taxonID	type	description	language	source
03BD87B2FFBF4539279C2EE2FB98121C.taxon	materials_examined	Material examined: Altogether 132 animals and 133 eggs. Specimens were mounted on microscope slides in Hoyer’s medium (84 animals + 123 eggs), fixed on SEM stubs (15 + 10), processed for DNA sequencing (three animals) and aceto-orcein staining (30 animals). Neotype locality: 78 ° 38 ’ 13 ’’ N, 16 ° 46 ’ 07 ’’ E; 15 m a. s. l.: Norway, Svalbard, Spitsbergen, Brucebyen, Billefjorden; moss on soil; coll. 7 July 2017 by Michala Bryndová. Type depositories: Neotype (slide NO. 385.81 with ten neoparatypes) and 56 paratypes (slides: NO. 385. *, where the asterisk can be substituted by any of the following numbers 24 – 25, 27, 62, 75 – 78) and 103 eggs (slides: NO. 385. *: 3, 23, 34, 63 – 71) are deposited at the Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30 – 387, Kraków, Poland and 17 neoparatypes (slides: NO. 385. *: 79 – 80) and 20 eggs (slides: NO. 385. *: 72 – 74) are deposited in the Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, DK- 2100 Copenhagen, Denmark.	en	Stec, Daniel, Krzywański, Łukasz, Zawierucha, Krzysztof, Michalczyk, Łukasz (2020): Untangling systematics of the Paramacrobiotus areolatus species complex by an integrative redescription of the nominal species for the group, with multilocus phylogeny and species delineation in the genus Paramacrobiotus. Zoological Journal of the Linnean Society 188 (3): 694-716, DOI: 10.1093/zoolinnean/zlz163, URL: https://academic.oup.com/zoolinnean/article/188/3/694/5706821
03BD87B2FFBF4539279C2EE2FB98121C.taxon	description	REDESCRIPTION OF PARAMACROBIOTUS AREOLATUS (MURRAY, 1907) Animals (measurements and statistics in Table 3): In live animals, body almost transparent in juveniles and white in adults; after fixation in Hoyer’s medium body transparent (Fig. 1 A). Eyes present in live animals but absent in Hoyer’s. Body cuticle smooth, i. e. without pores or sculpturing, but legs with visible granulation (Fig. 1 B, C, E, F). On legs I – III, the patch of granulation extends from the external through the posterior and to the internal surface of the legs (Figs 1 B, D, E, G, 2 A, C), whereas the granulation on legs IV spreads from the claws onto the entire dorsal surface of legs (Fig. 1 C, F). The leg granulation is composed of microgranule aggregations (Fig. 2 E). A cuticular bulge / fold (pulvinus) is present on the internal surface of legs I – III (Fig. 1 D, G). Claws slender, of the hufelandi type. Primary branches with distinct accessory points, a long common tract and with an evident stalk connecting the claw to the lunula (Fig. 2 A – D). The end of the common tract apparently thickened in all claws (Fig. 2 A – D). Lunulae on legs I – III smooth (Fig. 2 A, C), whereas on legs IV clearly dentate and larger (Fig. 2 B, D, F). Bars with irregular margins under claws I – III (Fig. 2 A, C). Paired muscle attachments just below cuticular bars on legs I – III often visible in both PCM / NCM and SEM (Fig. 2 A, C), whereas the horseshoe-shaped structure under claws IV visible only in PCM / NCM (Fig. 2 F). Mouth anteroventral. Buccopharyngeal apparatus of the Macrobiotus type, with the ventral lamina and ten small peribuccal lamellae. The oral cavity armature well developed and composed of three bands of teeth (Fig. 3 A – D). The first band of teeth is composed of numerous small granules arranged in several rows situated anteriorly in the oral cavity, just behind the bases of the peribuccal lamellae (Fig. 3 B – D). The second band of teeth is situated between the ring fold and the third band of teeth and comprised of ridges parallel to the main axis of the buccal tube and granules, bigger than those in the first band (Fig. 3 A – D). Several additional teeth are often present between the second and the third band, especially in larger animals (Fig. 3 B, D). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the buccal tube opening (Fig. 3 A – D). The third band of teeth is divided into the dorsal and the ventral portion. Under PCM / NCM, 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 or up to three smaller median teeth are visible (Fig. 3 A, B). Also, in SEM, both dorsal and ventral teeth are clearly distinct with indented / sharp margins (Fig. 3 C, D). Pharyngeal bulb spherical, with triangular apophyses, three rodshaped macroplacoids and without microplacoid (Fig. 3 E, F), but in 33 of 65 analysed individuals (51 %) we observed a small and irregular but evident cuticular thickening in the place where the microplacoid in individuals of the Paramacrobiotus richtersi complex is present (Fig. 3 E); thus this thickening could be seen as an rudimentary microplacoid. The macroplacoid length sequence is 2 <1 <3. The first macroplacoid is anteriorly narrowed and the third has a subterminal constriction (Fig. 3 E, F). Eggs (measurements and statistics in Table 4): Laid freely, white, spherical with elongated conical processes (Figs 4 A, B, 5 A). Process apices are sometimes bi- or trifurcated (Fig. 4 D). The labyrinthine layer between the process walls is visible under PCM / NCM as a reticular pattern with sinuous margins (Fig. 4 B – E). The elongated meshes decrease in size from the base to the processes top (Fig. 4 B – E). The surface of processes and their elongated apices smooth under SEM (Fig. 5 B – F). Eight to ten flat areoles around each process (Figs 4 B, C, 5 A, B). Areoles smooth inside under PCM / NCM (Fig. 4 B, C), but delicately wrinkled in SEM (Fig. 5 B, C). Only occasionally are the areoles underdevoloped (Fig. 4 C). REPRODUCTIVE MODE The examined population is most likely parthenogenetic. Aceto-orcein staining of 30 individuals revealed no testes filled with spermatozoa or spermathecae filled with spermatozoa. DNA SEQUENCES We obtained sequences for all four of the above m e n t i o n e d m o l e c u l a r m a r k e r s f r o m a l l t h r e e specimens destined for DNA extraction. All of them were represented by single haplotypes: 18 S rRNA (GenBank: MH 664931), 1013 - bp long; 28 S rRNA (MH 664948), 732 - bp long; ITS 2 (MH 666080), 373 - bp long; COI (MH 675998), 656 - bp long. PHENOTYPIC DIFFERENTIAL DIAGNOSIS Based on the areolatus type of egg shell ornamentation (sensu Kaczmarek et al., 2017), Paramacrobiotus areolatus sensu stricto is most similar to the following five species in the P. areolatus complex: Paramacrobiotus centesimus (Pilato, 2000), P. crenatus (Maucci, 1991); P. intii Kaczmarek et al., 2014 b, P. klymenki Pilato et al., 2012 and P. walteri (Biserov, 1997 – 98).	en	Stec, Daniel, Krzywański, Łukasz, Zawierucha, Krzysztof, Michalczyk, Łukasz (2020): Untangling systematics of the Paramacrobiotus areolatus species complex by an integrative redescription of the nominal species for the group, with multilocus phylogeny and species delineation in the genus Paramacrobiotus. Zoological Journal of the Linnean Society 188 (3): 694-716, DOI: 10.1093/zoolinnean/zlz163, URL: https://academic.oup.com/zoolinnean/article/188/3/694/5706821
03BD87B2FFBF4539279C2EE2FB98121C.taxon	description	• Paramacrobiotus centesimus, known only from Brazil and Ecuador (Pilato, 2000), by: lunulae IV morphology (evidently larger and clearly dentate in P. areolatus vs. smaller and smooth in P. centesimus), egg process apex shape (clearly elongated in P. areolatus vs. short in P. centesimus), a larger full egg diameter (148.7 – 195.5 μm in P. areolatus vs. 76 – 91 μm in P. centesimus) and by taller egg processes (26.8 – 44.5 μm in P. areolatus vs. 7 – 11 μm in P. centesimus). • Paramacrobiotus intii, known only from Peru (Kaczmarek et al., 2014 b), by: a better developed oral cavity armature (three bands of teeth in P. areolatus vs. the second and third band of teeth in P. intii identifiable under PCM), egg process apex shape (clearly elongated in P. areolatus vs. short in P. intii), larger full egg diameter (148.7 – 195.5 μm in P. areolatus vs. 78.8 – 137.3 μm in P. intii), a longer egg processes (26.8 – 44.5 μm in P. areolatus vs. 15.4 – 24.4 μm in P. intii) and by a larger number of egg processes on the egg circumference (12 – 15 in P. areolatus vs. 9 – 10 in P. intii). • Paramacrobiotus klymenki, known only from Belarus (Pilato et al., 2012), by: the macroplacoid length sequence (2 <1 <3 in P. areolatus vs. 2 <3 <1 in P. klymenki), egg process apex shape (clearly elongated in P. areolatus vs. short in P. klymenki), a larger full egg diameter (148.7 – 195.5 μm in P. areolatus vs. 101 – 109 μm in P. klymenki) and by taller egg processes (26.8 – 44.5 μm in P. areolatus vs. 14.5 – 18.5 μm in P. klymenki). • Paramacrobiotus walteri, known only from Russia (Biserov, 1997 – 98), by: egg process surface (smooth in P. areolatus vs. apically covered by irregular granulation in P. walteri), and by taller egg processes (26.8 – 44.5 μm in P. areolatus vs. 10 – 17 μm in P. walteri). GENOTYPIC DIFFERENTIAL DIAGNOSIS The ranges of uncorrected genetic p-distances between the neotype population and genotyped species of the genus Paramacrobiotus are as follows: • 18 S rRNA: 2.2 – 4.2 % (3.7 % on average), with the most similar being an undetermined P. areolatus complex species from Italy (MH 664937) and the least similar being an undetermined P. richtersi complex species from Kenya (EU 038081); • 28 S rRNA: 3.4 – 8.2 % (6.9 % on average), with the most similar being an undetermined P. areolatus complex species from Portugal (MH 664960) and the least similar being Paramacrobiotus lachowskae Stec et al., 2018 c from Colombia (MF 568533); • ITS 2: 7.5 – 26.8 % (20.9 % on average), with the most similar being P. areolatus complex species from Italy (MH 666085) and the least similar being P. lachowskae from Colombia (MF 568535); • COI: 16.9 – 24.3 % (21.7 % on average), with the most similar being an undetermined P. areolatus complex species from Portugal (MH 676013) and the least similar being Paramacrobiotus arduus Guidetti et al., 2019 from Italy (MK 041020 – 1). PHYLOGENY, SPECIES DELIMITATION AND GEOGRAPHIC DISTRIBUTION OF THE GENUS PARAMACROBIOTUS The phylogenetic analysis conducted on the concatenated dataset of the four DNA markers shows that species with a microplacoid (Paramacrobiotus richtersi complex) cluster in a monophyletic clade. However, the members of the Paramacrobiotus areolatus complex form a paraphyletic group at the base of the Paramacrobiotus phylogenetic tree (Fig. 6). Regardless of the employed genetic species delimitation method (PTP or ABGD), the ITS 2 analysis recovered ten and the COI suggested 13 putative species (Fig. 7). The difference in the number of genetically delineated species resulted from two groups of populations. In the ITS 2 analysis, populations PT. 048 + TN. 014 + FR. 077 + AU. 044 + NZ. 001 + HU. 012 appear as a single species, but in the COI analysis they are divided into three separate species (PT. 048 + TN. 014 + FR. 077 + AU. 044, NZ. 001, and HU. 012). Moreover, in the ITS 2 analysis, populations IT. 048 and PT. 006 appear as a single species, whereas in the COI analysis, they appear as two species. Although populations from five continents were analysed in this study, no evident geographic pattern of their clustering on the phylogenetic tree is observed. In one case, a species delineated using the mitochondrial marker is found in Europe, Africa and in Australia (PT. 048 + TN. 014 + FR. 077 + AU. 044). However, based on ITS 2 delimitation, this putative species has an even wider geographic range because two more populations, from Hungary (HU. 012) and New Zealand (NZ. 001), are considered as representing this species too. Another species, Paramacrobiotus fairbanksi Schill et al., 2010, was originally described from Alaska (USA) and it was found in the present study in two localities in Poland (PL. 018 and PL. 035), meaning that the species has at least a Holarctic distribution. All other Paramacrobiotus species found in this study are limited to single localities, except the European populations of P. areolatus complex species from Italy (IT. 048) and Portugal (PT. 006), which in the ITS 2 analysis are recovered as a single species. EXPERIMENTAL CROSSES As expected for such divergent genetic distances in ITS 2 and COI (1.4 % vs. 13.8 %, respectively), two morphologically identical gonochoristic populations of the P. areolatus complex from Portugal (PT. 006) and Italy (IT. 048) were recognized as two separate species in both PTP and ABGD delimitations based on the COI dataset and as a single species in the ITS 2 dataset. F 1 eggs were laid both in intrapopulation crosses (PT × PT and IT × IT) and interpopulation crosses (PT × IT). As expected, the proportion of fertile pairs in intrapopulation crosses was not statistically different from 50 %, as around 50 % of pairs were expected to be heterosexual (41 – 57 %; P = 0.604 in IT × IT, P = 0.763 in PT × PT; see Table 5 for details). Similarly, the proportion of fertile pairs in interpopulation crosses was also not statistically different from the expected 50 %, even though only 25 % of pairs produced eggs (25 %; P = 0.135; see Table 5 for details). In addition, there were no significant differences in the proportion of successfully reproducing pairs compared to the total number of observed pairs in any of the crosses at the adjusted α- level p BH <0.02 (PT × PT vs. IT × IT: P = 0.391; PT × PT vs. PT × IT: P = 0.037; IT × IT vs. PT × IT: P = 0.254). Moreover, the KruskalWallis ANOVA test showed no statistical differences in the total number of eggs laid in each treatment (H 2,29 = 2.40, P = 0.302; overall there were 2.7 ± 2.0 eggs laid per fertile pair). All eggs in all three treatments hatched and offspring were viable (i. e. survived until reproductive maturity). As expected, all offspring obtained from interpopulations crosses (PT × IT) were heterozygous, which was evidenced by double peaks in mutated loci in the ITS 2 sequence chromatogram (Fig. 8). The sequencing of the COI fragment from the offspring of interpopulation crosses showed that fertilization is possible in two directions: in 50 % of PT × IT pairs, the female originated from Portugal and in the other 50 % of PT × IT crosses, the female was from the Italian population. Virgin animals cultured individually in isolation did not produce eggs in the course of the experiment indicating they are not capable to reproduce parthenogenetically.	en	Stec, Daniel, Krzywański, Łukasz, Zawierucha, Krzysztof, Michalczyk, Łukasz (2020): Untangling systematics of the Paramacrobiotus areolatus species complex by an integrative redescription of the nominal species for the group, with multilocus phylogeny and species delineation in the genus Paramacrobiotus. Zoological Journal of the Linnean Society 188 (3): 694-716, DOI: 10.1093/zoolinnean/zlz163, URL: https://academic.oup.com/zoolinnean/article/188/3/694/5706821
