Two new species of the genus Milnesium Doyère, 1840 (Tardigrada, Apochela, Milnesiidae) from Madagascar

Abstract 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 matheusisp. 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 wrightaesp. nov., by the presence of four points on secondary branches of claws IV, is most similar to Mil. quadrifidum Nederström, 1919. However, Mil. wrightaesp. nov. differs from Mil. quadrifidum by claw configuration ([4-4]–[4-4] in Mil. quadrifidum vs. [3-3]–[4-4] in Mil. wrightaesp. 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. matheusisp. nov. is most similar to Milnesium sp. (28S rRNA), Mil. variefidum (COI) and Mil. t. tardigradum (ITS-2) while Mil. wrightaesp. 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.


Introduction
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 km 2 , 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 (de Witt 2003, Kusky et al. 2007. Madagascar is characterised by high biological endemism, estimated at >90% for terrestrial vertebrates and >80% for vascular plants Benstead 2003, Callmander et al. 2011). A great number of species occurring in Madagascar have restricted geographical ranges and are reported from only one or several localities (Wilmé et al. 2006). Several present taxa are assumed to be Gondwanan relicts. Most of the biota is believed to be derived from African and Asian colonizers (Yoder and Nowak 2006, Warren et al. 2010, Buerki et al. 2013Hong-Wa and Besnard 2013). Madagascar has a tropical climate with two main climatic and biogeographic zones characterised by a substantially different vegetation cover, i.e., evergreen humid forests and deciduous forests divided by a mountain range that extends from north to south in eastern-central Madagascar (Du Puy and Moat 1996). Both zones are divided into several regions, each of which has distinctive climatic features and a set of unique habitats.
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 (Wright and Houlihan 2017). This unusual geology strongly influences the species composition of the existing vegetation (Du Puy and Moat 1996).
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 (Miller and Williams 2012;Roszkowska et al. 2015Roszkowska et al. , 2016. Species in this genus reproduce parthogenetically and/or bisexually, and are characterised by sexual dimorphism (e.g., Suzuki 2003;Ciobanu et al. 2015). Thirty-eight species of the genus Milnesium have been reported mostly from mosses and lichens from many localities, ranging from the Antarctic through tropical and temperate to Arctic regions. Most have been described in recent years (Degma et al. 2009(Degma et al. -2018Kaczmarek et al. 2014Kaczmarek et al. , 2015Kaczmarek et al. , 2016McInnes et al. 2017). According to its unique morphology (and based on molecular data) the genus Milnesium is classified in the class Apotardigrada (Schuster, Nelson, Grigarick & Christenberry, 1980) (Guil et al. 2019). Until now, only three Milnesium species (Mil. dornensis Ciobanu, Roszkowska &Kaczmarek, 2015, Mil. t. tardigradum andMil. tetralamellatum Pilato & have been reported in the so-called African region (McInnes et al. 2017), in which Madagascar is placed. This paper describes two new species from Madagascar using integrative taxonomy.

Sample processing
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 Stec et al. (2015).

Microscopy and imaging
Specimens for light microscopy were mounted on microscope slides in a small drop of Hoyer's medium, prepared according to Ramazzotti and Maucci (1983) as in the English translation by Beasley (1995), and secured with a cover slip. The slides were then placed in an incubator and dried for two days at ca. 60 °C. Dried slides were sealed with a transparent nail polish and examined under an Olympus BX41 phase contrast light microscope (PCM) associated with an ARTCAM-300Mi digital camera (Olympus Corporation, Shinjuku-ku, Japan).
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.

Morphometrics and morphological nomenclature
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 Tumanov (2006). Buccal tube width was measured at three points as suggested by Michalczyk et al. (2012). The pt ratio is the ratio of the length of a given structure to the length of the buccal tube, expressed as a percentage (Pilato 1981). The pt values are always provided in [square brackets and in italics]. Configuration of the number of claw points on the secondary branches ("claw configuration") is given according to Michalczyk et al. (2012).

Genotyping
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 H 2 O and kept frozen at -80 °C until DNA isolation. DNA was extracted from individual animals following a modified Chelex100 resin (Bio-Rad) extraction method (Casquet et al. 2012), modified in order to obtain tardigrade exoskeletons, according to Zawierucha et al. (2016). DNA was extracted by incubating each specimen in 40 μl of 10% Chelex100 resin solution in sterile MQ H 2 O with the addition of 0.02 mg of Proteinase K (Genoplast) at 55 °C for 5h with shaking (500 RPM, Eppendorf Thermomixer 5436) and occasionally centrifuged. In the next step, Proteinase K was inactivated by incubating at 70 °C for 15 min. Subsequently, 20 μl of sterile MQ H 2 O was added to the tube and centrifuged for 2 min at 8000 G. For further analysis, ca. 40 μl of DNA extract (to the level of remaining Chelex beads at the bottom) was carefully transferred from each tube to a new 1.5 ml Eppendorf microcentrifuge tube. The tardigrade exoskeleton, present in a pellet after centrifugation, containing Chelex beads on the bottom of each tube, was extracted under stereomicroscope and then mounted in Hoyer's medium for further morphological analysis. Polymerase chain reaction (PCR) amplifications were carried out for three DNA fragments differing in mutation rates: mitochondrial cytochrome oxidase subunit I (COI), nuclear internal transcribed spacer 2 (ITS-2) and cytoplasmic ribosome large subunit component (28S rRNA) in a total volume of 15-50 μl (see Table  1 for primers, Table 2 for PCR cocktail recipes and Table 3 for PCR programmes). PCR products were verified by agarose gel electrophoresis (1-1.2% agarose) with ethidium bromide. Prior to sequencing, PCR products were purified with thermosensitive Exonuclease I and FastAP Alkaline Phosphatase (Fermentas, Thermo Scientific) to improve their quality. Properly prepared PCR products were sequenced bidirectionally with BigDye Terminator v3.1 on an ABI Prism 3130XL Analyzer (Applied Biosystems,  Table 3. PCR programmes used for the amplification of COI, ITS-2 and 28S rRNA. Step Foster City, CA, USA), according to the manufacturer's protocol. The sequences were edited and manually checked against non-conservative alignments using BioEdit, version 7.0.5 (Hall 1999), and submitted to GenBank (see Results section).

Comparative molecular analysis
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 (Thompson et al. 1994) implemented in BioEdit and trimmed to 510 (COI), 184 (ITS-2), 625 (28S rRNA) bp, respectively. Based on the recommendation of Srivathsan and Meier (2012), pairwise distances were calculated using MEGA7 in preference to the genetic distances corrected by the Kimura 2 parametric model (K2P). All positions with less than 95% site coverage were eliminated.  Table 4) with no modified claws I. Body light yellow before fixation and transparent afterwards, eyes present (in 89% of measured specimens). Dorsal cuticle sculptured with pseudopores, not arranged in bands, sparsely distributed and not forming a reticular design (Fig. 2). Six peribuccal papillae and six peribuccal lamellae present around the mouth opening. Two cephalic papillae positioned laterally. Peribuccal papillae slightly longer than lateral papillae.

Taxonomic account
The buccal apparatus of the Milnesium type (Figs 1, 3). The buccal tube wide and short (standard width, on average 46% of its length), and slightly funnel-shaped, wider  anteriorly (posterior diameter on average 89% of the anterior diameter) ( Table 4). The pharyngeal bulb elongated, pear-shaped and without placoids or septulum.
Claws of the Milnesium type, slender (Figs 4,5). Primary branches on all legs with small, but distinct accessory points detaching from the branch at its greatest curvature (Fig. 5, arrowhead). Secondary branches with rounded basal thickenings (Figs 4, 5). Table 4. 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).

All secondary branches on all legs with three points (claw configuration: [3-3]-[3-3]).
Single, long transverse, cuticular bars present under claws I-III (Fig. 4, arrow). Adult males (Table 5) with modified claws I. Similar to females but clearly smaller, with secondary branches of claws I modified into strong hooks and with a different proportion of peribuccal and lateral papillae length (peribuccal papillae clearly shorter than lateral), eyes present only in 33% of measured specimens.
Eggs oval, smooth and deposited in the exuvium as in all other known Milnesium species.
Etymology. The second author with great pleasure dedicates this species to her fiance -Mateusz Wojciechowski.
Genotypic differential diagnosis. 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 6 for details), are as follows:   (Fig. 6, Table 7) with no modified claws I. Body light yellow before fixation and transparent afterwards, eyes present only in 39% of measured specimens. Dorsal cuticle sculptured with pseudopores not arranged in bands, sparsely distributed and not forming reticular design (Fig. 7). Six peribuccal papillae and six peribuccal lamellae present around the mouth opening. Two cephalic papillae positioned laterally. Peribuccal papillae slightly longer than lateral papillae.
The buccal apparatus of the Milnesium type (Figs 6, 8). The buccal tube with standard width on average 62% of its length, and funnel-shaped, wider anteriorly (posterior diameter on average 91% of the anterior diameter) ( Table 7). The pharyngeal bulb elongated, pear-shaped and without placoids or septulum.
Claws of the Milnesium type, stout . Primary branches on all legs with small, but distinct accessory points detaching from the branch at its greatest curvature (Fig. 10, empty arrowhead). Secondary branches of claws similar in length to primary branches and sometimes even longer. Secondary branches with rounded basal thickenings (Figs 9-11). Secondary branches on legs I-III with three points, secondary claws of anterior and posterior claws IV with four points (claw configuration: [3-3]- ). The fourth point on secondary branches is always very small and located near the base of the claw (Figs 10-11, arrowheads). Single, long transverse, cuticular bars present under claws I-III (Fig. 9, arrow).
Males unknown. Eggs oval, smooth and deposited in the exuvium as in all other known Milnesium species.
Remarks. The fourth points on secondary branches of posterior claws can be barely visible or not visible at all in some positions of the specimens.
Etymology. 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.
Morphological differential diagnosis. 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 Table 7. 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).

]-[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. Genotypic differential diagnosis. 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 6 for details), are as follows: 1. 28S rRNA: 5.7-8.0% (6.7% on average), with the most similar being Milnesium sp. from North America (JX888585.