Paramacrobiotus areolatus, (MURRAY, 1907)

PARAMACROBIOTUS AREOLATUS ( MURRAY, 1907) View in CoL

( TABLES 3–4, FIGS 1–7 View Figure 1 View Figure 2 View Figure 3 View Figure 4 View Figure 5 View Figure 6 View Figure 7 )

Macrobiotus echinogenitus var. areolatus ( Murray, 1907) View in CoL ; Macrobiotus areolatus Murray ( Murray, 1910) View in CoL ; Macrobiotus crenatus Maucci, 1991 View in CoL .

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. GoogleMaps

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.

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. 1A View Figure 1 ). 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. 1B, C, E, F View Figure 1 ). On legs I–III, the patch of granulation extends from the external through the posterior and to the internal surface of the legs ( Figs 1B, D, E, G View Figure 1 , 2A, C View Figure 2 ), whereas the granulation on legs IV spreads from the claws onto the entire dorsal surface of legs ( Fig. 1C, F View Figure 1 ). The leg granulation is composed of microgranule aggregations ( Fig. 2E View Figure 2 ). A cuticular bulge/fold (pulvinus) is present on the internal surface of legs I–III ( Fig. 1D, G View Figure 1 ).

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. 2A–D View Figure 2 ). The end of the common tract apparently thickened in all claws ( Fig. 2A–D View Figure 2 ). Lunulae on legs I–III smooth ( Fig. 2A, C View Figure 2 ), whereas on legs IV clearly dentate and larger ( Fig. 2B, D, F View Figure 2 ). Bars with irregular margins under claws I–III ( Fig. 2A, C View Figure 2 ). Paired muscle attachments just below cuticular bars on legs I–III often visible in both PCM/NCM and SEM ( Fig. 2A, C View Figure 2 ), whereas the horseshoe-shaped structure under claws IV visible only in PCM/NCM ( Fig. 2F View Figure 2 ).

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. 3A–D View Figure 3 ). 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. 3B–D View Figure 3 ). 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. 3A–D View Figure 3 ). Several additional teeth are often present between the second and the third band, especially in larger animals ( Fig. 3B, D View Figure 3 ). 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. 3A–D View Figure 3 ). 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. 3A, B View Figure 3 ). Also, in SEM, both dorsal and ventral teeth are clearly distinct with indented/sharp margins ( Fig. 3C, D View Figure 3 ). Pharyngeal bulb spherical, with triangular apophyses, three rodshaped macroplacoids and without microplacoid ( Fig. 3E, F View Figure 3 ), 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. 3E View Figure 3 ); 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. 3E, F View Figure 3 ).

Eggs (measurements and statistics in Table 4): Laid freely, white, spherical with elongated conical processes ( Figs 4A, B View Figure 4 , 5A View Figure 5 ). Process apices are sometimes bi- or trifurcated ( Fig. 4D View Figure 4 ). The labyrinthine layer between the process walls is visible under PCM/NCM as a reticular pattern with sinuous margins ( Fig. 4B–E View Figure 4 ). The elongated meshes decrease in size from the base to the processes top ( Fig. 4B–E View Figure 4 ). The surface of processes and their elongated apices smooth under SEM ( Fig. 5B–F View Figure 5 ). Eight to ten flat areoles around each process ( Figs 4B, C View Figure 4 , 5A, B View Figure 5 ). Areoles smooth inside under PCM/NCM ( Fig. 4B, C View Figure 4 ), but delicately wrinkled in SEM ( Fig. 5B, C View Figure 5 ). Only occasionally are the areoles underdevoloped ( Fig. 4C View Figure 4 ).

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: 18S rRNA (GenBank: MH664931 View Materials ), 1013-bp long; 28S rRNA ( MH664948 View Materials ), 732-bp long; ITS2 ( MH666080 View Materials ), 373-bp long; COI ( MH675998 View Materials ), 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., 2014b , P. klymenki Pilato et al., 2012 and P. walteri ( Biserov, 1997 –98).

Paramacrobiotus crenatus : A morphological comparison of the neotype population with the original description, as well as with photomicrographs of the holotype, a paratype and an egg of P. crenatus , showed no morphological differences between the two species. Maucci (1991, 1996) compared P. crenatus , known just from Greenland and Russia (Irkutsk Oblast) ( Kaczmarek et al., 2005), only with the limited original description of P. areolatus View in CoL and specimens from Italy identified as ‘ P. areolatus View in CoL ’ using the original description. He differentiated P. crenatus from P. areolatus View in CoL by three characters: the absence of eyes, indented lunulae IV, and by longer egg processes. However, none of these differences can be considered valid. Specifically, Maucci (1991, 1996) did not specify whether his observation on the absence of eyes in P. crenatus was based on live or fixed individuals ( Murray, 1907, observed live animals as apart from the presence of eyes, he also described storage cells that are visible only in living specimens). This is important as we noted that eyes in the neotype population of P. areolatus View in CoL quickly dissolve in Hoyer’s medium, making this supposed difference used by Maucci (1991) questionable. Another character that allegedly differentiates these two species is the morphology of lunules on the hind legs [smooth in P. areolatus View in CoL vs. dentate in P. crenatus according to Maucci (1991)]. However, Murray (1907) notably did not mention anything about lunule morphology and they were not even presented on the drawings, either in the 1907 or in the 1910 description (peculiarly, he did not draw lunules in many other species). Thus, the morphology of hind lunulae in P. areolatus View in CoL is unknown and the supposition that lunulae in this species are smooth is unjustified. However, the fact that P. areolatus View in CoL was originally described as a variety of Macrobiotus echinogenitus Richters, 1904 View in CoL , a species with dentate lunulae, may suggest that P. areolatus View in CoL also exhibited indented lunulae (it is possible that Murray assumed that the indentation of lunulae in P. areolatus View in CoL was obvious given the species was supposed to be a variety of M. echinogenitus View in CoL and he omitted this trait). Finally, in contrast to what Maucci (1991) claims, there are no differences in egg process height between P. areolatus View in CoL and P. crenatus . Although Murray (1907) did not provide measurements of egg processes, he did provide maximal egg bare and full diameters (95 μm and 180 μm, respectively) and they are in line with those provided by Maucci (1991) for P. crenatus (92–128 μm and 136–180 μm, respectively). Importantly, all these also correspond well with the ranges in the neotype population (63–128 μm and 149–196 μm, respectively; see also Table 4). Moreover, it should be noted that a morphological comparison between the neotype population with two other P. areolatus View in CoL populations reported from Svalbard by Zawierucha et al. (2015) and a population from Bjørnøya analysed in the present study, confirmed their identifications as P. areolatus View in CoL , which suggests that the species is common in the European Arctic. Considering all the above mentioned arguments, it seems that Maucci (1991) apparently found a population of P. areolatus View in CoL that he described as P. crenatus using erroneous premises. Therefore, we propose to designate P. crenatus ( Maucci, 1991) as a junior synonym of P. areolatus ( Murray, 1907) View in CoL .

Of all the other species mentioned above, P. areolatus View in CoL s.s. differs specifically from:

• 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., 2014b), 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:

• 18S rRNA: 2.2–4.2% (3.7% on average), with the most similar being an undetermined P. areolatus complex species from Italy ( MH664937 View Materials ) and the least similar being an undetermined P. richtersi complex species from Kenya ( EU038081 View Materials );

• 28S rRNA: 3.4–8.2% (6.9% on average), with the most similar being an undetermined P. areolatus complex species from Portugal ( MH664960 View Materials ) and the least similar being Paramacrobiotus lachowskae Stec et al., 2018c from Colombia ( MF568533 View Materials );

• ITS2: 7.5–26.8% (20.9% on average), with the most similar being P. areolatus complex species from Italy ( MH666085 View Materials ) and the least similar being P. lachowskae from Colombia ( MF568535 View Materials );

• COI: 16.9–24.3% (21.7% on average), with the most similar being an undetermined P. areolatus complex species from Portugal ( MH676013 View Materials ) and the least similar being Paramacrobiotus arduus Guidetti et al., 2019 from Italy ( MK041020 View Materials –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 View Figure 6 ).

Regardless of the employed genetic species delimitation method (PTP or ABGD), the ITS2 analysis recovered ten and the COI suggested 13 putative species ( Fig. 7 View Figure 7 ). The difference in the number of genetically delineated species resulted from two groups of populations. In the ITS2 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 ITS2 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 ITS2 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 ITS2 analysis are recovered as a single species.

EXPERIMENTAL CROSSES

As expected for such divergent genetic distances in ITS2 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 ITS2 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 ITS2 sequence chromatogram ( Fig. 8 View Figure 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.