Zoosyst. Evol. 97 (1) 2021, 281-306 | DO! 10.3897/zse.97.65280 yg Musee TOR BERLIN Integrative descriptions of two new Macrobiotus species (Tardigrada, Eutardigrada, Macrobiotidae) from Mississippi (USA) and Crete (Greece) Matteo Vecchi!, Daniel Stec? 1 Department of Biological and Environmental Science, University of Jyvaskyla, PO Box 35, FI-40014 Jyvaskyla, Finland 2 Department of Invertebrate Evolution, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Gronostajowa 9, 30-387 Krakow, Poland http://zoobank.org/BCASCCAB-C578-4F EB-B053-F483D53922AD Corresponding authors: Matteo Vecchi (matteo.m.vecchi@Jyu.fi), Daniel Stec (daniel_stec@interia.eu) Academic editor: Pavel Stoev # Received 1 March 2021 # Accepted 29 April 2021 # Published 19 May 2021 Abstract In this paper, we describe two new Macrobiotus species from Mississippi (USA) and Crete (Greece) by means of integrative taxon- omy. Detailed morphological data from light and scanning electron microscopy, as well as molecular data (sequences of four genetic markers: 18S rRNA, 28S rRNA, ITS-2 and COI), are provided in support of the descriptions of the new species. Macrobiotus annew- intersae sp. nov. from Mississippi belongs to the Macrobiotus persimilis complex (Macrobiotus clade B) and exhibits a unique egg processes morphology, similar only to Macrobiotus anemone Meyer, Domingue & Hinton, 2014, but mainly differs from that species by the presence of eyes, granulation on all legs, dentate lunulae on legs IV, and of bubble-like structures within the tentacular arms that are present on the distal portion of the egg processes. Macrobiotus rybaki sp. nov. from Crete belongs to the Macrobiotus clade A and is most similar to Macrobiotus dariae Pilato & Bertolani, 2004, Macrobiotus noemiae Roszkowska & Kaczmarek, 2019, Mac- robiotus santoroi Pilato & D’ Urso, 1976, and Macrobiotus serratus Bertolani, Guidi & Rebecchi, 1996, but differs from them mainly in the morphological details of its egg processes and chorion reticulation, but also by a number of morphometric characters. In light of the specific morphology of the egg processes of Macrobiotus annewintersae sp. nov. and Macrobiotus anemone, that are equipped with tentacular arms instead of proper terminal disc, we also provide an updated definition of the Macrobiotus persimilis complex. Key Words egg ornamentation, integrative taxonomy, Macrobiotus persimilis complex, molecular phylogeny, species delineation, water bears Introduction of phylogenetic relationships within a larger group of or- ganisms. This was the case for the family Macrobiotidae, Tardigrades are a phylum of micrometazoans distributed worldwide, that inhabit marine and limno-terrestrial en- vironments (Schill 2019). Currently, there are more than 1300 formally recognised tardigrade species (Guidetti and Bertolani 2005; Degma and Guidetti 2007; Degma et al. 2009-2020). In recent years, the number of tardigrade species described with integrative taxonomy has steadily increased (e.g., Surmacz et al. 2019; Bochnak et al. 2020; Kayastha et al. 2020; Tumanov et al. 2020a, b; Guidetti et al. 2021). The accumulation of data from such integra- tive studies allows at some point for broader examination one of the most speciose and diverse groups among tar- digrades, which was recently extensively revised (Stec et al. 2021) and which is partially in focus in this study. Faunistic and taxonomic studies on the tardigrades of North America are numerous and both local and conti- nental species lists have been compiled (Meyer 2013; Kaczmarek et al. 2016). It is, however, clear from new species in the USA being described (see for example Nel- son et al. 2020a), that we are still far from a complete knowledge of the taxonomic diversity of tardigrades in this country. In particular, the tardigrade fauna in the state Copyright Matteo Vecchi, Daniel Stec. 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. 282 Vecchi, M., Stec, D.: Two new Macrobiotus species from USA and Greece of Mississippi (USA) has been investigated only once by Hinton and Meyer (2009) who reported only 9 species (from 20 samples). In contrast, the tardigrade fauna in the neighbouring states have been more thoroughly in- vestigated and consequently more than 20 species have been recorded for Alabama, Louisiana and Arkansas, and about 100 species in Tennessee (Bartels and Nelson 2007; Meyer 2013; Kaczmarek et al. 2016; Nelson et al. 2020b). The first information on Greek tardigrades was provided 85 years ago (Marcus 1936), and since then only a cou- ple of studies have been explicitly devoted to assessing the diversity in this country (Durante Pasa and Maucci 1979; Maucci and Durante Pasa 1982). On the island of Crete, 28 species (from more than 150 samples) have been listed based on two sampling campaigns alone (Maucci and Du- rante Pasa 1982). Taking into consideration recent progress in tardigrade taxonomy and faunistic studies brought about by the integrative approach, it is more than likely that the region exhibits higher species diversity and additional sam- pling effort may reveal more species (Vuori et al. 2020). In this paper, we provide descriptions of two new Mac- robiotus species: Macrobiotus annewintersae sp. nov. from Mississippi (USA) and Macrobiotus rybaki sp. nov. from Crete (Greece) and show their phylogenetic position within the genus Macrobiotus. Detailed morphological and morphometric data were obtained using phase con- trast and scanning electron microscopy (PCM and SEM, respectively) supported by DNA sequences for four mo- lecular markers (three nuclear — 18S rRNA, 28S rRNA, and ITS-2 — and one mitochondrial — COI). Materials and methods Samples and specimens A mixed leaf litter sample containing M. annewintersae sp. nov. was collected in a garden in a suburban area of Jackson, Mississippi (32°21'05"N, 89°56'30"W; 106 m asl; Jyvasky- la University (JSYU) sample code S207, Jagellonian Univer- sity (JAG) sample code US.084), and a moss sample from a rock in a xeric shrubland containing M. rybaki sp. nov. was collected in Omalos, Crete (35°15'00"N, 23°49'28"E, 30 m asl; JAG sample code GR.O11). The samples were examined for tardigrades using the protocol by Dastych (1980), with modifications described in detail in Stec et al. (2015). Live animals and eggs of M. annewintersae sp. nov. were placed into culture. Specimens were reared in plastic Petri dishes according to the protocol by Stec et al. (2015). Tardigrades were fed ad /ibitum with unicellular freshwater algae (Chlorococcum sp. and Chlorella sp.; 1:1, Sciento, UK) and Lecane inermis Bryce, 1892 (Rotifera) and kept at 16C under a 2:22 light:dark photoperiod. In order to perform the taxonomic analysis, animals and eggs were either extracted from culture (VM. annewin- tersae ssp. nov.), or directly from the sample (M. rybaki Sp. nov.) and split into several groups for specific analy- ses 1.e., morphological analysis in PCM and SEM, as well zse.pensoft.net as DNA sequencing (for details see sections “Material ex- amined” provided below in the results section for each species description). Microscopy and imaging Specimens for light microscopy were mounted on mi- croscope slides in a small drop of Hoyer’s medium and secured with a cover slip, following protocol by Morek et al. (2016). Slides were examined under an Olympus BX53 light microscope with PCM, associated with an Olympus DP74 digital camera or under a Zeiss Axio- scope A2 light microscope associated with a MiniVID digital camera. Immediately after mounting, the speci- mens were checked under PCM for the presence of males and females in each of the studied populations, as the spermatozoa in testes and vasa deferentia are visible for several hours after mounting (Coughlan and Stec 2019; Coughlan et al. 2019). To obtain clean and extended spec- imens for SEM analysis, tardigrades were processed ac- cording to the protocol by Stec et al. (2015). Specimens were examined under high vacuum in a Versa 3D Dual- Beam SEM at the ATOMIN facility of the Jagiellonian University, Krakow, Poland or in a Raith e-LINE E-beam SEM at Nanoscience Center of University of Jyvaskyla, Jyvaskyla, Finland. 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 micro- scope photograph, a stack of 2-6 images were taken with an equidistance of ca. 0.2 um and assembled manually into a single deep-focus image in Corel Photo-Paint X6. Morphometrics and morphological nomenclature All measurements are given in micrometres (um). Sam- ple size was adjusted following the recommendations by Stec et al. (2016). Structures were measured only if their orientation was suitable. Body length was measured from the anterior extremity to the posterior end of the body, excluding the hind legs. The terminology used to describe oral cavity armature and eggshell morphology follows Michalczyk and Kaczmarek (2003) and Kaczmarek and Michalczyk (2017). Macroplacoid length sequence is given according to Kaczmarek et al. (2014). 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 ratio (Pilato 1981). Measurements of buccal tube widths, heights of claws and eggs follow Kaczmarek and Michalczyk (2017). Morphometric data were handled using the “Parachela” ver. 1.7 template available from the Tardigrada Register (Michalczyk and Kaczmarek 2013). The raw morphomet- ric data are provided as Suppl. materials 1, 2. Tardigrade taxonomy follows Bertolani et al. (2014) and Stec et al. Zoosyst. Evol. 97 (1) 2021, 281-306 (2021). Thorpe’s normalisation was performed with the R software (R Core Team 2020) on the morphometric traits following Bartels et al. (2011) (SM.03). Additional material Individuals of Macrobiotus aff. polonicus (JYU sam- ple code S165; 58°52'42"N, 17°55'60"E; 23 m asl: Nyndashamn, Sweden; lichen growing on rock on a road- side in a coastal area; coll. Sept. 2019 by MV and Sara Calhim) were genotyped for all the four markers and added to the phylogenetic reconstruction to increase the number of species included in the phylogenetic analysis. Photographs of eggs from the type series of Macrobious anemone Meyer, Domingue & Hinton, 2014 (slides 9551 and 9552) were kindly provided by Harry A. Meyer (Mc- Neese State University, Louisiana, USA). Photographs of eggs from the type series of dariae Pilato & Berto- lani, 2004 (slides PC45s1 and PC45s3) and M. serratus Bertolani, Guidi & Rebecchi, 1996 (slides C1907s17 and C1907s30) from the Bertolani collection were kindly provided by Roberto Guidetti (University of Modena and Reggio Emilia, Italy). Additional photos of the paratypes and eggs of Macrobiotus andinus Maucci, 1988 were kindly taken for us by Witold Morek and Piotr Gasiorek (Jagiellonian University, Poland) from the Maucci collec- tion (Natural History Museum of Verona). Genotyping DNA was extracted from individual animals follow- ing a Chelex 100 resin (BioRad) extraction method by Casquet et al. (2012) with modifications described in de- tail in Stec et al. (2020a). Each specimen was mounted in water and examined under a light microscope prior to DNA extraction. We sequenced four DNA fragments, three nuclear (18S rRNA, 28S rRNA, ITS2) and one mitochondrial (COI). All fragments were amplified and sequenced according to the protocols described in Stec et al. (2020a); primers with original references are list- ed 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, Krakow, Poland. Sequences were processed in MEGA7 (Kumar et al. 2016) and submitted to NCBI GenBank (Table 2). 283 Phylogenetic analysis The phylogenetic analyses were conducted using con- catenated 18S rRNA+28S rRNA+ITS-2+COI sequences from Macrobiotidae, with Richtersius coronifer (Rich- ters, 1903) and Dactylobiotus parthenogeneticus Bertola- ni, 1982 as outgroups. GenBank accession numbers of all sequences used in the analysis are listed in Table 2. Only species/populations with at least 3 markers were included in the analysis. The 18S rRNA, 28S rRNA and ITS-2 sequences were aligned using MAFFT ver. 7 (Katoh et al. 2002; Ka- toh and Toh 2008) with the G-INS-i method (thread=4, threadtb=5, threadit=0, reorder, adjust direction, any symbol, max iterate=1000, retree 1, global pair input). The COI sequences were aligned according to their ami- no acid sequences (translated using the invertebrate mi- tochondrial code) with the MUSCLE algorithm (Edgar 2004) in MEGA7 with default settings (1.e., all gap pen- alties=0, max iterations=8, clustering method=UPGMB, lambda=24). Alignments were visually inspected and trimmed 1n MEGA7. Model selection and phylogenetic reconstructions were undertaken using the CIPRES Sci- ence Gateway (Miller et al. 2010). Model selection was performed for each alignment partition (6 in total: 18S tRNA, 28S rRNA, ITS-2 and three COI codons) using PartitionFinder2 (Lanfear et al. 2016), partitions and model selection process together with results are con- tained in Suppl. material 4. Bayesian inference (BI) phy- logenetic reconstruction was performed using MrBayes v3.2.6 (Ronquist et al. 2012) without BEAGLE. Two runs (one cold chain and three heated chains each) of 20 million generations were used with a burn-in of 2 mil- lion generations, sampling a tree every 1000 generations. Posterior distribution sanity was checked using Tracer v1.7 (Rambaut et al. 2018). The MrBayes input file with the input alignment is available as Suppl. material 5, and the MrBayes output consensus tree is available as Sup- pl. material 6. The phylogenetic tree was visualised with FigTree v1.4.4 (Rambaut 2007) and the image was edited with Inkscape 0.92.3 (Bah 2011). Results Taxonomic account Phylum: Tardigrada Doyere, 1840 Table 1. Primers with their original references used for amplification of the four DNA fragments sequenced in the study. DNA marker Primer name Primer direction 18S rRNA 18S_Tar_Ffl forward 18S_Tar_Rrl reverse 28S rRNA 28S_Eutar_F forward 28SRO0990 reverse ITS-2 ITS2_Eutar_Ff forward ITS2_Eutar_Rr reverse COl LCO1490-JJ forward HCO2198-JJ reverse Primer source Stec et al. (2017a) Primer sequence (5’-3’) AGGCGAAACCGCGAATGGCTC GCCGCAGGCTCCACTCCTGG ACCCGCTGAACT TAAGCATAT CCTTGGTCCGTGTTTCAAGAC CGTAACGTGAATTGCAGGAC TCCTCCGCTTATTGATATGC CHACWAAYCATAAAGATATYGG Gasiorek et al. (2018) Mironov et al. (2012) Stec et al. (2018a) Astrin and Stuben (2008) AWACTTCVGGRTGVCCAAARAATCA zse.pensoft.net 284 Table 2. GenBank accession numbers of sequences downloaded from GenBank and used in the present study. Newly generated sequences are bolded. Dactylobiotus parthenogeneticus Macrobiotus aff. pseudohufelandi PL Macrobiotus aff. pseudohufelandi ZA Macrobiotus aff. polonicus SE Macrobiotus annewintersae sp. nov. Macrobiotus basiatus Macrobiotus caelestis Macrobiotus canaricus Macrobiotus cf. pallarii Fl Macrobiotus cf. pallarii ME Macrobiotus cf. pallarii PL Macrobiotus cf. pallarii US Macrobiotus cf. recens Macrobiotus crustulus Macrobiotus engbergi Macrobiotus glebkai Macrobiotus hannae Macrobiotus kamilae Macrobiotus macrocalix Macrobiotus noongaris Macrobiotus papel Macrobiotus paulinae Macrobiotus polonicus AT Macrobiotus polonicus SK Macrobiotus polypiformis Macrobiotus porifini Macrobiotus rybaki sp. nov. Macrobiotus scoticus Macrobiotus shonaicus Macrobiotus sottilei Macrobiotus viadimiri Macrobiotus wandae Mesobiotus harmsworthi Mesobiotus radiatus Mesobiotus romani Minibiotus joculator Minibiotus pentannulatus Paramacrobiotus areolatus Paramacrobiotus fairbanksi Paramacrobiotus lachowskae Paramacrobiotus tonollii Richtersius coronifer Sisubiotus spectabilis Fl Sisubiotus spectabilis NO Tenuibiotus danilovi Tenuibiotus tenuiformis Tenuibiotus zandrae zse.pensoft.net 18S MT373693 MN888373 MN888374 MW588026 MW588027 MW588024 MW588025 MT498094 MK737073 MHO063925 MN888366 MN888365 MN888367 MN888368 MHO063927 MT261912 MN443039 MW247177 MH063922 MK737070 MHO063926 MK737069 MHO063881 KT935502 MN888369 MN888370 KX810008 MT241900- MT241901 MW588028 MW588029 KY797265 MG757132 MW247178 MN888375 MN435112 MH197146 MH197153 MH197158 MT023999 MT023998 MH664931 MH664942 MF568532 MH664946 MH681760 MN888371 MN888372 MN888377 MN888378 MN443040 Vecchi, M., Stec, D.: Two new Macrobiotus species from USA and Greece 28S MT373699 MN888358 MN888359 MW588032 MW588033 MW588030 MW588031 MT488397 MK737071 MHO063934 MN888352 MN888351 MN888353 MN888354 MHO063936 MT261903 MN443034 MW247176 MH063924 MK737064 MHO063935 MK737063 MHO63880 KT935501 MN888355 MN888356 KX8 10009 MT241897- MT241898 MW588034 MW588035 KY.797266 MG757133 MW247175 MN888360 MN435116 MH197264 MH197152 MH197151 MT024041 MT024042 MH664948 MH664959 MF568533 MH664963 MH681757 MN888357 MN888364 MN888362 MN888363 MN443035 COl MT373803 MN888325 MN888326 MW593929 MW593930 MW593927 MW593928 MT502116 MK737922 MHO57765 MHO57766 MN888312 MN888316 MN888313 MN888314 MN888315 MH057768 MHO057769 MT260371 MN444824 MN444825 MN444826 MW246134 MH057764 MK737920 MK737921 MHO57767 MK737919 MHO57763 KT951668 MN888317 MN888318 MN888319 MN888320 MN888321 KX810011 KX810012 MT246659 MT246661 MW593931 MW593932 KY797267 MG757136 MG757137 MW246133 MN888327 MN482684 MH195150 MH195147 MH195149 MT023412 MT023413 MH675998 MH676012 MF568534 MH676018 MH676053 MN888322 MN888323 MN888324 MN888329 MN888330 MN444827 ITS2 MT374190 MN888345 MN888346 MW588020 MW588021 MW588018 MW588019 MT505165 MK737072 MH063928 MH063929 MN888343 MN888342 MN888335 MN888336 MN888341 MN888339 MN888340 MHO063932 MH063933 MT261907 MN443036 MN443037 MW247180 MH063923 MK737067 MH063931 MK737065 MK737066 MHO063921 KT935500 MN888337 MN888338 MN888332 MN888333 MN888334 KX810010 MW588022 MW588023 KY797268 MG757134 MG757135 MW247179 MN888347 MN435120 MH197154 MH197267 MH197150 MTO24000 MTO24001 MH666080 MH666091 MF568535 MH666096 MH681763 MN888331 MN888344 MN888349 MN888350 MN443038 Reference Pogwizd and Stec (2020) Stec et al. (2021) Stec et al. (2021) This study This study Nelson et al. (2020) Coughlan et al. (2019) Stec et al. (2018b) Stec et al. (2021) Stec et al. (2021) Stec et al. (2021) Stec €tal.(2021) Stec et al. (2018b) Stec et al. (2020c) Stec et al. (2020b) Kiosya et al. (2021) Nowak and Stec (2018) Coughlan and Stec (2019) Stec et al. (2018b) Coughlan and Stec (2019) Stec et al. (2018c) Stec et al. (2015) Stec et al. (2021) Stec et al. (2021) Roszkowska et al. (2017) Kuzdrowska et al. (2021) This study Stec et al. (2017b) Stec et al. (2018d) Kiosya et al. (2021) Stec et al. (2021) Kayastha et al. (2020a) Kaczmarek et al. (2018a) Stec et al. (2018¢) Roszkowska et al. (2018) Stec et al. (2020a) Stec et al. (2020a) Stec et al. (2020d) Stec et al. (2020d) Stec et al. (2018f) Stec et al. (2020d) Stec et al. (2020e) Stec et al. (2021) Stec et al. (2021) Stec et al. (2021) Stec et al. (2021) Stec et al. (2020b) Zoosyst. Evol. 97 (1) 2021, 281-306 Class: Eutardigrada Richters, 1926 Order: Parachela Schuster et al., 1980 (restored by Morek et al. 2020) Superfamily: Macrobiotoidea Thulin, 1928 (in Mar- ley et al. 2011) Family: Macrobiotidae Thulin, 1928 Genus: Macrobiotus Schultze C.A.S., 1834 Macrobiotus annewintersae Vecchi & Stec, sp. nov. http://zoobank.org/OSEFF40C-9238-49B8-9D79-7986979F674D Tables 3, 4, Figures 1-8, Suppl. material 1 Etymology. We dedicate this species to MV friend and colleague Dr. Anne Winters, evolutionary ecologist, who collected the sample in which the new species was found. Material examined. 146 animals and 56 eggs. Speci- mens mounted on microscope slides in Hoyer’s medium (93 animals + 38 eggs), fixed on SEM stubs (51+18), and processed for DNA sequencing (2+0). Type locality. 32°21'05"N, 89°56'30"W; 106 m asl: suburban area of Jackson, Mississippi, USA; mixed leaf litter on ground; coll. December 2019 by Anne Winters. Type depositories. Holotype 9 (slide US.084.01 with 10 paratypes) and 63 paratypes (slides: US.084.*, 285 where the asterisk can be substituted by any of the fol- lowing numbers: 02—05) and 20 eggs (slides US.084.*: 06-08) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University (Gronos- tajowa 9, 30-387, Krakow, Poland). Additional para- types (71 animals + 29 eggs) (slides: S207 SL*: 1-15; SEM stubs: S207 Stub*:1—4) are deposited at the Department of Biological and Environmental Sci- ences, University of Jyvaskyla (Survontie 9C, 40500, Jyvaskyla, Finland). Description of the new species. Animals (measure- ments and statistics in Table 3): In live animals, body translucent in smaller specimens and opaque whitish in larger animals: transparent after fixation in Hoyer’s medium (Figure 1). Eyes present in live animals and after fixation in Hoyer’s medium. Small roundish cuticular pores on the dorsal and lateral cuticle, as well as on the external cuticle of all legs (0.2-0.6 um in diameter), visible under both PCM and SEM (Fig- ures 1B, C, 2D). On the dorsal surface, pores are absent between cuticle folds and arranged in loose belts (Fig- ure 1C). Pores sparse on the ventral surface and visible only under SEM (Figure 8C). Patches of fine granulation, on the external surface of legs I-III as well as on the dor- sal and dorso-lateral sides of legs IV, visible in PCM (Fig- Table 3. Measurements [in um] of selected morphological structures of individuals of Macrobiotus annewintersae 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). Character N Range Mean Sd Holotype yum pt um pt yum pt yum Pt Body length 29 287 - 441 934 - 1226 371 1074 46 84 434 1226 Buccal tube Buccal tube length 28 27.1 - 40.4 - 34.3 - oul - 35.4 - Stylet support insertion point 28 21:2) = - 3210 76:8 .—- 61:6 272 79.4 2.4 1.3 ZY iO = VOT Buccal tube external width 29 34 - 6.1 125 = 17.0 4.7 13.8 0.6 1.0 5.4 15:3 Buccal tube internal width 29 19 - 4.5 6.8 —- 11.5 3.2 9.4 0.6 cal wo 9:3 Ventral lamina length 22 16.0 - 26.1 49.4 - 64.5 20.1 58.8 22 3.0 21.9 69:9 Placoid lengths Macroplacoid 1 28 GG- = (10:3. ©2059 — 28.9 8.3 24.4 16 1.8 9.4 26.6 Macroplacoid 2 30 3.6 - 6.8 12.6 - 18.5 5.3 15.2 0.8 1.6 5.6 15.8 Microplacoid 30 16 - 4.1 4.7 - 11.5 2.6 7.7 0.6 1.6 29 8.2 Macroplacoid row 26 10.9 - 176 388 - 49.4 14.8 43.6 1.8 2.8 16.6 46.9 Placoid row 26 13:77) =~ "22:3 ASB =~. 62:6. 185° 54.5 2 3.6 207 58-5 Claw 1 heights External primary branch 24 7.4: oe PIO: «22,7— = 304 9.5 27.6 0.8 2.0 10.4 29.4 External secondary branch 22 5.7 = 8.7 18.6 - 24.2 7.6 21.6 0.7 2.0 8.5 24.0 Internal primary branch 25 7a = 10:5 21.8 - 284 8.7 25.5 0.7 1.9 9.6 27.1 Internal secondary branch 23 54 - 8.6 LOR = ZS. 7.0 20.1 0.7 1.4 1:5 Ai Claw 2 heights External primary branch 26 72 = Ile “25:6 = 32:5 10.0. 294 1.0 1.9 LEO Salad External secondary branch 25 6.3 - ee) 18.9 - 26.3 8.0 23.0 0.8 2.0 Ox 26.3 Internal primary branch 28 70 - 116 23.8 - 30.8 9.4 Pht QS 1.9 9.8 Zi oy Internal secondary branch 26 54 - 920 15.6 - 24.3 Find Abas 0.9 Pe 8.6 24.3 Claw 3 heights External primary branch 25 83 - 114 25.8 - 31.0 a9 28.8 0.9 jive 10.9 30.8 External secondary branch 24 5.9 - oS 19.1 - 272 7.8 22.6 1.0 De hie) 26.3 Internal primary branch 26 70> = Wet “203° = ~28:8 9.0 26.3 0.9 1.8 9.4 26.6 Internal secondary branch 24 5.20 - 8.4 16.5 - 23.1 7.1 2087, 0.9 1.8 7.7 218 Claw 4 heights Anterior primary branch 26 82 - 12.5 25.0 - 35.3 10.4 30.6 il ane Lote Bod Anterior secondary branch 25 5.2 - 9.4 14.3 - 26.3 Fine 2exf 0.8 2:5 ae 2613 Posterior primary branch 25 92 - 14.5 29 59" 37.6: TTY" 3835 Ln 2.4 Lea me ™ -B5Lo. Posterior secondary branch 23 69 - 10.4 19.9 - 31.6 8.4 24,7 0.9 2.8 ? ? zse.pensoft.net Vecchi, M., Stec, D.: Two new Macrobiotus species from USA and Greece Figure 1. Macrobiotus annewintersae sp. nov. — habitus and cuticular pores: A. Dorso-ventral view of the body (Holotype 9@;, PCM); B, C. Cuticular pores on the dorsal part of the body under PCM and under SEM, respectively. Arrowheads indicate pores and empty arrows indicate places on dorsal cuticle without pores. Scale bars in um. Figure 2. Macrobiotus annewintersae sp. nov. — cuticular structures on legs: A. External granulation on leg III under PCM; B. A cuticular bulge (pulvinus) on the internal surface of leg II] under PCM; C. Granulation on leg [TV under PCM; D. External granulation on leg III under SEM; E. A cuticular bulge (pulvinus) on the internal surface of leg II] under SEM. Filled flat arrowheads indicate the granulation patch, empty flat arrowheads indicate pulvinus and filled indented arrowheads indicate muscle attachments. C assembled from several photos. Scale bars in um. zse.pensoft.net Zoosyst. Evol. 97 (1) 2021, 281-306 ge = a ’ ~ \4 »” a9 Ria. a ; ae 287 Figure 3. Macrobiotus annewintersae sp. nov. — claws: A, B. Claws III and IV, respectively, under PCM; C, D. Claws III and IV, respectively, under SEM. Filled indented arrowheads indicate double muscle attachments under the claws, empty indented arrow- heads indicate a faintly visible divided cuticular bar. A and B assembled from several photos. Scale bars in wm. ure 2A, C) and SEM (Figure 2D). A pulvinus is present on the internal surface of legs I-III (Figure 2B, E). Claws Y-shaped, of the hufelandi type. Primary branches with distinct accessory points, a common tract, and an evident stalk connecting the claw to the lunula (Figure 3). The lunulae I-III are smooth (Figure 3A, C), whereas lunulae IV are dentate (Figure 3B, D). A divided cuticular bar with double muscle attachments are poorly visible under PCM (Figure 3A). Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type (Figure 4) with ventral lam- ina and ten peribuccal lamellae. The stylet furcae typi- cally-shaped, the basal portion is enlarged and has two caudal branches with thickened, swollen, rounded apices. Under PCM, the oral cavity armature is of the patagon- icus type, i.e., with only the second and third bands of teeth visible (Figure 4B, C). However, under SEM the first band of teeth 1s visible and composed of one row of very small cones situated anteriorly in the oral cavity, just behind the bases of the peribuccal lamellae (Figure 5). The second band of teeth is situated between the ring fold and the third band of teeth and composed of 3—4 rows of teeth visible in PCM as granules (Figure 4B, C). The third band of teeth is divided into a dorsal (Figure 4B) and a ventral portion (Figure 4C). Under PCM, the dorsal teeth are seen as three distinct transverse ridges whereas the ventral teeth appear as two separate lateral transverse ridges between which one big tooth (sometimes circular in PCM) is visible (Figure 4B, C). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a drop-shaped mi- croplacoid (Figure 4A, D, E). The macroplacoid length sequence is 2<1. The first and the second macroplacoid have a central and a subterminal constriction, respective- ly (Figure 4D, E). Eggs (measurements and statistics in Table 4): The surface between processes is of the persimilis type, 7.e., with a continuous smooth chorion, never with pores or reticulum (Figures 6, 7). Under PCM the surface between the processes is covered with wrinkles that ap- pear as dark thickenings/striae, whereas under SEM the surface appears clearly wrinkled (Figures 6, 7). Processes zse.pensoft.net 288 Vecchi, M., Stec, D.: Two new Macrobiotus species from USA and Greece Figure 4. Macrobiotus annewintersae sp. nov. — buccal apparatus and the oral cavity armature under PCM: A. Dorso-ventral view of the entire buccal apparatus; B, C. Oral cavity armature in dorsal and ventral view, respectively; D, E. Placoid morphology in dorsal and ventral view, respectively. Empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth in the oral cavity, and empty indented arrowheads indicate central constriction in the first macroplacoid and subterminal constriction in the second macroplacoid. A, D and E assembled from several photos. Scale bars in um. are of a modified hufelandi type (Figures 6, 7). The prop- er terminal disc is absent and instead 2—8 thick tentacular arms (typically 5—6) are present in the distal part of the process (Figures 6, 7). The tentacular arms present bub- ble-like structures (visible in PCM). Under SEM, each tentacular arm is distally divided into many irregular digi- tations that are sometime covered with micro-granulation (Figure 7C—F). Also, under SEM micro-pores can be seen on the egg surface between the processes and around the process bases (Figure 7C, E). zse.pensoft.net Reproduction / Sexual dimorphism. The species is dioecious. Spermathecae in females as well as testis in males, clearly visible under PCM up to 24 hours after mounting in Hoyer’s medium, have been found to be filled with spermatozoa (Figure 8A, B). The species ex- hibits secondary sexual dimorphism in the form of clearly visible lateral gibbosities on the hind legs in males (Fig- ure 8B, C). DNA sequences. 18S rRNA: GenBank: MW588024— MW588025; 659 and 664 bp long. Zoosyst. Evol. 97 (1) 2021, 281-306 Figure 5. Macrobiotus annewintersae sp. nov. — anterior view of the mouth opening under SEM. Filled flat arrowhead indicates the first band of teeth. Scale bar in um. — Figure 6. Macrobiotus annewintersae sp. nov. — egg chorion morphology under PCM: A, B. Egg surface; C, D. Midsection of the processes. Filled flat arrowheads indicate bubble-like structures within tentacular arms in the distal portion of the egg processes and empty flat arrowheads indicate dark thickenings/striae on the egg surface between processes. Scale bars in um. zse.pensoft.net Vecchi, M., Stec, D.: Two new Macrobiotus species from USA and Greece OS Figure 7. Macrobiotus annewintersae sp. nov. — egg chorion morphology under SEM: A, B. Entire egg; C—E. Details of the egg processes and egg surface between them; F. Details of the tentacular arms in the distal portion of each egg process. Filled indented arrowheads indicate micropores and empty indented arrowheads indicate lobes in tentacular arms covered by micro-granulation. Scale bars in um. zse.pensoft.net Zoosyst. Evol. 97 (1) 2021, 281-306 291 Figure 8. Macrobiotus annewintersae sp. nov. — reproduction: A. Female under PCM; B. Male under PCM; C. Male under SEM. Filled indented arrowhead indicates spermathecae filled with spermatozoa, empty indented arrowhead indicates male’s testis, arrows indicate lateral gibbosities on legs IV and filled flat arrowhead indicates cuticular pore on the ventral side of the body. Scale bars in um. Table 4. Measurements [in um] of selected morphological structures of the eggs of Macrobiotus annewintersae sp. nov. mounted in Hoyer’s medium (N-number of eggs/structures measured, RANGE refers to the smallest and the largest struc- ture among all measured specimens; SD—standard deviation). Character N Range Mean Sd egg bare diameter 207" 59:84 j= MOY a 665-8 Sy Egg full diameter 20 69.8 - 87.1 75.7 4.6 Process height 63 4.2 - KSo | SERGE FO Process base width 63. 2.4 - 59 41 #£4Q.7 Process base/height ratio 63 52% - 100% 71% 10% Terminal disc width 63 2.8 - G7, 44 09 Inter-process distance 63.5 £253 - 69 42 0.9 Number of processes on 20 ah - 28 24.4 1.7 the egg circumference 28S rRNA: GenBank: MW588030-MW588031; 679 and 703 bp long. ITS-2: GenBank: MW588018—MW588019; 298 bp long. COI: GenBank: MW593927—-MW593928: 532 and 535 bp long. Phenotypic differential diagnosis. By having an egg chorion of the persimilis type (smooth or wrinkled cho- rion) and by having thick tentacular arms instead of a proper terminal disc on the distal part of egg processes, M. annewintersae sp. nov. resembles only one species: Macrobiotus anemone Meyer, Domingue & Hinton, 2014 from USA. However, the new species differs spe- cifically from: e M. anemone by having eyes (absent in M. anem- one), by the presence of granulation on all legs (absent in I. anemone), by having the oral cavity armature (OCA) of the patagonicus type (macula- tus type — only the third band of teeth visible under light microscope — in M. anemone), by the presence of dentate lunulae in legs IV (smooth lunulae in legs IV in M. anemone), by having the thick tentacular arms in the distal part of the processes filled with bubble-like structures (tentacular arms solid in M anemone, Figure 17) and by lacking a cavity be- tween the process trunk and tentacular arms that zse.pensoft.net 292 Vecchi, M., Stec, D.: Two new Macrobiotus species from USA and Greece appears in PCM as a clearly refracting dot (the cav- ity present in M. anemone, Figure 17). Macrobiotus rybaki Stec & Vecchi, sp. nov. http://zoobank.org/FC73B03E-E5BF-4597-822F-BBAC95F 1 FFEB Tables 5, 6, Figures 9-16, SM.02 Etymology. We dedicate this species to the singer, com- poser, musician, actor and the 2009 Eurovision Song Contest winner, Alexander Rybak. Material examined. 173 animals and 37 eggs. Speci- mens mounted on microscope slides in Hoyer’s medium (156 animals + 32 eggs), fixed on SEM stubs (15+5), and processed for DNA sequencing (2+0). Type locality. 35°15'00"N, 23°49'28"E; 30 m asl: Om- alos, Crete, Greece; moss on rock in a xeric shrubland; coll. June 2015 by Malgorzata Mitan and Malgorzata Osielczak. Type depositories. Holotype 3 (slide GR.O11.11 with 11 paratypes) and 160 paratypes (slides: GR.O11.*, where the asterisk can be substituted by any of the following numbers: 02—08, 10-13, 15-16; SEM stub: 18.10) and 37 eggs (slides GR.O11.*: 01, 09, 14; SEM stub: 18.10) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University (Gronostajowa 9, 30- 387, Krakow, Poland). Description of the new species. Animals (measure- ments and statistics in Table 5): In live animals, body translucent in smaller specimens and opaque whitish in larger animals; transparent after fixation in Hoyer’s medium (Figure 9A). Eyes present in live animals and after fixation in Hoyer’s medium. Ellipti- cal cuticular pores (0.6—1.5 um in length) present all over the body and clearly visible under both PCM and SEM (Figures 9B—D, 10). Patches of fine granulation on the external surface of legs I-III as well as on the dorsal and dorso-lateral sides of legs IV clearly visible under both PCM and SEM (Figure 10A, B, E, F). A pulvinus is pres- ent on the internal surface of legs I-III (Figure 10C, D). Claws Y-shaped, of the hufelandi type. Primary branches with distinct accessory points, a common tract, and an evident stalk connecting the claw to the lunula (Figure 11). The lunulae J-III are smooth (Figure 11A, D, E), whereas lunulae IV are dentate (Figure 11B, C, F). A divided cuticular bar and doubled muscle attachments are visible under PCM (Figures 10C, D, 11A, D, E). Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type (Figure 12), with ventral lamina and ten peribuccal lamellae (Figure 13A). The stylet furcae Table 5. Measurements [in um] of selected morphological structures of individuals of Macrobiotus rybaki 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). Character N Range um Body length 80°, 320 - 520. 915 Buccal tube Buccal tube length 30 349 - 44.4 Stylet support insertion point 30 258 - Sook. “F320 Buccal tube external width 30 44 - Gor tes Buccal tube internal width 30 28 - 5:5 7.0 Ventral lamina length 2t 20.5 - 28.9 59.4 Placoid lengths Macroplacoid 1 30 33:2 - Tl. +2355 Macroplacoid 2 206 5:8 - eiO- MLS:s Microplacoid 30, 1.9 - 3.8 4.3 Macroplacoid row 30 154 - Zeal. -4216 Placoid row 30> AS.Z - 20 oS. Claw 1 heights External primary branch 272 AOS - Te 22638 External secondary branch 26 «68.0 - 121 21g Internal primary branch 27 94 - 14.8 26.1 Internal secondary branch 27 hae - 10.8 18.6 Claw 2 heights External primary branch 30 105 - 15.0" SO: External secondary branch 28 8x2 - 12.8 22.9 Internal primary branch 30- Ox - 14.6 26.6 Internal secondary branch SO.) 25 - 11.8 19.4 Claw 3 heights External primary branch 28 11.5 - 15,8 § 2556 External secondary branch 25. 3&5 - ISB. 2324 Internal primary branch 29 106 - 15,2 2859 Internal secondary branch 29 hie - 1.8, 20°76 Claw 4 heights Anterior primary branch 28 12.5 - 17.4 34.2 Anterior secondary branch 23. te - 129: 20-6 Posterior primary branch 26 13.2 - 18.8 35.4 Posterior secondary branch 25° FOG - Sas ae zse.pensoft.net Mean Sd Holotype pt ym pt ym pt ym pt 1190 424 1054 3g 67 436 1093 40.2 - 23 - So9 = 75.4 29.7 73.9 1g 0.6 30.1 75.4 E56: as 13.7 0.5 0.8 Bile GiAS 1323 4.6 11.4 0.5 1.0 2.8 7.0) 65.9 25-6 63.7 1.8 1.7 24.5 61.4 30.1 10.8 26.8 1 1.8 9:5. 2358 19.5 6.9 Lhd 0.6 1.1 Gro PP 5T5: 9:2 29 6.8 0.4 £0 275 6.3 Pale, 18.7 46.5 LF Ans 17.0 42.6 61.0 2251 Soa) 1.8 ag 20.4 51.1 Bor? P25 31.0 12 2.1 EPs BOr6 28.9 a9 24.5 1.0 1.9 9.4 23.6 33.9 11.9 2OE5 1.2 1.8 11.8 29.6 26.9 OF 22.8 yl ad Ge 8226. 37.4 11 ous F 1.0 1.7 12.4 31.1 31.4 LS.5 26.0 sal 2A IOI “2468 35.4 12.6 Sig 1.0 igh 11.8 2956 29.6 9.9 24.6 1.1 2.5 Spo! 2 18 38.2 13.4 355 LZ Mae ai 1S) ESOS 32,1 10.6 26.7 1.2 245 9.8 24.6 36.2 ee 3S 32.2 iba Ted: Le AZO 29.8 10.0 24.9 val 2.3 9.4 23.6 44,9 15.7 age? 1.4 ae 15.4 38.6 31.4 10.7 26.6 1.4 S77 L129 28.1 46.3 16.8 41.8 1.4 Sit 17.3 43.4 B18 dele 29.2 dal 2.6 LG 2958 Zoosyst. Evol. 97 (1) 2021, 281-306 Figure 9. Macrobiotus rybaki sp. nov. — habitus and cuticular pores: A. Dorso-ventral view of the body (Holotype @; Hoyer’s me- dium, PCM); B. Cuticular pores on the dorsal part of the body under SEM; C, D. Cuticular pores on the dorsal and ventral part of the body under PCM, respectively. Filled arrows indicate lateral gibbosities. Arrowheads indicate elliptical pores. Scale bars in um. typically-shaped, the basal portion is enlarged and has two caudal branches with thickened, swollen, rounded apices. Under PCM, the oral cavity armature is of the pa- tagonicus type, i.e., with only the second and third bands of teeth visible (Figure 12B, C). However, under SEM the first band of teeth is visible as a row of irregularly distributed small teeth situated anteriorly in the oral cav- ity, just behind the bases of the peribuccal lamellae (Fig- ure 13A, B). The second band of teeth is situated between the ring fold and the third band of teeth and comprised of 3-4 rows of teeth faintly visible in PCM (Figure 12B, C) and visible as cones in SEM (Figure 13A). Teeth of the second band are larger than those in the first band. The teeth of the third band are located within the poste- rior portion of the oral cavity, between the second band of teeth and the buccal tube opening (Figures 12B, C, 13A, B). The third band of teeth is divided into a dorsal and the ventral portion. Under both PCM and SEM, the dorsal teeth are seen as three distinct transverse ridges (Figures 12B, 13A). The ventral teeth appear as two sep- arate lateral transverse ridges between which one conical medial tooth (roundish in PCM) is visible (Figures 12C, 13B). Lateral cribrose area present in the buccal tube behind the third band of teeth (Figure 13B). Pharyngeal bulb spherical, with triangular apophyses, three anteri- or cuticular spikes (typically only two are visible in any given plane), two rod-shaped macroplacoids and a drop- shaped microplacoid (Figures 12A, D, E). The macropla- coid length sequence is 2<1. The first macroplacoid has a weak central constriction, whereas the second is weakly constricted only subterminally (Figures 12D, E). Eggs (measurements and statistics in Table 6): Table 6. Measurements [in um] of selected morphological structures of the eggs of Macrobiotus rybaki sp. nov. mounted in Hoyer’s medium (N-number of eggs/structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD—standard deviation). Character N Range Mean Sd Egg bare diameter 14 68.7 - 93.4 76.2 7.6 Egg full diameter 14 836 - 107.9 94.1 7.9 Process height Sao ES WSeder OZ) 16 Process base width 42 44 - G76" <69> 1:0 Process base/height ratio 42. 52% - 99% 76% 12% Terminal disc width 42 13 - 4.2 2.37 O37 Inter-process distance 42 14 - 4.5 2¢ 308 Number of processesonthe 14 25 - 34 28.1 3.0 egg circumference The surface between processes is of the hufelandi type, i.e., covered with a reticulum (Figures 14A, B, 15A—E). Peribasal meshes of slightly larger diameter compared to interbasal meshes (Figures 14A, B, IS5A—D). Typi- cally, the reticulation between neighbouring processes is composed of two rows of peribasal meshes and with a third row of smaller mashes interposed (the third row sometimes missing) (Figures 14A, B, 15A—D). Mesh di- ameter is usually larger than the mesh walls and nodes (Figures 14A, B, 15A—D). The meshes are 0.4—1.4 um in diameter, with roundish irregular shape. The pillars con- necting the reticulum with the chorion surface are visible only under SEM (Figure 15C). The bases of the processes are surrounded by cuticular thickenings that merge into the bars and nodes of the reticulum (Figure 15C, D). These basal thickenings appear under PCM as short dark projections around the process bases (Figure 14A, B). zse.pensoft.net Vecchi, M., Stec, D.: Two new Macrobiotus species from USA and Greece Figure 10. Macrobiotus rybaki sp. nov. — cuticular structures on legs: A, B. External granulation on leg HI and I under PCM and SEM, respectively; C, D. A cuticular bulge (pulvinus) on the internal surface of legs III under PCM and SEM, respectively; E, F. Granulation on legs TV under PCM and SEM, respectively. Filled flat arrowheads indicate the granulation patch, empty flat arrowheads indicate pulvinus and filled indented arrowheads indicate muscle attachments. A and E assembled from several photos. Scale bars in um. zse.pensoft.net Zoosyst. Evol. 97 (1) 2021, 281-306 295 r ne x Figure 11. Macrobiotus rybaki sp. nov. — claws: A, B. Claws III and IV, respectively, under PCM; C. Magnification of lunulae IV of a different specimen; D—F. Claws II, HI and IV respectively, under SEM. Filled indented arrowheads indicate double muscle attachments under the claws, empty indented arrowheads indicate a divided cuticular bar. A and B assembled from several photos. Scale bars in um. Processes are of the hufelandi type with very elongated concave trunk and extremely reduced (narrow), round and convex terminal discs with irregularly jagged edges (Figures 14C—F, 15). Under SEM the surface of the con- vex terminal discs is covered by small irregular granules and tubercles (Figures 15C—F). Reproduction / Sexual dimorphism. The species is di- oecious. Testis in males, which were clearly visible under PCM up to 24 hours after mounting in Hoyer’s medium, have been found to be filled with spermatozoa, (Fig- ure 16). In females spermathecae filled with spermatozoa were not observed. The species exhibits secondary sexual dimorphism in the form of small lateral gibbosities on the hind legs of males (Figure 16). DNA sequences. 18S rRNA: GenBank: MW588028- MW588029; 1018 bp long. 28S rRNA: GenBank: MW588034-MW588035; 783 bp long. ITS-2: GenBank: MW588022—MW588023; 391 bp long. COI: GenBank: MW593931-MW593932; 658 bp long. Phenotypic differential diagnosis. By having the OCA of the patagonicus type (only the 2™ and 3" bands of teeth visible under light microscopy), egg chorion of the hufelandi type (covered with a reticulum), and egg processes with reduced (narrow) terminal disc, Macro- biotus rybaki sp. nov. is most similar to four species: Macrobiotus dariae Pilato & Bertolani, 2004, Macrobio- tus noemiae Roszkowska & Kaczmarek, 2019, Macro- biotus santoroi Pilato & D’Urso, 1976 and Macrobiotus serratus Bertolani, Guidi & Rebecchi, 1996. The new species differs specifically from: ¢ M. dariae by having a more anteriorly placed stylet Support insertion point (pt 73—75.5 in the new spe- cies vs. 77.2—77.9 in M. dariae), a narrower buccal tube external diameter (pt /2.3—/5.6 in the new spe- cies vs. 15.6—25.7 in M. dariae), a smaller number of processes on the egg circumference (25—34 in the new species vs. 34-38 in M. dariae), a different egg process morphology (processes with very elongated concave trunks and extremely reduced — narrow — convex terminal discs in the new species vs. conical processes with flexible distal portion without termi- nal discs in M. dariae; Figure 18A—-C). e M. noemiae by having a more anterior stylet sup- port insertion point (pt 73.0—75.5 in the new species vs. 78.3—81.8 in M. noemiae), by a smaller number of processes on the egg circumference (25—34 in the new species vs. 35—36 in M. noemiae), by well-de- fined reticulation on the chorion surface with the peribasal mesh larger than the interbasal mesh and mesh diameter larger than the walls and nodes of the reticulum (very delicate and faint reticulation with mesh of uniform size distributed randomly on the zse.pensoft.net 296 Vecchi, M., Stec, D.: Two new Macrobiotus species from USA and Greece Figure 12. Macrobiotus rybaki sp. nov. — buccal apparatus and the oral cavity armature under PCM: A. Dorso-ventral view of the entire buccal apparatus; B, C. Oral cavity armature in dorsal and ventral view, respectively; D, E. Placoid morphology in dorsal and ventral view, respectively. Empty flat arrowheads indicate the second band of teeth, filled indented arrowheads indicate the third band of teeth in the oral cavity, empty indented arrowheads indicate central constriction in the first macroplacoid and subterminal constriction in the second macroplacoid and arrows indicate cuticular spikes between end of the buccal tube and anterior portion of the bulbus. A, D, E assembled from several photos. Scale bars in um. Figure 13. Macrobiotus rybaki sp. nov. — anterior view of the oral cavity armature under SEM: A, B. Dorsal and ventral view, respectively. Filled flat arrowheads indicate the first band of teeth, empty flat arrowhead indicates the second band of teeth, filled indented arrowheads indicate the third band of teeth in the oral cavity. Scale bars in um. zse.pensoft.net Zoosyst. Evol. 97 (1) 2021, 281-306 297 Figure 14. Macrobiotus rybaki sp. nov. — egg chorion morphology under PCM: A, B. Egg surface; C—F. Midsection of the pro- cesses. Filled flat arrowheads indicate cuticular thickenings around the processes base that merge into the bars and nodes of the reticulum. Scale bars in ym. egg surface between the processes in M. noemiae), a different egg processes morphology (processes with very elongated concave trunks and extremely reduced — narrow — convex terminal discs without flexible filaments in the new species vs. conical pro- cesses without terminal discs but with hair-like, and flexible filaments in M. noemiae). ¢ M. santoroi by having taller egg processes (6.7— 13.4 um in the new species vs. 4 um or less in M. santoroi), by a smaller number of processes on the egg circumference (25—34 in the new species vs. 37-40 in M. santoroi), by processes with very elon- gated concave trunks (processes peg-shaped in M. santoroi), by well-defined reticulation on the cho- rion surface with the peribasal mesh larger than the interbasal mesh and mesh diameter larger than walls and nodes of the reticulum (very fine mesh with ev- ident and wide walls and nodes, giving the false im- pression of a granulated surface in M. santoroi). ¢ M. serratus by having amore anterior stylet support insertion (pt 73.0—75.5 in the new species vs. 75.6— 77.7 in M. serratus), by a taller egg process height (6.7—13.4 um in the new species vs. 5.5—6.0 um in M. serratus) and by well-defined reticulation on the chorion surface with the peribasal mesh larger than the interbasal mesh and mesh diameter larger than zse.pensoft.net Vecchi, M., Stec, D.: Two new Macrobiotus species from USA and Greece ra ey Figure 15. Macrobiotus rybaki sp. nov. — egg chorion morphology under SEM: A, B. Entire egg; C—E. Details of the egg processes and egg surface between them; F. Details of the reduced terminal disc. Filled flat arrowheads indicate cuticular thickenings around the processes base that merge into the bars and nodes of the reticulum. Scale bars in um. zse.pensoft.net Zoosyst. Evol. 97 (1) 2021, 281-306 299 Figure 16. Macrobiotus rybaki sp. nov. — reproduction: male under PCM. Empty indented arrowhead indicates male’s testis and arrows indicate lateral gibbosities on legs IV. Scale bar in um. Figure 17. Macrobiotus anemone Meyer, Domingue & Hinton, 2014 (type series) — egg chorion morphology under PCM: A, B. Egg surface (slides 9551 and 9552 respectively). Filled flat arrowheads indicate a cavity between the process trunk and tentacular arms that appears in PCM as a clearly refracted dot. Scale bars in um. walls and nodes of the reticulum (very delicate and faint reticulation with mesh of similar sizes distrib- uted uniformly on the egg surface between process- es in M. serratus; Figure 18D, E). Phylogenetic analysis. The phylogenetic reconstruc- tion (Figure 19) recovered the genus Macrobiotus as well as the three clades found by Stec et al. (2021) and by Kiosya et al. (2021) to be monophyletic. All three clades have high support values (pp=1). The new species Mac- robiotus annewintersae sp. nov. belongs to subclade B, within the Macrobiotus persimilis complex, even though the monophyly of this complex was not strongly sup- ported (pp=0.73). Macrobiotus engbergi Stec, Tumanov & Kristensen, 2020 was recovered as the closest relative of M. annewintersae sp. nov. (Figure 19). The second species analysed in this study, Macrobiotus rybaki sp. nov., belongs to subclade A with its closest relatives be- ing Macrobiotus wandae Kayastha, Berdi, Miaduchows- ka, Gawlak, Lukasiewicz, Goltdyn & Kaczmarek, 2020 and Macrobiotus vladimiri Bertolani, Biserov, Rebecchi & Cesari, 2011 (Figure 19). The newly found Swedish population identified in this study as Macrobiotus aff. polonicus, as could have been predicted from its mor- phological similarity with that species, clusters together with two populations of Macrobiotus polonicus Pilato, Kaczmarek, Michalczyk & Lisi, 2003 from Austria and Slovakia (Figure 19). zse.pensoft.net 300 Vecchi, M., Stec, D.: Two new Macrobiotus species from USA and Greece ¢<=Bs , tee #- ‘ - ¥ fe af " ad . . - . : : y * q plied 5 a c . 4 Figure 18. Macrobiotus dariae Pilato & Bertolani, 200 Las a 7 4 . : - “+ : , + = ; {: hae bn ¥ ‘* - f . 4 bs . \ . he’ Pi _ - 9 ° aa n . : fo , SS ath Be —™ 4 and Macrobiotus serratus Bertolani, Guidi & Rebecchi, 1996 (type series) — egg chorion morphology under PCM: A—C. Egg surface (A) and midsections of the processes (B, C) of M. dariae (slides PC45s1 and PC45s3 respectively); D, E. Egg surface of M. serratus (slides C1907s17 and C1907s30 respectively). Scale bars in um. Discussion We identified two new tardigrade species in the genus Macrobiotus using an integrative taxonomy approach combining the analyses of detailed morphological and genetic data. Thanks to the phylogenetic analysis per- formed in this study we confirmed Macrobiotus annew- intersae sp. nov. to belong to the Macrobiotus persimilis complex (as defined by Stec et al. 2021). Nevertheless, the morphological definition provided by Stec et al. (2021) does not encompass the extraordinary egg pheno- type exhibited by Macrobiotus annewintersae sp. nov., indicating the need for further amendment of the charac- ters describing this monophyletic group of species. The definition of that complex, regarding the egg processes, states “[...] single-walled egg processes [...] in the shape of truncated cones terminated with a well-developed disc and with solid chorion surface [...]”, It 1s therefore clear that as M. annewintersae sp. nov. possesses 2—8 tentacu- lar arms on the distal part of its egg processes, as opposed to ‘well-developed discs’, it falls outside the current defi- nition of the group. Very similar egg processes are also present in M. anemone, which was previously included in the M. persimilis complex by Stec et al. (2021) with- out any elaboration on that issue (please see Table 5 in zse.pensoft.net Stec et al. (2021) for the list of species included there in the complex). Therefore, to avoid inconsistency in ac- commodating these two species within the M. persimilis complex, we propose an upgraded definition that reads: species with white body, hufelandi type claws and with single-walled egg processes (without the labyrinthine lay- er = not reticulated) in the shape of truncated cones termi- nated with a well-developed disc or tentacular arms and with a solid chorion surface (the surface can be wrinkled and sometimes with faintly visible micropores but never properly porous or reticulated). Furthermore, we propose to tentatively include Macrobiotus andinus Maucci, 1988 within the M. persimilis complex. The species meet now all the criteria except the porous cuticle, (hence it was not considered as a member of the hufelandi group sensu Kaczmarek and Michalczyk (2017), but it is likely that these pores could be visible only under SEM similarly as in same species of the Macrobiotus pseudohufelandi complex (Stec et al. 2021). In their faunistic study devoted to Greek tardigrades Maucci and Durante Pasa (1982) reported Macrobiotus anderssoni Richters, 1907, specifically from the island of Crete. According to the description provided by Maucci and Durante Pasa (1982), their Macrobiotus anderssoni population from Crete is very similar to M. rybaki sp. Zoosyst. Evol. 97 (1) 2021, 281-306 0.91 Macrobiotus canaricus 1 Macrobiotus canaricus 2 Macrobiotus glebkai Macrobiotus basiatus Macrobiotus rybaki sp. nov. 2 Macrobiotus wandae Macrobiotus vladimiri Macrobiotus cf. recens 1 Macrobiotus cf. recens 2 Macrobiotus macrocalix 301 Macrobiotus rybaki sp. nov. 1 VW apejd snjzoigoiepW Macrobiotus crustulus Macrobiotus hannae Macrobiotus polonicus AT 1 Macrobiotus polonicus AT 2 Macrobiotus polonicus SK 1 0.99 * . Macrobiotus aff. polonicus SE 1 Macrobiotus aff. polonicus SE 2 *— Macrobiotus caelestis Macrobiotus engbergi 1 0.73 *_| Macrobiotus engbergi 2 x 0.99 Macrobiotus annewintersae sp. nov. 1 Macrobiotus annewintersae sp. nov. 2 O88. *<<_| Macrobiotus pallarii complex Macrobiotus polonicus SK 2 Macrobiotus engbergi 3 Xa|dwod sijiwisiad snjoiqoey g apejd snjzoigosep :