feeding mainly on rotifers, nematodes and other tardigrades, but single reports show that they can also feed on amoebas (
Two moss and lichen samples from tree and rocks were collected in the Ivohibory forest on June 4, 2017 (permits No 122/17/MEEF/SG/DGF/DSAP/SCB.Re and 150N-EV06/MG17). The samples were packed in paper envelopes, dried at a temperature of ca. 30 °C and delivered to the laboratory at the Faculty of Biology, Adam Mickiewicz University, Poznań, Poland. Tardigrades were extracted from the samples and studied following the protocol of
Specimens for light microscopy were mounted on microscope slides in a small drop of Hoyer’s medium, prepared according to
All figures were assembled in Corel Photo-Paint 2017. For deep structures that could not be fully focused in a single photograph, a series of 2–10 images were taken every ca. 0.5 μm and then manually assembled into a single deep-focus image in Corel Photo-Paint 2017.
All measurements are given in micrometres [μm]. Structures were measured only if their orientation was suitable. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. All measurements (except buccal tube width) followed protocols in
Morphometric data were handled using the “Apochela” ver. 1.1 template available from the Tardigrada Register (
Species were identified using the key in
All specimens were preliminarily identified using light microscopy (LM) before DNA extraction. Later, each specimen was placed individually in a 1.5 ml Eppendorf microcentrifuge tube in 20 µl of sterile MQ H2O and kept frozen at -80 °C until DNA isolation. DNA was extracted from individual animals following a modified Chelex100 resin (Bio-Rad) extraction method (
DNA fragment | Direction | Code | Sequence (5’-3’) | Reference |
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COI | Forvard | bcdF01 | CATTTTCHACTAAYCATAARGATATTGG |
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Reverse | bcdR04 | TATAAACYTCDGGATGNCCAAAAAA |
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ITS-2 | Forvard | ITS2_Eutar_Ff | CGTAACGTGAATTGCAGGAC |
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Reverse | ITS2_Eutar_Rr | TGATATGCTTAAGTTCAGCGG | ||
28S rRNA | Forvard | 28SF0001 | ACCCVCYNAATTTAAGCATAT |
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Reverse | 28SR0990 | CCTTGGTCCGTGTTTCAAGAC |
Component | Concentration | Additional note |
---|---|---|
H2O | – | sterile MQ |
buffer | 1× | 5X Phusion HF Buffer; Thermo Scientific |
dNTPs | 200 µM | dNTP Mix; Thermo Scientific |
forward primer | 0.5 µM | – |
reverse primer | 0.5 µM | – |
polymerase | 0.02 U/µl | Phusion High-Fidelity DNA Polymerase; Thermo Scientific |
DNA | – | – |
Step | COI | ITS-2 and 28S rRNA | ||||
---|---|---|---|---|---|---|
Cycles | Time [min.:sec.] | Temp. [°C] | Cycles | Time [min:sec] | Temp. [°C] | |
initial denaturation | – | 05:00 | 98 | – | 05:00 | 98 |
denaturation | 5 | 00:30 | 98 | – | – | – |
annealing | 00:30 | 45 | – | – | – | |
extension | 01:00 | 72 | – | – | – | |
denaturation | 30 | 00:30 | 98 | 35 | 00:30 | 98 |
annealing | 00:30 | 50 | 00:30 | 50 | ||
extension | 01:00 | 72 | 01:00 | 72 | ||
final extension | – | 07:00 | 72 | – | 07:00 | 72 |
In the first step, the sequences of Mil. wrightae sp. nov. and Mil. matheusi sp. nov. were analysed by Standard Nucleotide BLAST to confirm their uniqueness. Then, a comparison was performed with COI, ITS-2 and 28S rRNA sequences of the genus Milnesium, deposited in GenBank, using only the sequences of good quality and length. All sequences were aligned with the ClustalW Multiple Alignment tool (
Class Apotardigrada (Schuster, Nelson, Grigarick & Christenberry, 1980)
Order Apochela Schuster, Nelson, Grigarick & Christenberry, 1980
Family Milnesiidae Ramazzotti, 1962
Genus Milnesium Doyère, 1840
Holotype and 18 paratypes, all from sample No 139: Ivohibory forest, Madagascar, lichen sample from quartz rocks, coll. Marta Kepel and Andrzej Kepel.
Adult females
(Fig.
The buccal apparatus of the Milnesium type (Figs
Measurements and pt values of selected morphological structures of adult females of Milnesium matheusi sp. nov. mounted in Hoyer’s medium (N – number of specimens/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD – standard deviation, 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 | 6 | 630 | – | 766 | – | – | – | 691 | – | 45 | – | 766 | – |
Peribuccal papillae length | 5 | 10.0 | – | 12.0 | 18.6 | – | 22.1 | 11.0 | 19.9 | 0.8 | 1.5 | 11.8 | 18.9 |
Lateral papillae length | 7 | 9.4 | – | 10.7 | 16.5 | – | 19.7 | 10.0 | 18.1 | 0.4 | 1.2 | 10.3 | 16.5 |
Buccal tube | |||||||||||||
Length | 9 | 51.3 | – | 62.5 | – | – | – | 56.6 | – | 3.8 | – | 62.5 | – |
Stylet support insertion point | 9 | 34.5 | – | 42.3 | 66.1 | – | 69.4 | 38.4 | 67.8 | 2.4 | 1.3 | 41.5 | 66.4 |
Anterior width | 9 | 25.2 | – | 35.9 | 47.6 | – | 57.9 | 28.9 | 51.0 | 3.2 | 3.1 | 31.4 | 50.2 |
Standard width | 9 | 23.1 | – | 31.1 | 42.4 | – | 50.8 | 26.3 | 46.5 | 2.7 | 3.0 | 29.4 | 47.0 |
Posterior width | 9 | 23.0 | – | 30.2 | 41.1 | – | 50.3 | 25.7 | 45.3 | 2.6 | 3.1 | 28.9 | 46.2 |
Standard width/length ratio | 9 | 42% | – | 51% | – | – | – | 46% | – | 3% | – | 47% | – |
Posterior/anterior width ratio | 9 | 84% | – | 94% | – | – | – | 89% | – | 4% | – | 92% | – |
Claw 1 lengths | |||||||||||||
External primary branch | 9 | 17.2 | – | 21.8 | 30.2 | – | 35.2 | 18.9 | 33.3 | 1.5 | 1.6 | 21.8 | 34.9 |
External base + secondary branch | 9 | 13.3 | – | 16.7 | 23.5 | – | 27.9 | 15.0 | 26.5 | 1.2 | 1.5 | 16.6 | 26.6 |
External spur | 7 | 3.5 | – | 5.3 | 6.4 | – | 9.6 | 4.4 | 7.8 | 0.7 | 1.3 | ? | ? |
External branches length ratio | 9 | 76% | – | 82% | – | – | – | 80% | – | 2% | – | 76% | – |
Internal primary branch | 9 | 16.0 | – | 21.1 | 30.2 | – | 34.5 | 18.3 | 32.3 | 1.6 | 1.6 | 21.1 | 33.8 |
Internal base + secondary branch | 9 | 13.3 | – | 16.6 | 24.5 | – | 27.3 | 14.8 | 26.2 | 1.1 | 1.0 | 16.3 | 26.1 |
Internal spur | 9 | 3.3 | – | 5.5 | 6.1 | – | 10.5 | 4.4 | 7.7 | 0.8 | 1.4 | 5.5 | 8.8 |
Internal branches length ratio | 9 | 77% | – | 88% | – | – | – | 81% | – | 4% | – | 77% | – |
Claw 2 lengths | |||||||||||||
External primary branch | 8 | 17.4 | – | 21.2 | 32.9 | – | 36.5 | 19.5 | 34.9 | 1.4 | 1.4 | 21.2 | 33.9 |
External base + secondary branch | 7 | 13.7 | – | 17.0 | 24.5 | – | 27.5 | 15.0 | 26.7 | 1.1 | 1.2 | 17.0 | 27.2 |
External spur | 3 | 3.9 | – | 4.9 | 7.2 | – | 7.8 | 4.4 | 7.6 | 0.5 | 0.4 | 4.9 | 7.8 |
External branches length ratio | 7 | 72% | – | 81% | – | – | – | 77% | – | 3% | – | 80% | – |
Internal primary branch | 8 | 16.8 | – | 20.5 | 31.1 | – | 35.7 | 18.7 | 33.3 | 1.3 | 1.5 | 20.2 | 32.3 |
Internal base + secondary branch | 9 | 13.0 | – | 16.3 | 25.0 | – | 27.9 | 14.7 | 26.0 | 1.1 | 0.9 | 16.3 | 26.1 |
Internal spur | 9 | 3.4 | – | 5.8 | 6.1 | – | 10.3 | 4.4 | 7.8 | 0.8 | 1.5 | 4.7 | 7.5 |
Internal branches length ratio | 8 | 74% | – | 81% | – | – | – | 78% | – | 3% | – | 81% | – |
Claw 3 lengths | |||||||||||||
External primary branch | 5 | 19.7 | – | 21.0 | 32.3 | – | 38.3 | 20.5 | 35.7 | 0.6 | 2.5 | ? | ? |
External base + secondary branch | 6 | 14.2 | – | 16.3 | 24.5 | – | 28.4 | 15.4 | 27.1 | 0.7 | 1.5 | ? | ? |
External spur | 5 | 3.5 | – | 5.2 | 6.4 | – | 9.3 | 4.4 | 7.7 | 0.7 | 1.1 | ? | ? |
External branches length ratio | 5 | 72% | – | 82% | – | – | – | 75% | – | 4% | – | ? | – |
Internal primary branch | 5 | 18.9 | – | 20.4 | 31.3 | – | 36.5 | 19.7 | 34.3 | 0.6 | 2.0 | ? | ? |
Internal base + secondary branch | 6 | 13.7 | – | 16.0 | 23.7 | – | 28.2 | 14.9 | 26.2 | 0.8 | 1.9 | ? | ? |
Internal spur | 5 | 3.8 | – | 5.6 | 7.0 | – | 9.7 | 4.8 | 8.3 | 0.7 | 1.1 | ? | ? |
Internal branches length ratio | 5 | 70% | – | 79% | – | – | – | 75% | – | 4% | – | ? | – |
Claw 4 lengths | |||||||||||||
Anterior primary branch | 7 | 19.6 | – | 23.0 | 35.1 | – | 39.8 | 20.9 | 37.2 | 1.3 | 1.5 | 23.0 | 36.8 |
Anterior base + secondary branch | 7 | 14.6 | – | 17.2 | 26.3 | – | 29.4 | 15.8 | 28.2 | 0.9 | 1.1 | 17.2 | 27.5 |
Anterior spur | 6 | 4.1 | – | 6.3 | 7.5 | – | 11.5 | 5.4 | 9.7 | 0.9 | 1.6 | 6.0 | 9.6 |
Anterior branches length ratio | 7 | 71% | – | 80% | – | – | – | 76% | – | 4% | – | 75% | – |
Posterior primary branch | 7 | 20.5 | – | 24.0 | 38.1 | – | 41.3 | 21.8 | 38.9 | 1.1 | 1.1 | 24.0 | 38.4 |
Posterior base + secondary branch | 7 | 15.2 | – | 17.7 | 26.9 | – | 29.6 | 16.1 | 28.6 | 0.8 | 1.0 | 17.7 | 28.3 |
Posterior spur | 7 | 4.4 | – | 5.8 | 7.6 | – | 10.3 | 5.2 | 9.3 | 0.6 | 1.1 | 5.5 | 8.8 |
Posterior branches length ratio | 7 | 70% | – | 76% | – | – | – | 74% | – | 2% | – | 74% | – |
Claws of the Milnesium type, slender (Figs
Adult males
(Table
Eggs oval, smooth and deposited in the exuvium as in all other known Milnesium species.
Measurements and pt values of selected morphological structures of adult males (with modified claws I) of Milnesium matheusi sp. nov. mounted in Hoyer’s medium (N – number of specimens/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD – standard deviation, 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 | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|
µm | pt | µm | pt | µm | pt | ||||||
Body length | 2 | 409 | – | 428 | – | – | – | 419 | – | 13 | – |
Peribuccal papillae length | 3 | 3.0 | – | 3.9 | 8.9 | – | 11.3 | 3.5 | 10.2 | 0.5 | 1.2 |
Lateral papillae length | 3 | 5.6 | – | 6.0 | 16.2 | – | 17.8 | 5.9 | 17.1 | 0.2 | 0.8 |
Buccal tube | |||||||||||
Length | 3 | 33.8 | – | 34.5 | – | – | – | 34.2 | – | 0.4 | – |
Stylet support insertion point | 2 | 21.2 | – | 22.3 | 62.7 | – | 64.6 | 21.8 | 63.7 | 0.8 | 1.4 |
Anterior width | 3 | 9.4 | – | 11.2 | 27.8 | – | 32.6 | 10.5 | 30.8 | 1.0 | 2.6 |
Standard width | 3 | 9.1 | – | 9.8 | 26.9 | – | 28.5 | 9.5 | 27.8 | 0.4 | 0.8 |
Posterior width | 3 | 9.4 | – | 10.2 | 27.8 | – | 29.6 | 9.8 | 28.7 | 0.4 | 0.9 |
Standard width/length ratio | 3 | 27% | – | 28% | – | – | – | 28% | – | 1% | – |
Posterior/anterior width ratio | 3 | 88% | – | 100% | – | – | – | 94% | – | 6% | – |
Claw 1 lengths | |||||||||||
External primary branch | 2 | 15.8 | – | 16.3 | 45.9 | – | 48.2 | 16.1 | 47.1 | 0.4 | 1.6 |
External base + secondary branch | 3 | 14.1 | – | 15.0 | 41.7 | – | 43.5 | 14.6 | 42.7 | 0.5 | 0.9 |
External spur | 2 | 3.2 | – | 3.4 | 9.3 | – | 9.9 | 3.3 | 9.6 | 0.1 | 0.4 |
External branches length ratio | 2 | 87% | – | 94% | – | – | – | 90% | – | 5% | – |
Internal primary branch | 3 | 14.9 | – | 15.7 | 43.2 | – | 46.4 | 15.4 | 45.1 | 0.5 | 1.7 |
Internal base + secondary branch | 3 | 14.0 | – | 14.5 | 40.6 | – | 42.9 | 14.2 | 41.4 | 0.3 | 1.3 |
Internal spur | 3 | 3.0 | – | 3.7 | 8.9 | – | 10.7 | 3.4 | 9.9 | 0.4 | 0.9 |
Internal branches length ratio | 3 | 89% | – | 94% | – | – | – | 92% | – | 2% | – |
Claw 2 lengths | |||||||||||
External primary branch | 2 | 16.9 | – | 17.9 | 49.0 | – | 53.0 | 17.4 | 51.0 | 0.7 | 2.8 |
External base + secondary branch | 1 | 13.2 | – | 13.2 | 39.1 | – | 39.1 | 13.2 | 39.1 | ? | ? |
External spur | 1 | 3.5 | – | 3.5 | 10.4 | – | 10.4 | 3.5 | 10.4 | ? | ? |
External branches length ratio | 1 | 74% | – | 74% | – | – | – | 74% | – | ? | – |
Internal primary branch | 3 | 16.4 | – | 16.9 | 47.5 | – | 50.0 | 16.7 | 48.8 | 0.3 | 1.2 |
Internal base + secondary branch | 2 | 12.7 | – | 12.8 | 37.2 | – | 37.6 | 12.8 | 37.4 | 0.1 | 0.3 |
Internal spur | 2 | 3.5 | – | 5.0 | 10.4 | – | 14.5 | 4.3 | 12.4 | 1.1 | 3.0 |
Internal branches length ratio | 2 | 75% | – | 76% | – | – | – | 76% | – | 1% | – |
Claw 3 lengths | |||||||||||
External primary branch | 3 | 16.2 | – | 17.4 | 47.1 | – | 51.5 | 16.8 | 49.0 | 0.6 | 2.3 |
External base + secondary branch | 2 | 12.1 | – | 12.8 | 35.2 | – | 37.9 | 12.5 | 36.5 | 0.5 | 1.9 |
External spur | 1 | 3.9 | – | 3.9 | 11.3 | – | 11.3 | 3.9 | 11.3 | ? | ? |
External branches length ratio | 2 | 74% | – | 75% | – | – | – | 74% | – | 1% | – |
Internal primary branch | 3 | 14.8 | – | 17.0 | 43.0 | – | 50.3 | 16.0 | 46.7 | 1.1 | 3.6 |
Internal base + secondary branch | 2 | 12.7 | – | 13.0 | 37.6 | – | 37.8 | 12.9 | 37.7 | 0.2 | 0.2 |
Internal spur | 2 | 2.9 | – | 4.0 | 8.4 | – | 11.8 | 3.5 | 10.1 | 0.8 | 2.4 |
Internal branches length ratio | 2 | 75% | – | 88% | – | – | – | 81% | – | 9% | – |
Claw 4 lengths | |||||||||||
Anterior primary branch | 3 | 16.3 | – | 17.0 | 47.4 | – | 49.3 | 16.6 | 48.5 | 0.4 | 1.0 |
Anterior base + secondary branch | 2 | 12.4 | – | 12.9 | 36.7 | – | 37.5 | 12.7 | 37.1 | 0.4 | 0.6 |
Anterior spur | 1 | 3.8 | – | 3.8 | 11.0 | – | 11.0 | 3.8 | 11.0 | ? | ? |
Anterior branches length ratio | 2 | 75% | – | 79% | – | – | – | 77% | – | 3% | – |
Posterior primary branch | 3 | 17.7 | – | 18.8 | 51.3 | – | 54.7 | 18.3 | 53.4 | 0.6 | 1.8 |
Posterior base + secondary branch | 3 | 12.7 | – | 13.7 | 37.1 | – | 39.8 | 13.1 | 38.2 | 0.6 | 1.5 |
Posterior spur | 2 | 3.0 | – | 4.1 | 8.7 | – | 11.9 | 3.6 | 10.3 | 0.8 | 2.3 |
Posterior branches length ratio | 3 | 69% | – | 73% | – | – | – | 72% | – | 2% | – |
Madagascar, 22°37'07.7"S, 46°43'14.5"E, ca. 1187 m asl, Fianarantsoa Province, Ivohibory forest.
The second author with great pleasure dedicates this species to her fiance – Mateusz Wojciechowski.
The holotype and 13 paratypes (slides: MAD139/14, MAD139/16, MAD139/18, MAD139/19, MAD139/34, MAD139/35, MAD139/42, MAD139/56, MAD139/72) are deposited at the Department of Animal Taxonomy and Ecology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6, Poznań, Poland; five paratypes (slides: MAD139/12, MAD139/13, MAD139/15) are deposited at the Natural History Museum, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark.
The new species with three points on the secondary branches of all claws (claw configuration [3-3]–[3-3]) and a rather wide buccal tube, in relation to its length, is most similar to: Mil. beatae Roszkowska, Ostrowska & Kaczmarek, 2015, Mil. bohleberi Bartels, Nelson, Kaczmarek & Michalczyk, 2014, Mil. eurystomum Maucci, 1991, Mil. shilohae Meyer, 2015 and Mil. tumanovi Pilato, Sabella & Lisi, 2016, but it differs from:
1. Milnesium beatae, only reported from Argentina and USA (
2. Milnesium bohleberi, only recorded from North Carolina and Tennessee (USA) (
3. Milnesium eurystomum reported from a few localities in Argentina, Chile, Greenland, Mongolia and USA (see review by
4. Milnesium shilohae, only reported from the type locality in Hawaii (USA) (
5. Milnesium tumanovi, only recorded from the type locality in the Crimea (Ukraine) (
The ranges of uncorrected genetic p-distances between Mil. matheusi sp. nov. and species of the genus Milnesium, for which molecular marker sequences are available from GenBank (see Table
Sequences of 28S rRNA, COI and ITS-2 of Milnesium taxa available in GenBank and used in differential diagnosis.
DNA marker | Taxon | Accession number | Source |
---|---|---|---|
28S rRNA | Milnesium sp. | JX888585.1 | Adams et. al. unpublished |
JX888586.1 | Adams et. al. unpublished | ||
JX888587.1 | Adams et. al. unpublished | ||
Milnesium tardigradum | JX888541.1 | Adams et. al. unpublished | |
JX888540.1 | Adams et. al. unpublished | ||
KC138808.1 | Zawierucha unpublished | ||
KC138809.1 | Zawierucha unpublished | ||
Milnesium sp. | AY210826.1 |
|
|
Milnesium tardigradum | FJ435780.1 |
|
|
FJ435779.1 |
|
||
Milnesium berladnicorum | KT951661.1 |
|
|
Milnesium variefidum | KT951665.1 |
|
|
COI | Milnesium sp. | KX306950.1 | Fox et al. unpublished |
Milnesium tardigradum | EU244603.1 | Schill unpublished | |
EU244604 | Schill unpublished | ||
FJ435810.1 |
|
||
Milnesium t. tardigradum | JN664950.1 |
|
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Milnesium cf. tardigradum | JX683824.1 |
|
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JX683823.1 |
|
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JX683822.1 |
|
||
Milnesium sp. | KJ857002.1 |
|
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KJ857001.1 |
|
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Milnesium cf. alpigenum | KU513422.1 |
|
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Milnesium variefidum | KT951663.1 |
|
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Milnesium berladnicorum | KT951659.1 |
|
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Milnesium sp. | EF632553.1 | Sands et. al unpublished | |
Milnesium cf. granulatum | MH751517.1 |
|
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Milnesium lagniappe | MH751518.1 |
|
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Milnesium tardigradum | MG923558.1 |
|
|
MG923559.1 |
|
||
MG923560.1 |
|
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MG923561.1 |
|
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MG923562.1 |
|
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MG923563.1 |
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MG923564.1 |
|
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MG923565.1 |
|
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Milnesium dornensis | MG923566.1 |
|
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ITS-2 | Milnesium alpigenum | MH000382.1 | Morek et al. unpublished |
Milnesium sp. | MH000386.1 | Morek et al. unpublished | |
MH000387.1 | Morek et al. unpublished | ||
Milnesium tardigradum | HM150648.2 |
|
|
GQ403682.1 |
|
||
GQ403681.1 |
|
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Milnesium t. tardigradum | JF951049 |
|
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Milnesium variefidum | KT951667.1 |
|
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KT951666.1 |
|
||
Milnesium berladnicorum | KT951662.1 |
|
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Milnesium cf. granulatum | MK681875.1 |
|
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MK681876.1 |
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MK681877.1 |
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MK681878.1 |
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MK681879.1 |
|
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MK681880.1 |
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MK681881.1 |
|
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MK681882.1 |
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MK681883.1 |
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MK681884.1 |
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MK681885.1 |
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MK681886.1 |
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1. 28S rRNA: 4.5–6.7% (5.4% on average), with the most similar being Milnesium sp. from North America (JX888585.1, JX888586.1, JX888587.1) (unpublished) and the least similar being Mil. wrightae sp. nov. (MN191504.1) (present studies);
2. COI: 20.1–38.8% (23.3% on average), with the most similar being Mil. variefidum Morek, Gąsiorek, Stec, Blagden & Michalczyk, 2016 from UK (KT951663.1) (
3. ITS-2: 17.8–31.1% (23.7% on average), with the most similar being Mil. t. tardigradum from Germany (JF951049.1) (
Holotype and 28 paratypes, all from sample No 109: Ivohibory forest, Madagascar, moss sample from tree, coll. Marta Kepel and Andrzej Kepel.
Adult females
(Fig.
The buccal apparatus of the Milnesium type (Figs
Measurements and pt values of selected morphological structures of females of Milnesium wrightae sp. nov. mounted in Hoyer’s medium (N – number of specimens/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD – standard deviation, 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 | 17 | 329 | – | 553 | – | – | – | 448 | – | 60 | – | 515 | – |
Peribuccal papillae length | 12 | 6.8 | – | 10.4 | 13.2 | – | 16.6 | 9.0 | 14.9 | 1.1 | 1.0 | 9.3 | 15.3 |
Lateral papillae length | 8 | 5.1 | – | 8.4 | 10.0 | – | 13.0 | 6.7 | 11.3 | 1.0 | 0.9 | 6.1 | 10.0 |
Buccal tube | |||||||||||||
Length | 17 | 44.8 | – | 65.6 | – | – | – | 58.4 | – | 6.5 | – | 60.9 | – |
Stylet support insertion point | 15 | 31.2 | – | 45.8 | 69.3 | – | 73.2 | 40.8 | 70.6 | 4.7 | 1.1 | 43.9 | 72.1 |
Anterior width | 16 | 14.0 | – | 23.0 | 29.5 | – | 37.9 | 19.0 | 32.7 | 2.5 | 2.4 | 20.1 | 33.0 |
Standard width | 14 | 13.0 | – | 20.7 | 27.5 | – | 36.4 | 17.7 | 31.0 | 2.3 | 2.6 | 19.8 | 32.5 |
Posterior width | 14 | 12.7 | – | 20.1 | 26.6 | – | 33.5 | 16.9 | 29.6 | 2.2 | 2.3 | 18.9 | 31.0 |
Standard width/length ratio | 14 | 28% | – | 36% | – | – | – | 31% | – | 3% | – | 33% | – |
Posterior/anterior width ratio | 14 | 88% | – | 97% | – | – | – | 91% | – | 3% | – | 94% | – |
Claw 1 lengths | |||||||||||||
External primary branch | 16 | 11.0 | – | 15.2 | 19.5 | – | 24.6 | 13.0 | 21.9 | 1.2 | 1.6 | 13.8 | 22.7 |
External base + secondary branch | 15 | 9.6 | – | 14.9 | 19.5 | – | 23.4 | 12.6 | 21.2 | 1.4 | 1.0 | 13.8 | 22.7 |
External spur | 7 | 2.8 | – | 3.7 | 4.7 | – | 5.6 | 3.2 | 5.2 | 0.3 | 0.3 | ? | ? |
External branches length ratio | 14 | 87% | – | 103% | – | – | – | 97% | – | 5% | – | 100% | – |
Internal primary branch | 16 | 10.9 | – | 14.0 | 19.4 | – | 24.6 | 12.4 | 20.9 | 0.9 | 1.4 | 12.7 | 20.9 |
Internal base + secondary branch | 16 | 9.0 | – | 14.0 | 18.9 | – | 21.7 | 12.1 | 20.4 | 1.4 | 0.8 | 12.8 | 21.0 |
Internal spur | 13 | 2.8 | – | 3.6 | 4.7 | – | 6.5 | 3.1 | 5.2 | 0.3 | 0.5 | 3.2 | 5.3 |
Internal branches length ratio | 15 | 83% | – | 103% | – | – | – | 98% | – | 7% | – | 101% | – |
Claw 2 lengths | |||||||||||||
External primary branch | 15 | 10.6 | – | 15.3 | 20.5 | – | 26.0 | 13.3 | 22.4 | 1.2 | 1.5 | 14.6 | 24.0 |
External base + secondary branch | 14 | 9.3 | – | 13.7 | 18.8 | – | 21.5 | 12.2 | 20.5 | 1.4 | 0.7 | 12.5 | 20.5 |
External spur | 8 | 3.1 | – | 4.1 | 4.9 | – | 6.7 | 3.4 | 5.5 | 0.3 | 0.6 | 4.1 | 6.7 |
External branches length ratio | 13 | 78% | – | 103% | – | – | – | 92% | – | 7% | – | 86% | – |
Internal primary branch | 14 | 10.9 | – | 15.0 | 19.2 | – | 24.3 | 12.5 | 21.3 | 1.1 | 1.7 | 13.5 | 22.2 |
Internal base + secondary branch | 15 | 9.0 | – | 14.2 | 18.0 | – | 22.3 | 12.1 | 20.2 | 1.5 | 1.0 | 12.9 | 21.2 |
Internal spur | 12 | 2.6 | – | 4.6 | 4.3 | – | 6.9 | 3.4 | 5.7 | 0.6 | 0.7 | 3.7 | 6.1 |
Internal branches length ratio | 13 | 82% | – | 103% | – | – | – | 95% | – | 8% | – | 96% | – |
Claw 3 lengths | |||||||||||||
External primary branch | 17 | 10.8 | – | 15.2 | 19.0 | – | 26.5 | 13.2 | 22.6 | 1.4 | 1.7 | ? | ? |
External base + secondary branch | 16 | 9.5 | – | 15.7 | 19.1 | – | 24.7 | 12.0 | 20.7 | 1.6 | 1.4 | ? | ? |
External spur | 7 | 3.0 | – | 4.0 | 4.9 | – | 6.8 | 3.3 | 5.5 | 0.4 | 0.7 | ? | ? |
External branches length ratio | 16 | 79% | – | 103% | – | – | – | 92% | – | 6% | – | ? | – |
Internal primary branch | 17 | 10.7 | – | 14.1 | 19.2 | – | 25.0 | 12.4 | 21.3 | 1.1 | 1.7 | ? | ? |
Internal base + secondary branch | 16 | 9.0 | – | 14.1 | 17.8 | – | 21.8 | 11.5 | 19.7 | 1.5 | 1.1 | ? | ? |
Internal spur | 10 | 2.4 | – | 4.0 | 4.1 | – | 6.8 | 3.3 | 5.7 | 0.5 | 0.9 | ? | ? |
Internal branches length ratio | 16 | 80% | – | 102% | – | – | – | 93% | – | 7% | – | ? | – |
Claw 4 lengths | |||||||||||||
Anterior primary branch | 12 | 12.6 | – | 18.4 | 23.7 | – | 28.9 | 15.2 | 25.8 | 1.6 | 1.9 | 15.8 | 25.9 |
Anterior base + secondary branch | 12 | 11.2 | – | 17.4 | 22.6 | – | 27.5 | 14.7 | 25.0 | 1.8 | 1.6 | 16.5 | 27.1 |
Anterior spur | 7 | 2.7 | – | 5.2 | 5.6 | – | 8.2 | 3.7 | 6.3 | 0.8 | 0.9 | 4.2 | 6.9 |
Anterior branches length ratio | 11 | 85% | – | 104% | – | – | – | 97% | – | 6% | – | 104% | – |
Posterior primary branch | 12 | 11.7 | – | 20.0 | 23.7 | – | 31.4 | 16.0 | 27.3 | 2.2 | 2.0 | 17.5 | 28.7 |
Posterior base + secondary branch | 11 | 12.1 | – | 18.5 | 24.0 | – | 28.9 | 15.6 | 26.5 | 2.2 | 1.8 | 17.5 | 28.7 |
Posterior spur | 7 | 2.9 | – | 5.2 | 5.3 | – | 8.2 | 3.8 | 6.7 | 0.9 | 1.0 | 4.4 | 7.2 |
Posterior branches length ratio | 10 | 92% | – | 103% | – | – | – | 98% | – | 4% | – | 100% | – |
Claws of the Milnesium type, stout (Figs
Milnesium wrightae sp. nov. 9 Claws I (paratype), arrow indicates bar under claw 10 claws IV (holotype), empty arrowhead indicates small accessory point, filled arrowheads indicate the fourth points on secondary branches near the base of the claw 11 focus on the fourth points on secondary branches near the base of the claw IV (holotype, filled arrowheads). All in PCM.
Males unknown.
Eggs oval, smooth and deposited in the exuvium as in all other known Milnesium species.
The fourth points on secondary branches of posterior claws can be barely visible or not visible at all in some positions of the specimens.
Madagascar, 22°37'04.5"S, 46°43'14.1"E, ca. 1198 m asl, Fianarantsoa Province, Ivohibory forest.
This species is named after Patricia Chapple Wright, an American primatologist and conservationist, best known for her studies on lemurs. She contributed to the establishment of the Ranomafana National Park in Madagascar. She also organized and led the expedition to the Ivohibory forest, during which several new species of tardigrades were found, including this species.
The holotype and 23 paratypes (slides: MAD109/1, MAD109/3, MAD109/4, MAD109/5, MAD109/7) are deposited at the Department of Animal Taxonomy and Ecology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6, Poznań, Poland, five paratypes (slides: MAD109/2) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9,30-387, Kraków, Poland.
The new species, by the presence of four points on secondary branches of claws IV, is most similar to Mil. quadrifidum Nederström, 1919, which is the only valid Milnesium species with four points on secondary branches of all claws. However, Mil. wrightae sp. nov. differs from Mil. quadrifidum not only by claw configuration ([4-4]–[4-4] in Mil. quadrifidum vs. [3-3]–[4-4] in Mil. wrightae sp. nov.), but also by the position of fourth points on secondary branches of claws IV (located near the base of the claw in the new species vs. near the top of the claw in Mil. quadrifidum). Additionally, all secondary branch points have similar length in Mil. quadrifidum, whereas the fourth points are very clearly smaller than the others in Mil. wrightae sp. nov.
The ranges of uncorrected genetic p-distances between the Mil. wrightae sp. nov. and species of the genus Milnesium, for which molecular marker sequences are available from GenBank (see Table
1. 28S rRNA: 5.7–8.0% (6.7% on average), with the most similar being Milnesium sp. from North America (JX888585.1, JX888586.1, JX888587.1) (unpublished) and the least similar being Mil. t. tardigradum from Poland (KC138808.1 and KC138809.1) (unpublished);
2. COI: 17.7–38.4% (22.0 % on average), with the most similar being Mil. variefidum from UK (KT951663.1) (
3. ITS-2: 25.6–36.3% (31.5% on average), with the most similar being Mil. matheusi sp. nov. (present studies) and the least similar being Mil. cf. granulatum from USA (MK681879.1) (
Milnesium matheusi sp. nov. and Mil. wrightae sp. nov. are new for science taxa, based on morphological as well as molecular characteristics. Until now, five Milnesium taxa have been reported from the African region, including Madagascar (i.e. Mil. dornensis, Mil. matheusi sp. nov. Mil. tardigradum s.s., Mil. tetralamellatum and Mil. wrightae sp. nov.). The presence of Mil. tardigradum s.s. in Madagascar needs confirmation and currently this record should be considered dubious.
Field study was the result of the collaboration and support of many people and institutions, especially ICTE/MICET in Antatananarivo and Centre ValBio in Ranomafana (Madagascar). The main funding for the expedition was obtained from the National Geographic Society’s Committee (USA) for Research and Exploration and Rainforest Trust exploratory grants.
The study was conducted on the basis of the authorization No. 122/17/MEEF/SG/DGF/DSAP/SCB.Re of Le Ministère de l’Environnement, de l’Ecologie et des Forêts (MEEF) of the Republic of Madagascar. Export of samples from Madagascar was authorized with the MEEF permit No 150N-EV06/MG17.
Studies (excluding field research) have been conducted in the framework of activities of BARg (Biodiversity and Astrobiology Research group at Adam Mickiewicz University, Poznań, Poland).
Milena Roszkowska is a scholarship holder of the AMU Foundation for the academic year 2018/2019.