Metaradiophrya speculorum, Obert & Vďačný, 2021

Obert, Tomáš & Vďačný, Peter, 2021, Multi-gene phylogeny of the subclass Astomatia (Protista: Ciliophora) refreshed with two rare astome ciliates from the digestive tube of endogeic earthworms, Organisms Diversity & Evolution (New York, N. Y.) 21 (1), pp. 59-77 : 70-73

Comparison of Metaradiophrya speculorum with Metaradiophrya falcifera

Metaradiophrya speculorum is morphologically most similar to M. falcifera ( Stein 1861) , a highly conspicuous species that has been, however, not reported for almost half a century. Stein (1861) originally described M. falcifera under the name Hoplitophrya falcifera Stein 1861 . Cépède (1910) mentioned this values for maximum likelihood conducted in IQTrees as well as posterior probabilities for Bayesian inferences conducted in Phycas and MrBayes were mapped onto the 50% majority rule Phycas tree. Dashes indicate bootstrap values below 50% or posterior probabilities below 0.50. Sequences in boldface were obtained during this study. For GenBank accession numbers, see Supplementary Table S2. The scale bar denotes two substitutions per one hundred nucleotide positions species under its original name, but his description was based only on documents provided by E. Maupas and not on his own observations. Heidenreich (1935) transferred H. falcifera to a new genus, Metaradiophrya , which was a nomen nudum because established without designation of type species ( Aescht 2001). Jankowski (2007) re-established the genus Metaradiophrya , fixed M. lumbrici ( Dujardin 1841) as the type species, and validly transferred H. falcifera into the genus Metaradiophrya . The present molecular data support that M. speculorum belongs to the genus Metaradiophrya ( Figs. 5 , 6 , 7 , and 8).

Metaradiophrya speculorum and M. falcifera share a similar body size, nuclear apparatus, contractile vacuole pattern and, most importantly, the peculiar arched skeletal ridge. More specifically, M. speculorum is 135 × 105 μm in size, and populations of M. falcifera have a size of 125 × 85 μm in the morphostatic specimens while 75 × 65 μm directly after the division ( Cépède 1910), 120 –150 × 70–90 μm ( Heidenreich 1935), 68–124 × 57–93 μm ( Williams 1942), 120 × 70 μm ( de Puytorac 1954), 120–170 × 60–120 μm ( de Puytorac 1960), and 78–135 × 32–82 μm ( Lom 1961). Both species exhibit a single micronucleus and an elongated macronucleus whose anterior portion is slightly bent leftwards forming a Γ- shaped pattern. They also possess two staggered rows of contractile vacuoles, one row extends along the right side and the other row along the left side of the macronucleus. However, M. speculorum differs from M. falcifera by the orientation and length of the skeletal ridge. In the new species,

onto the 50% majority rule IQTree. Dashes indicate posterior probabilities below 0.50. Sequences in boldface were obtained during this study. For GenBank accession numbers, see Supplementary Table S3. The scale bar denotes four substitutions per one hundred nucleotide positions the ridge extends rightwards from the point where shorter and longer hook arms adjoin and terminates at the level of the posterior end of the hook. In M. falcifera , the ridge extends leftwards and terminates about in the mid-body. The course of the ridge was explicitly described by Stein (1861) and confirmed by all subsequent researchers by drawings and sometimes also by photographs ( de Puytorac 1954, 1960, 1972; Heidenreich 1935; Lom 1961; Williams 1942). Moreover, M. speculorum is distinguished from M. falcifera also by the number of ciliary rows. The former species has about 170 ciliary rows in total in the mid-body, specifically, 95 on the ventral side and 75 on the dorsal side. De Puytorac (1960) mentioned about 215 ciliary rows in total, 149–154 on the ventral side, and 61–66 on the dorsal side in the latter species. Similarly, Lom (1961) stated that there are 205–221 ciliary rows in total, but there were about 77 rows on the ventral side while about 137 rows on the dorsal side. This conspicuous

50% majority rule IQTree. Nodes without support values had bootstrap values below 50% and were not recognized by at least one Bayesian method. Dash indicates posterior probabilities below 0.50. Sequences in boldface were obtained during this study. For GenBank accession numbers, see Supplementary Table S4. The scale bar denotes seven substitutions per one hundred nucleotide positions difference between the numbers of ciliary rows on the ventral and dorsal sides indicates that populations studied by de Puytorac (1960) and Lom (1961) might have belonged to different M. falcifera -like species.

Metaradiophrya speculorum very likely differs from M. falcifera also by the earthworm host. However, the matter is very complex and we also cannot exclude that various M. falcifera -like species were mixed, as indicated by the comparatively wide range of body size and the number of ciliary rows (see above). This taxonomic problem might have led to an overestimation of the host range of M. falcifera . The type host of M. speculorum is Aporrectodea tuberculata , which belongs to the Aporrectodea caliginosa complex ( Pérez-Losada et al. 2009). Stein (1861) discovered M. falcifera in the intestine of the earthworm “ Lumbricus anatomicus ” (very likely Aporrectodea caliginosa ( Savigny 1826) according to Heidenreich 1935 and de Puytorac 1960). However, according to Christoffersen (2011), reports of L. anatomicus could be assigned also to two other endogeic lumbricids, Allolobophora chlorotica ( Savigny 1826) and Aporrectodea rosea . Thus, the type host of M. falcifera is unclear. Heidenreich (1935) mentioned that M. falcifera is very rare, and he detected it only once in a single specimen of the anecic earthworm Lumbricus terrestris . Williams (1942) reported M. falcifera from the endogeic earthworms “ Helodrilus chlorotica ” (= Allolobophora chlorotica ) and “ H. caliginosus (?)” (= Aporrectodea caliginosa ). Lom (1961) detected M. falcifera in the intestine of “ Allolobophora caliginosa ” (= Aporrectodea caliginosa ). He also found it in two new host organisms, the endogeic “ Octolasium lacteum ” [= Octolasion lacteum Örley 1885 )] and the epigeic Lumbricus rubellus Hoffmeister 1845 . De Puytorac (1954) reported M. falcifera also from the earthworm “ Allolobophora caliginosa ” and de Puytorac (1960) from the endogeic “ Octolasium cyaneum ” [= Octolasion cyaneum ( Savigny 1826) ]. Dixon (1975) found M. falcifera in Allolobophora chlorotica and Aporrectodea caliginosa . However, detailed morphological and molecular data from ciliates identified as M. falcifera isolated from various ecological groups of earthworms are needed to confirm the determination and to test for the validity of the comparatively huge host range.

Figures

Fig. 5 Phylogenetic tree based on the 18S rRNA gene, showing the systematic positions of Metaradiophrya speculorum sp. n. and Maupasella mucronata within the class Oligohymenophorea. The subclass Peniculia was used to a posteriori root the tree. Bootstrap values for maximum likelihood conducted in IQTrees as well as posterior probabilities for Bayesian inferences conducted in Phycas and

Fig. 6 Phylogenetic tree based on the 18S rRNA gene, showing the systematic positions of Metaradiophrya speculorum sp. n. and Maupasella mucronata isolated from endogeic lumbricid earthworms. The phylogenetic tree suggests that the evolution of endosymbiotic astome ciliates has proceeded through specialization to ecological groups of their host earthworms. Haptophrya planariarum and Dexiotricha spp. were used to a posteriori root the tree. Bootstrap

Fig.7 Phylogenetic tree based on the 18S rRNA gene and the ITS region, showing the systematic positions of Metaradiophrya speculorum sp. n. and Maupasella mucronata within the subclass Astomatia. The tree was a posteriori rooted according to Fig. 6. Bootstrap values for maximum likelihood conducted in IQTrees as well as posterior probabilities for Bayesian inferences conducted in Phycas and MrBayes were mapped