Зоологический журнал, 2021, T. 100, № 3, стр. 256-263

MATERIALS AND METHODS

Planarians representing different species were collected by E.P. Zaytseva in July 2016 from the depth of 5–7 m in the Boguchany Reservoir. Histological sections were made following the standard protocol. The slides were stained according to Mallory (Romeis, 1953). Schematic reconstructions of the copulatory organ were performed using the serial photos obtained under the Axio Imager M2 Carl Zeiss microscope. The histological reconstructions were based on the medial sections of adult planarian specimens; the slides and samples are stored at the Department of Zoology and General Biology, Kazan Federal University (Table 1).

Phylogenetic analysis was carried out on Baikal planarians from six genera (12 species in total). The planarian specimen identified as a representative of the family Planariidae was taken as an outgroup. Species names and sampling sites are given in table. DNA extraction, PCR of the ITS1-5.8S-ITS2 ribosomal cluster region, and sequencing were performed as described previously (Porfiriev et al., 2018).

The nucleotide sequences were aligned using the MAFFT v. 7.42 (Katoh, Standley, 2013) offered by EMBL-EBI service (Madeira et al., 2019). The genetic distances were calculated in the MEGA v. 7 (Kumar et al., 2016) using the K2P model (Kimura, 1980); the phylogenetic relationships were analyzed with the help of NJ (Saitou, Nei, 1987) in the MEGA v. 7 and GTR + I + G model in the MrBayes v. 3.1.2 (Huelsenbeck, Ronquist, 2001). The significance of nodes in the tree was tested by posterior probability and bootstrap test (Felsenstein, 1985).

RESULTS

Genus Baikalobia Kenk 1930

Material: four series of sagittal sections (cop1–cop4), planarians sampled on July 7, 2016, sample no. 7, Boguchany Reservoir, section 1, depth 5 m.

Based on the external (Fig. 1) and internal features, these triclads were identified as B. raddei. The dorsal body part of B. raddei is dark brown. The structure of the copulatory organ is characteristic of B. raddei: the canal of copulatory bursa runs alongside the copulatory organ; the latter has a single adenodactyl oriented posteriorly and slightly laterally, which is distinguishing for this species (Fig. 2). B. raddei was earlier found near the mouth of the Angara River, on stones in the offshore area and at small depths (Porfirieva, 1977). Therefore, our data suggest that the habitat range of this species is significantly larger.

Fig. 1.

External appearance of Baikalobia raddei: А – dorsal view, B – ventral view.

Fig. 2.

Baikalobia raddei: А – copulatory organ reconstruction, B – a series of sagittal sections through copulatory organ.

Phylogenetic analysis

A total of 20 sequences were obtained for the ITS1-5.8S-ITS2 region (982–1217 bp). The sequences were deposited in the NCBI database under the accession numbers MK426771-MK426791. The analysis also included the sequences for B. elochinensis Porfiriev et Timoshkin 2018 (E1) and B. guttata (Gerstfeldt, 1858) (EL1) from our previous research (KY848669 and KY848669, respectively; Porfiriev et al., 2018). After the alignment, 903 positions were analyzed without indels; 401 positions were found to be informative. The genetic distances (d) within the non-endemic genus Bdellocephala and between the remaining endemic genera were in the range of 0.01–0.1 and 0.007–0.13, respectively. Therefore, the intraspecific differences in the genus Bdellocephala are generally at the same level as those between species of different genera of other Baikal planarians. If the sequences for Bdellocephala planarians are compared to the latter ones, the d values increase up to 0.35 on average. The genetic distances between B. angarensis planarians of Baikal and Boguchany origin were zero. B. raddei from the Boguchany Reservoir was closest to B. copulatrix (d = = 0.007) among all Baikal planarians. Other representatives of this genus (B. elochinensis, B. guttata, and B. variegata (Korotneff 1912)) were more different from B. raddei (d = 0.021, 0.026, and 0.041, respectively).

The inferred phylogenetic tree is shown in Fig. 3 (Fig. 3). All the studied planarians fall into two distinct clusters – the genus Bdellocephala and other endemic genera. The genus Bdellocephala is represented in our study by three species: B. melanocinerea (Korotneff 1912) and B. angarensis, including a specimen from the Boguchany Reservoir, are closely related; B. baikalensis (Sabussov 1903) forms a separate branch of this cluster. Another cluster contains several clades with high support values – M. stringulatus; the clade of giant planarians, including B. valida (Korotneff 1912) and R. pulvinar (Grube 1872); A. decoloratus; and planarians of the genus Baikalobia. The relationships between these clades are not clear. In the clade of Baikalobia, B. raddei found in the Boguchany Reservoir is closely related to B. copulatrix.

Fig. 3.

Phylogenetic tree of Baikal planarians reconstructed from the sequences of the ribosomal cluster ITS1-5.8S-ITS2 (903 bp). The individuals found in the Boguchansky reservoir are marked with a circle. The nodes of the tree indicate the values of a posteriori probability (MrBayes v.3.1.2) and bootstrap test (MEGA 7). The nodes without values have low support. The scale bar shows genetic distances. Full names of species are given in Table 1.

DISCUSSION

The findings of Baikal planarians outside the lake margins have been reported by a number of researches (Rubtsov, 1928; Zabusova-Zhdanova, 1955; Golyshkina, 1963, 1966, 1969, 1970; Kozhov, 1972; Porfirieva, 1973, 1977) during the 20th century. From this perspective, planarian species belonging to the genera Baikalobia, Archicotylus, and Microarchicotylus, which were registered in the Boguchany Reservoir, are of special interest.

The migration of these triclads can be explained by the fact that the hydrochemical regime of the Angara River in the past was generally the same as that of Lake Baikal (low water temperatures, high concentrations of dissolved oxygen). The geological age of the Angara flow is estimated around 60 000 years (Kononov, Mats, 1986): it can be taken as the time when the above-mentioned planarian species of Baikal origin must have distributed downstream along the river bed.

Particular attention should be paid to B. raddei, for which the morphological (Figs. 1, 2) and molecular (Fig. 3) data were obtained. B. raddei was first described by Zabusov (1911) from G.I. Radde’s collection. However, the exact area of finding was not specified. It was only mentioned that the specimens were sampled in Eastern Siberia. Later, Porfirieva (1977) recorded this species near the headwaters of the Angara River, where it occurred regularly until the late 1960s (the latest sections of B. raddei are stored in the collection of the Zoological Museum of Kazan Federal University, the specimens were sampled in the mouth of the Angara River on June 3, 1969 by R. Mansurov). The problem of the origin of B. raddei is controversial. It has been still widely discussed whether B. raddei first appeared in the lake and then left it or its ancestor evolved in the river system after the migration. N.A. Porfirieva assumed that the ancestral form of all Baikalobia planarians resembled B. raddei, had a single adenodactyl (similarly to the modern species), and settled in Lake Baikal separately from other Baikal triclads (Porfirieva, 1977). Our molecular data suggest that B. raddei is similar to B. copulatrix with the number of adenodactyls ranging from 7 to 9, being unrelated to the ancestral form, from which all Baikalobia planarians were later derived (Fig. 3). Previously, it was revealed that Baikalobia planarians evolved from adenodactyl-deprived dendrocoelids inside Lake Baikal (Kuznedelov et al., 1996).

Bdellocephala representatives inhabiting the same water body are widespread in the entire Palearctic region. The fact that the ITS1-5.8S-ITS2 sequences of B. angarensis of Baikal and Boguchany origin match each other perfectly (Fig. 3) can be attributed to the conservatism of the analyzed region of the ribosomal cluster, because there is no evidence of intraspecific polymorphism within it. Another reason can be the young age of B. angarensis population from the Boguchany Reservoir. The natural migration of B. angarensis from Lake Baikal along the Angara River bed probably ended after the Irkutsk HPS was commissioned in 1956.

As for the evolution of other Baikal planarians, our phylogenetic reconstruction based on the ITS1-5.8S-ITS2 region is generally consistent with that one for the short 18S rRNA fragment (Kuznedelov et al., 1996, 2000) and the actin gene region (Koroleva et al., 2013). Two large clusters can be distinguished – Bdellocephala (cluster I) and other Baikal planarians (cluster II), among which the above-described Baikalobia form a separately evolving clade (Fig. 3). The highest similarity was observed between B. copulatrix, B. raddei, B. elochinensis, and B. guttata, whereas B. variegata holds a basal position in this genus. The giant B. valida (the largest freshwater planarian in the world reaching 20–40 cm in length (Timoshkin, 1994)) and R. pulvinar planarians living in deep waters nested in a separate clade close to other small planarians of cluster II, which again confirms the hypothesis that giant planarians originated from small-sized littoral forms (Porfirieva, 1977; Timoshkin, 1994). Some giant planarians have creeping folds and numerous compound suckers; they are also characterized by a more complex structure of the muscular-glandular sac and copulatory organ (Porfirieva, 1977; Timoshkin, 1994). It is interesting that M. stringulatus, which is characterized by “dwarfness” (5–6 mm in length), bright coloration, and specific structure of the copulatory organ (Porfiriev, Timoshkin, 2016), resides in the same cluster (Fig. 3). Such diverse morphological features could have occurred in the process of evolution as result of adaptive or non-adaptive radiation (Czekanski-Moir, Rundell, 2019).

Since the morphological evolution of Baikal planarians proceeded in different directions, new planarian species – capable of expanding their habitat range beyond the lake – emerged, as seen in the case with the subendemics of Baikal origin. Despite the construction of the Angara chain of power plants, a barrier hampering the distribution of Baikal endemics downstream of the river, the populations of subendemics from Lake Baikal are still resitered in the Boguchany Reservoir.

Abbreviations in figures: ad – adenodactyl, bp – bulb of penis, ca – common atrium, cb – copulatory bursa, ccb – canal of copulatory bursa, cod – common oviduct, gp – gonopore, m – mouth, ma – male atrium, ph – pharynx, pp – penis papilla, sd – seminal duct.