Two new species of the Macrobiotus hufelandi complex (Tardigrada: Eutardigrada: Macrobiotidae) from Australia and India, with notes on their phylogenetic position

. In this paper we describe two new tardigrade species belonging to the Macrobiotus hufelandi complex: Macrobiotus noongaris sp. nov. from Perth, Australia, and Macrobiotus kamilae sp. nov. from Mussoorie, India. Live specimens extracted from moss samples were used to establish laboratory cultures in order to obtain additional animals and eggs needed for their integrative descriptions. These descriptions are based on traditional morphological and morphometric data collected using both light and scanning electron microscopy, which, at the same time, were associated with DNA sequences of four genetic markers, three nuclear (18S rRNA, 28S rRNA and ITS-2) and one mitochondrial (COI). The use of DNA sequences allowed for a more accurate verification of the taxonomic status of M. noongaris sp. nov. and M. kamilae sp. nov as independent species of the hufelandi group. Although they both exhibit typical inverted goblet-shaped processes, they represent a recently discovered clade, which was thought to group species with modified morphology of egg processes. Thus, this contribution expands the definition of the mentioned clade and constitutes another link that will be helpful for future studies on the evolution of the M. hufelandi complex. new species Macrobiotus hufelandi (Eutardigrada: Macrobiotidae) on their phylogenetic


Introduction
Tardigrades, also known as water bears or moss piglets, are a phylum of micrometazoans closely related to arthropods and onychophorans, although their exact placement within the Ecdysozoa remains unclear (Campbell et al. 2011). Since their discovery in the 18 th century by the German zoologist Johan Goeze, nearly 1300 species have been described Degma & Guidetti 2007;Degma et al. 2009Degma et al. -2019. Tardigrades have a global distribution and can be found in both marine and terrestrial

R e s e a r c h a r t i c l e
urn:lsid:zoobank.org:pub:7619772F-2300-442E-8950-69559172360E habitats. However, most of the described species have been discovered from mosses and lichen, where at least periodic hydration is required in order for them to survive. They have been found to exist in the most extreme environments on Earth, from the ocean depths to mountain tops and are present in all biomes and on every continent including Antarctica (Nelson et al. 2015).
Here, we describe two species within the Macrobiotus hufelandi group, a species complex considered to be among the most common group of limnoterrestrial tardigrades on the planet McInnes 1994;Kaczmarek et al. 2014aKaczmarek et al. , 2015Kaczmarek et al. , 2016Kaczmarek & Michalczyk 2017a;McInnes et al. 2017). Macrobiotus hufelandi Schultze, 1834 was the first ever formally described tardigrade species, and for over a century this species was believed to be cosmopolitan, but also exhibited some intraspecific variability. Subsequently, the discovery of very similar species, such as Macrobiotus hibiscus de Barros, 1942 and several more over the next three decades, led to the introduction of the term "Macrobiotus hufelandi group" which was first used by Durante Pasa & Maucci (1979), with Biserov (1990aBiserov ( , 1990b as the first to attempt to formally define the Macrobiotus hufelandi group. A revision of the criteria for inclusion in the hufelandi group by , along with a redescription of the nominal species, listed 17 species contained within this species complex. The most recent revision of the group, Kaczmarek & Michalczyk (2017a), recorded 48 species and predicted at least six more to be described by 2020, a prediction which has already been surpassed. To date six formal descriptions of new species within the hufelandi complex have been published and with this paper we add two more. The majority of these descriptions were prepared by means of integrative taxonomy, which together with previous integrative studies on the M. hufelandi complex Cesari et al. 2009;Bertolani et al. 2011aBertolani et al. , 2011bGuidetti et al. 2013) have enabled for the first time more detailed insights into the evolution of this eutardigrade group. Specifically, Stec et al. (2018aStec et al. ( , 2018b) discovered two well supported evolutionary lineages within this complex. This diversification was also congruent with morphology, since one of the discovered clades comprised species with a whitish body and mostly typical inverted goblet shaped processes, whereas the second one comprised species with a yellowish body and eggs with processes having a modified morphology.
Although the first studies of Indian tardigrades were conducted as early as in the beginning of the 20 th century (Murray 1907) and again in the second part of the century (Iharos 1969), very little is known about the terrestrial tardigrade fauna of the Indian subcontinent. Tumanov (2018) listed only eight published papers on the subject, including the two mentioned above, but also noted that these "should be considered obsolete compared to the current levels of morphological data on this taxon". Apart from larger studies based on islands far from the Indian mainland such as Maucci & Durante Pasa (1980) and Roa (1972), the other published studies, namely Maucci (1979), Kristensen (1987), Abe & Takeda (2000), Tumanov (2006Tumanov ( , 2018 and Jørgensen et al. (2007), are based on data for single species obtained mostly from solitary samples collected occasionally. A review study conducted as part of the Zoological survey of India counted 41 species of tardigrades known to India, with 23 species listed as being found in the Indian Himalaya (Dey & Mandal 2018). Although that study listed M. hufelandi hufelandi as being present in India, considering the immense progress in our understanding of the diversity and taxonomy of the hufelandi group made since the Indian record by Murray (1907), this record most likely represents a different species.
In contrast to India, the tardigrade fauna of Australia has been much more studied through the years with early expeditions being undertaken by Richters (1908), who observed M. hufelandi in the Blue European Journal of Taxonomy 573: 1-38 (2019) Mountains (again, most likely a different species in the group), and Murray (1910) who recorded 31 species (including 6 new to science) in the states of New South Wales and Queensland. Notably, however, in the hufelandi complex only two species have previously been described from Australia, namely Macrobiotus joannae Pilato & Binda, 1983 from Bright in the state of Victoria and M. santoroi Pilato & D'Urso, 1976 from near Sydney in the state of New South Wales. With regard to tardigrade diversity in Australia, the largest study undertaken to date was that of Claxton (2004) as part of her Ph.D. thesis, in which she found 141 species from 132 samples collected in Eastern Australia. Two other papers are, however, of particular relevance as they pertain to tardigrade species found in Western Australia, specifically around the city of Perth, where the first of the new species described below has been found. Morgan & Nicholls (1986) described Apodibius serventyi from moss samples collected in the Perth Zoo, which was later considered as a synonym of A. nuntius Binda, 1984(Van Rompu et al. 1995. A re-examination of the slides with 'types' of A. serventyi by Pilato & Lisi (2004) led to the discovery and description of two additional species, Doryphoribius neglectus Pilato & Lisi, 2004 and Parascon nichollsae Pilato & Lisi, 2004. An additional study by Gąsiorek & Michalczyk (2019) described Echiniscus siticulosus discovered in Western Australia, albeit it from an area 650 km to the north of Perth.
This study combines modern molecular techniques with classical morphometric and morphological methods in an integrative approach to describe two new species within the M. hufelandi complex. Using phase contrast and scanning electron microscopy (PCM and SEM, respectively), we have described the phenotypic characteristics of the new species, whereas the sequencing of DNA markers (three nuclear, 18S rRNA, 28S rRNA and ITS-2, and one mitochondrial, COI) allowed for an assessment of the phylogenetic position of the new species within the M. hufelandi complex and also provided barcodes for their genetic identification.

Sample processing and tardigrade culturing
One of the two moss samples analysed in this study was collected from King's Park, an urban park of bushland in the city of Perth situated in the state of Western Australia, Australia (31°57′30″ S, 115°50′09″ E). The moss was growing on soil at an altitude of 46 m a.s.l. and was collected on 22 March 2015 by Łukasz Michalczyk. The second sample of moss was collected from an urban area along Camel's Back Road in the town of Mussoorie in the Dehradin District of the state of Uttarakhand, India, which is situated at the foothills of the Garhwal Himalayan mountain range (30°27′28″ N, 78°04′41″ E). The moss was growing on rock at an altitude of 2001 m a.s.l. and was collected on 10 November 2017 by Krzysztof Miller.
The samples were examined for tardigrades using the protocol by Dastych (1980) with modifications described in detail in Stec et al. (2015). A total of 13 and 28 live individuals and no eggs of the new species were extracted from the Australian and Indian samples, respectively. They were subsequently used to establish laboratory cultures to obtain additional animals and eggs for further analysis. Tardigrades were reared in plastic Petri dishes according to the protocol by Stec et al. (2015) and are still maintained in the lab culture. In order to perform the taxonomic analysis, animals and eggs were isolated from the culture and split into three groups for specific analyses: morphological analysis with phase contrast light microscopy, morphological analysis with scanning electron microscopy and DNA sequencing (for details, please see section "Material examined" provided below for each description).

Microscopy and imaging
Specimens for light microscopy were mounted on microscope slides in a small drop of Hoyer's medium and secured with a cover slip, following the protocol by Morek et al. (2016). Slides were examined under an Olympus BX53 light microscope with phase contrast (PCM), associated with an Olympus COUGHLAN K. & STEC D., Two new Macrobiotus species from India and Australia DP74 digital camera. Subsequently, after mounting, the specimens in the medium slides where also checked under PCM for the presence of males and females in the studied population, as the spermatozoa in testis and spermathecae are visible for several hours after mounting. In order to obtain clean and extended specimens for SEM, tardigrades were processed according to the protocol of Stec et al. (2015). Specimens were examined under high vacuum in a Versa 3D DualBeam Scanning Electron Microscope (SEM) at the ATOMIN facility of the Jagiellonian University, Kraków, Poland. All figures were assembled in Corel Photo-Paint X6, ver. 16.4.1.1281. For structures that could not be satisfactorily focused in a single light microscope photograph, a stack of 2-6 images were taken with an equidistance of ca 0.2 μm and assembled manually into a single deep-focus image in Corel Photo-Paint X6, ver. 16.4.1.1281.

Morphometrics and morphological nomenclature
All measurements are given in micrometres (μm). Sample size was adjusted following recommendations by Stec et al. (2016a). 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. The terminology used to describe oral cavity armature and egg shell morphology follows Michalczyk & Kaczmarek (2003) and Kaczmarek & Michalczyk (2017a). The type of buccal apparatus and claws are given according to Pilato & Binda (2010). Macroplacoid length sequence is given according to Kaczmarek et al. (2014b). Buccal tube length and the level of the stylet support insertion point were measured according to Pilato (1981). The pt index is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage (Pilato 1981). All other measurements and nomenclature follow Kaczmarek & Michalczyk (2017a). Morphometric data were handled using the 'Parachela' ver. 1.6 template available from the Tardigrada Register (Michalczyk & Kaczmarek 2013). Raw morphometric data for each analysed species are provided as supplementary materials (SM.01 and SM.02) Tardigrade taxonomy follows Guil et al. (2019).

Genotyping
The DNA was extracted from individual animals following a Chelex ® 100 resin (Bio-Rad) extraction method by Casquet et al. (2012) with modifications described in detail in Stec et al. (2015). Before the extraction, live specimens were mounted in water slides and checked under the microscope to confirm their identification. We sequenced four DNA fragments: the small ribosome subunit (18S rRNA, nDNA), the large ribosome subunit (28S rRNA, nDNA), the internal transcribed spacer (ITS-2, nDNA) and the cytochrome oxidase subunit I (COI, mtDNA). All fragments were amplified and sequenced according to the protocols described in Stec et al. (2015); primers and original references for specific PCR programs are listed in Table 1. Sequencing products were read with the ABI 3130xl sequencer at the Molecular Ecology Lab, Institute of Environmental Sciences of the Jagiellonian University, Kraków, Poland. Sequences were processed in BioEdit ver. 7.2.5 (Hall 1999) and submitted to GenBank.

Comparative molecular and phylogenetic analysis
For molecular comparisons, all published sequences of the four above-mentioned markers for species of the hufelandi complex were downloaded from GenBank (Appendix 1). The sequences were aligned using the default settings (in the case of ITS-2 and COI) and the Q-INS-I method (in the case of ribosomal markers: 18S rRNA, 28S rRNA) of MAFFT ver. 7 (Katoh et al. 2002;Katoh & Toh 2008) and manually checked against non-conservative alignments in BioEdit. Then, the aligned sequences were trimmed to 763 (18S rRNA), 715 (28S rRNA), 352 (ITS-2) and 618 (COI) bp. All COI sequences were translated into protein sequences in MEGA7 ver. 7.0 (Kumar et al. 2016) to check against pseudogenes. Uncorrected pairwise distances were calculated using MEGA7 and are provided as supplementary materials (SM.03).  Vecchi et al. (2016), Mesobiotus philippinicus (accession number: KX129796) described by Mapalo et al. 2016, Mesobiotus insanis (accession number: MF441491) described by Mapalo et al. 2017 and Mesobiotus ethiopicus (accession number: MF678794) described by Stec & Kristensen (2017). Since the COI is a protein coding gene, before partitioning, we divided our alignment into 3 data blocks constituting the three separate codon positions using PartitionFinder ver. 2.1.1 (Lanfear et al. 2016) under the Bayesian Information Criterion (BIC). The best scheme of partitioning and substitution models were chosen for posterior phylogenetic analysis. We ran the analysis to test all possible models implemented in the program. As best-fit partitioning scheme, PartitionFinder suggested to retain three predefined partitions separately. The best-fit models for these partitions were: SYM+I+G for the first codon position, GTR+I+G for the second codon position and HKY+G for the third codon position. Bayesian inference (BI) marginal posterior probabilities were calculated using MrBayes ver. 3.2 (Ronquist & Huelsenbeck 2003). Random starting trees were used and the analysis was run for eight million generations, sampling the Markov chain every 1000 generations. An average standard deviation of split frequencies of < 0.01 was used as a guide to ensure the two independent analyses had converged. The program Tracer ver. 1.3 (Rambaut et al. 2018) was then used to ensure Markov chains had reached stationarity and to determine the correct 'burn-in' for the analysis, which was the first 10% of generations. A consensus tree was obtained after summarising the resulting topologies and discarding the 'burn-in'.

Etymology
The name refers to the indigenous Australians who live in the region where the new species was found. These are the Noongar peoples, 14 different but related language groups that occupied these lands before western settlement, including the modern city of Perth where the sample was collected. In their languages, the term Noongar means 'a person of the southwest of Western Australia'.

Description
Animals (measurements and statistics in Table 2) Body transparent in juveniles and white in adults but transparent after fixation in Hoyer's medium (Fig. 1A). Eyes present in live animals as well as in specimens mounted in Hoyer's medium. Small round and oval cuticular pores (0.3-0.8 μm in diameter), visible under both PCM and SEM, scattered randomly on entire body ( Fig. 1B-C). Granulation present on all legs ( Fig. 2A-F). A patch of clearly visible granulation present on external surface of legs I-III ( Fig. 2A-B). A cuticular bulge/fold (pulvinus) present on internal surface of legs I-III, with a faint cuticular fold covered with faint granulation and paired muscles attachments just above the claws ( Mouth antero-ventral followed by ten peribuccal lamellae and a circular sensory lobe (Figs 4A, 5A). Bucco-pharyngeal apparatus of Macrobiotus type (Fig. 4A). Under PCM, oral cavity armature of the patagonicus type, i.e., with only 2 nd and 3 rd bands of teeth visible ( Fig. 4B-C). However, in SEM all three bands of teeth visible, with first band being situated at base of peribuccal lamellae and composed of a single row of small fused cone-shaped teeth connected to form a continuous, slightly serrated ring ridge around oral cavity ( Fig. 5B-C). Second band of teeth situated between ring fold and third band of teeth and comprises 3-6 rows of small cone-shaped teeth (Figs 4B-C, 5B-C). Teeth of third band located within posterior portion of oral cavity, between second band of teeth and buccal tube opening (Figs 4B-C, 5B-C). Third band of teeth discontinuous and divided into dorsal and ventral portions. Under PCM, dorsal teeth appear as three distinct transverse ridges, whereas ventral teeth appear as  4B-C). In SEM, both dorsal and ventral teeth also clearly distinct ( Fig. 5B-C). Under SEM, margins of medio-dorsal tooth slightly serrated (Fig. 5B), whereas the medio-ventral tooth slightly anterior to lateral teeth (Fig. 5C). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a small triangular microplacoid (Fig. 4A, D-E). Macroplacoid length sequence 2 < 1. First macroplacoid exhibits central constriction, whereas second macroplacoid sub-terminally constricted (Fig. 4A, D-E).
Eggs (measurements and statistics in Table 3) Laid freely, white, spherical or slightly ovoid (Fig. 6A). Surface between processes is of the hufelandi type, i.e., covered with a reticulum (Figs 6E, 7B-D, F). Meshes of reticulum small (0.1-0.6 µm) and rounded, regular in size and with blurred rims in PCM (Fig. 6E), irregular in size and with thick borders in SEM (meshes in SEM appear as pores; Figs 7B-D, F). Interbasal meshes larger than peribasal meshes, but peribasal meshes do not form rings around process bases (Figs 6E, 7B-D, F). Eggs have 22-30 processes on circumference, 26 on average (Fig. 6A). Processes are of inverted goblet shape, with slightly concave trunks and concave terminal discs (Figs 6C-E, 7B-E). Terminal discs are round   European Journal of Taxonomy 573: 1-38 (2019) and strongly serrated (Fig. 7C-E). Each terminal disc has a distinct concave central area which may contain some scattered granulation within, which is also always present on the margin (visible only under SEM; Fig. 7C-E).

Reproduction
The new species is dioecious. No spermathecae filled with sperm have been found in gravid females on the freshly prepared slides. However, the testis in males, filled with spermatozoa, is clearly visible under PCM up to 24 hours after mounting in Hoyer's medium (Fig. 6F). The new species does not exhibit male secondary sexual dimorphism traits such as lateral gibbosities on legs IV.

DNA sequences
We obtained sequences for all four of the above mentioned DNA markers. All sequenced fragments were represented by single haplotypes except the ITS-2, in which two distinct haplotypes were present: The 18S

Etymology
We take great pleasure in dedicating this new species to the friend of the second author, Kamila Zając, who is a young malacologist and a PhD student at the Institute of Environmental Sciences, Jagiellonian University, Kraków, Poland.

Description
Animals (measurements and statistics in Table 4) Body transparent in juveniles and yellowish in adults, but transparent after fixation in Hoyer's medium (Fig. 8A). Eyes present in live animals as well as in specimens mounted in Hoyer's medium. Small round and oval cuticular pores (0.3-0.8 μm in diameter), visible under both PCM and SEM, scattered randomly on entire body (Fig. 8B-C). Granulation present on all legs (Fig. 9A-F). A patch of clearly visible granulation present on external surface of legs I-III (Fig. 9A-B). A cuticular bulge/fold (pulvinus) COUGHLAN K. & STEC D., Two new Macrobiotus species from India and Australia present on internal surface of legs I-III, with a faint cuticular fold and a patch of granulation between them (Fig. 9C-D). Both structures visible only if legs fully extended and properly oriented on slide. Cuticular granulation on legs IV always clearly visible and consisting of a single large granulation patch on each leg (Fig. 9E-F). In addition to granulation on legs, three patches of granulation on body located dorso-laterally between legs III and IV, with granule size and density increasing from 1 st to 3 rd patch ( Fig. 10A-E). Claws long and slender, of the hufelandi type ( Fig. 11A-D). Primary branches with distinct accessory points, a long common tract and with an evident stalk connecting the claw to the lunula (Fig. 11A-D). Lunulae I-III smooth (Fig. 11A, C), whereas lunulae IV clearly dentate (Fig. 11B, D). Cuticular bars under claws are absent. Double muscle attachments are faintly marked under PCM but clearly visible under SEM (Fig. 11A, C, respectively). A faintly marked horseshoe structure connecting the anterior and the posterior claw is visible only in PCM (Fig. 11B, D).
Mouth antero-ventral with ten peribuccal lamellae and a circular sensory lobe (Figs 12A, 13A). Buccopharyngeal apparatus of the Macrobiotus type (Fig. 12A). Under PCM, the oral cavity armature is of the patagonicus type, i.e., with only the 2 nd and 3 rd bands of teeth visible (Fig. 12B-E). However, in SEM all three bands of teeth are visible, with the first band being situated at the base of peribuccal lamellae and composed of a single row of small cone-shaped teeth. The second band of teeth is situated between the ring fold and the third band of teeth and comprises 2-4 rows of small cone-shaped teeth, slightly larger than those in the first band (Figs 12B-E, 13B-C). Under PCM the second band is faintly visible in large as well as small specimens (Fig. 12B-E). The teeth of the third band are located within the posterior Please also note a cribriform area, which is external evidence of a muscle attachment in the centre of Fig. 10D. Scale bars in μm.

COUGHLAN K. & STEC D., Two new Macrobiotus species from India and Australia
portion of the oral cavity, between the second band of teeth and the buccal tube opening (Figs 12B-E, 13B-C). The third band of teeth is discontinuous and divided into the dorsal and ventral portions. Under PCM, the dorsal teeth are fused and seen as one distinct transverse ridge, whereas the ventral teeth appear as two separate lateral transverse ridges and a median tooth which is sometimes divided into two roundish teeth (Fig. 12B-E). In SEM, both dorsal and ventral teeth are also clearly distinct (Fig. 13B-C). Under SEM, the margins of the dorsal portion of the third band are slightly serrated with two clearly visible peaks (Fig. 13B), whereas the ventral teeth are separated with a medio-ventral tooth slightly anterior to the lateral teeth (Fig. 13C). Pharyngeal bulb spherical, with triangular apophyses, two rodshaped macroplacoids and a small triangular microplacoid (Fig. 12A, F-G). The macroplacoid length sequence 2 < 1. The first macroplacoid exhibits a central constriction, whereas the second macroplacoid is faintly sub-terminally constricted (Fig. 12F-G).

COUGHLAN K. & STEC D., Two new Macrobiotus species from India and Australia
Eggs (measurements and statistics in Table 5) Laid freely, yellowish, spherical or slightly ovoid (Figs 14A, 15A). The surface between processes is of the hufelandi type, i.e., covered with a reticulum (Figs 14E, 15B-F). Meshes of the reticulum small and rounded, irregular in size (mesh diameter 0.3-0.8 µm), with interbasal meshes slightly larger than peribasal meshes but peribasal meshes do not form rings around process bases (Figs 14E, 15B-F). The   Fig. 13. Macrobiotus kamilae sp. nov., mouth opening and the oral cavity armature seen in SEM (paratype, IZiBB). A. Mouth opening with peribuccal sensory lobes and ten peribuccal lamellae. B-C. The oral cavity armature of a single paratype seen in SEM from different angles, in dorsal (B) and ventral (C) views, respectively. Empty flat arrowheads indicate the first band of teeth in the oral cavity, empty indented arrowheads indicate the second band of teeth in the oral cavity and filled indented arrowheads indicate the third band of teeth in the oral cavity. Scale bars in μm.
European Journal of Taxonomy 573: 1-38 (2019) nodes of reticulum are often narrower than the mesh diameters visible in PCM and SEM (Figs 14E,  15F). Eggs have 26-32 processes on the circumference, 29 on average (Fig. 14A). Processes are of the inverted goblet shape with slightly concave trunks and concave terminal discs (Figs 14C-D, 15B-E). Terminal discs round, with faintly indented margins (Fig. 15B-E). Each terminal disc has a distinct European Journal of Taxonomy 573: 1-38 (2019) concave central area which may contain some scattered granulation within, which is also always present on the margin (visible only under SEM; Fig. 15E).

Reproduction
The new species is dioecious. No spermathecae filled with sperm have been found in gravid females on the freshly prepared slides. However, in males the testis, filled with spermatozoa, is clearly visible under PCM up to 24 hours after mounting in Hoyer's medium (Fig. 14F). The new species does not exhibit male secondary sexual dimorphism traits such as lateral gibbosities on legs IV.

Phylogenetic analysis
The phylogenetic analysis, based on available COI sequences of M. hufelandi spp., conducted in our study showed that M. noongaris sp. nov. and M. kamilae sp. nov. indeed belong to this group. The analysis recovered two highly supported clades (Fig. 16). The first grouping (blue nodes) is of species with typical processes of inverted goblet shape and whitish body (with the only exception being M. cf. recens, which has processes in the shape of thin cones devoid of terminal discs). In contrast to the first clade, the second group (red nodes) is of species with yellowish body and morphological modifications of egg processes (flexible filaments on the terminal discs or processes without terminal discs). Interestingly, the two new species described within this study, which both exhibit typical inverted goblet-shaped processes, have been found to cluster together with the species which have modified egg processes. However, M. kamilae sp. nov. has a yellowish body, which conforms to the second characteristic of this clade, whereas M. noongaris sp. nov. has a whitish body.

Discussion
Phenotypic differential diagnosis of Macrobiotus noongaris sp. nov.
In terms of the morphology of the animals, M. noongaris sp. nov., by having only the 2 nd and 3 rd bands of teeth in the oral cavity visible under PCM, belongs to the patagonicus subgroup within the M. hufelandi complex. However, regarding egg shell ornamentation, by having the egg shell surface between the processes covered with a reticulum, it represents the hufelandi subgroup. These two traits combined with the typical concave terminal discs with serration/dentation make M. noongaris sp. nov. most similar to the following species: M. horningi Kaczmarek & Michalczyk, 2017, M. sandrae Bertolani & Rebecchi, 1993, M. sottilei Pilato, Kiosya, Lisi & Sabella, 2012, M. terminalis Bertolani & Rebecchi, 1993and M. vladimiri Bertolani, Biserov, Rebecchi & Cesari, 2011 it differs specifically in the following aspects.   (2019) It differs from M. horningi, reported only from its type locality in New Zealand (Kaczmarek & Michalczyk 2017b), by the presence of granulation on all legs (poorly visible granulation present only on legs IV in M. horningi), the presence of clearly visible subterminal constrictions in the second macroplacoid (only a poorly defined latero-terminal globular projection in the second macroplacoid in M. horningi), the morphology of lunules IV (dentate in the new species vs smooth in M. horningi), a smaller mesh size in the reticulum on the egg surface (mesh diameter: 0.1-0.6 µm in the new species vs 1.0-1.8 μm in M. horningi), a different location of larger meshes within the reticulum (interbasal meshes larger than peribasal meshes in the new species vs peribasal meshes slightly larger than the interbasal mesh in M. horningi), the morphology of terminal disc margins (strongly serrated in the new species vs welldefined indentations in M. horningi) and by smaller egg process dimensions (height 4.5-8.4 μm, base width 3.4-6.6 μm, disc diameter 2.3-4.8 μm in the new species vs height 11.8-13.3 μm, base width 8.2-8.6 μm and disk diameter 6.0-6.6 μm in M. horningi).
It differs from M. sandrae, reported from its type locality in Germany and also Italy  and from Belarus (Pilato et al. 2012), by the presence of a clearly visible subterminal constriction in the second macroplacoid (no constriction in M. sandrae), the presence of microgranulation on the margins of terminal discs of egg processes (the microgranulation absent in M. sandrae) and by a different morphology of the reticulation on the egg surface (slightly smaller mesh size (0.1-0.6 μm), several rows of meshes in the reticulum between processes, mesh rims often wider than pore diameter, meshes almost circular in the new species vs bigger mesh size (0.6-1.0 μm), often only up to three rows of pores in the reticulum between processes, mesh rims clearly thinner than pore diameter and meshes more ovoid in M. sandrae).
It differs from M. sottilei, known from its type locality in Belarus (Pilato et al. 2012) but also from Poland (Kaczmarek et al. 2018) and Italy (Roszkowska et al. 2019), by the presence of three distinct teeth/ridges in the dorsal portion of the third band of teeth (the teeth/ridges of the dorsal portion fused and form a continuous arc in M. sottilei) and by a slightly more posterior stylet support insertion point (pt = 76.7-81.6 in the new species vs 75.3-76.6 in M. sottilei).
It differs from M. terminalis, known from its type locality in Italy  and from Belarus (Pilato et al. 2012), by the morphology of lunules I-III (smooth in the new species vs dentate in M. terminalis), a different morphology of the reticulation on the egg surface (smaller mesh size (0.1-0.6 μm), several rows of meshes in the reticulum between processes, mesh rims often wider than pore diameter and meshes almost circular in the new species vs bigger mesh size (0.8-1.3 μm), often only up to three rows of pores in the reticulum between processes, mesh rims clearly thinner than pore diameter and meshes more ovoid in M. terminalis) and by the presence of microgranulation on the margins of terminal discs of egg processes (absent in M. terminalis).
It differs from M. vladimiri, known from its type locality in Italy and from Germany (Bertolani et al. 2011b; originally listed as M. cf. terminalis in ), Poland (Nowak & Stec 2017) and Spain (Bertolani et al. 2011a), by a different morphology of the reticulation on the egg surface (smaller mesh size (0.1-0.6 μm), several rows of meshes between processes, meshes distributed uniformly, mesh rims often wider than mesh diameter and meshes almost circular in the new species vs bigger mesh size (0.8-1.1 μm), often only up to three rows of meshes between processes, clear peribasal of larger meshes, mesh rims clearly thinner than mesh diameter and meshes more ovoid in M. vladimiri), the presence of microgranulation on the margins of terminal discs of egg processes (absent in M. vladimiri) and by reproductive mode (dioecism in the new species vs parthenogenesis in M. vladimiri).

COUGHLAN K. & STEC D., Two new Macrobiotus species from India and Australia
Genotypic differential diagnosis of Macrobiotus noongaris sp. nov.
The ranges of uncorrected genetic p-distances between the new species and species of the M. hufelandi complex, for which sequences are available from GenBank, are as follows (from the most to the least conservative):  Genotypic differential diagnosis of Macrobiotus kamilae sp. nov.
The ranges of uncorrected genetic p-distances between the new species and species of the M. hufelandi complex, for which sequences are available from GenBank, are as follows (from the most to the least conservative): •

Phylogeny of the Macrobiotus hufelandi complex
The first attempt to investigate the phylogeny of the M. hufelandi complex was presented by Guidetti et al. (2013) along with the description of the new species, Macrobiotus kristenseni Guidetti, Peluffo, Rocha, Cesari & Moly de Peluffo, 2013, which exhibits egg processes atypical for this group. First, they provided an unrooted neighbour joining dendrogram based on COI sequences showing high genetic divergences between eight M. hufelandi species for which these fragments had been available Cesari et al. 2009;Bertolani et al. 2011aBertolani et al. , 2011b. Second, they also presented phylogeny based on a conservative marker (18S rRNA), showing undoubtedly that, although the new species possesses modified egg processes, it still belongs to the M. hufelandi complex. Thus, Guidetti et al. (2013) hypothesised that within this group, animal morphology is more conserved than the morphology of egg ornamentation. Since then, several new species with modified egg process morphology have been discovered and described by means of integrative taxonomy (Stec et al. 2015(Stec et al. , 2017bRoszkowska et al. 2017). Soon after this, along with the description of another new species, M. shonaicus, Stec et al. (2018a) provided an upgraded COI phylogeny of the complex and discovered two well-supported evolutionary lineages. This diversification was congruent with the morphology, as one clade comprised species with a whitish body and the typical inverted goblet-shaped processes whereas the second one grouped species with a yellowish body and egg processes with a modified morphology (conical processes or processes with filaments growing out of terminal discs). The presence of these two evolutionary lineages was then confirmed by Stec et al. (2018b), who provided three congruent phylogenies based on different data sets for the M. hufelandi complex (1: 18S rRNA; 2: 18S rRNA+28S rRNA+ITS2+COI; 3: COI). However, Macrobiotus cf. recens (with a whitish body and conical egg processes) analysed in that study, was found to be embedded within the clade with species exhibiting the typical egg morphology. Nevertheless, these two clades still could be morphologically differentiated by the body colour, whitish vs yellowish, respectively. Although M. noongaris sp. nov. and M. kamilae sp. nov., described in our study, do not exhibit modified egg processes, both of them have been recovered as members of the clade with such eggs. Notably, however, only M. kamilae sp. nov. by having a yellowish body conforms to the second characteristic of this clade, whereas M. noongaris sp. nov. contradicts the hypothesis proposed by Stec et al. (2018aStec et al. ( , 2018b. These results indicate explicitly that diversification of egg shell ornamentation is definitely faster evolving than animal morphology, a hypothesis which was already presented by Guidetti et al. (2013). Furthermore, this is also in line with a previous study conducted on a more detailed and targeted scale by Stec et al. (2016b), who showed congruence between genetic and phenotypic traits of the eggs within a single parthenogenetic tardigrade species. The authors stated that this divergence between two reproductively isolated lineages could be seen as an example of very early incipient speciation. Hopefully, in the near future the phylogenetic data set will be extended by the addition of more M. hufelandi complex species, which can definitely contribute to our understanding of the morphological evolution within this group.

Conclusions
Our study integratively describes two new species of the cosmopolitan tardigrade group, the M. hufelandi complex, and contributes to the understanding of its evolution. The phylogenetic analysis showed that these new species, having the typical inverted goblet-shaped processes on the eggs, cluster together COUGHLAN K. & STEC D., Two new Macrobiotus species from India and Australia with species exhibiting morphological modifications of the egg processes. This contradicts the previous hypothesis that two well-supported evolutionary lineages within the M. hufelandi complex differ by egg chorion ornamentation. However, our results are in line with the hypothesis presented by Guidetti et al. (2013) that animal morphology is much more conserved that egg chorion ornamentation, where the latter provide the most important characters for species diagnosis and identification. Finally, we would like to note that M. noongaris sp. nov. is the third and M. kamilae sp. nov. the first formally described species of M. hufelandi complex from Australia and India, respectively.