The diversity of the Baikal lineage of Hydra oligactis Pallas, 1766: molecular and morphological evidence

Abstract In this paper, molecular analyses of Baikal hydras from the ‘oligactis group’, based on COI and ITS1–5.8S–ITS2, and morphological analysis of their holotrichous isorhizas, were performed. Low genetic diversity and shared haplotypes were found between Hydra oligactis Pallas, 1766 and Hydra baikalensis Swarczewsky, 1923 specimens, which is evidence of the mixing of these lineages. Genetic distances among all Baikal hydras (0.006) were less than the interspecific distances of other hydras. The size of hydras and proportions of their holotrichous isorhizas varied depending on microhabitat and environmental conditions. Our combined molecular and morphological approach proves that H. baikalensis is synonymous with H. oligactis


Introduction
Hydra is a member of the ancient phylum Cnidaria, class Hydrozoa, order Hydroida, family Hydridae. Freshwater representatives of Hydra inhabit virtually all zoogeographical regions except the Pacific Ocean Islands and Antarctica (Jankowski et al. 2008), living in lenthic and lothic waters such as ponds, lakes, and rivers, and prefering meso-eutrophic conditions. Lake Baikal is one of the deepest (1642 m) oligotrophic freshwater lakes in the world, but the habitat conditions in its bays are close to mesoeutrophic. Climate warming is currently considered as a key factor capable of stimulating the eutrophication of freshwater ecosystems (Cohen et al. 2016;Paerl and Otten 2013). According to the World Meteorological Organization, 2010 was one of the hottest years in the history of meteorological observations. Global climate changes are affecting the ecosystem of Lake Baikal as well. Structural rearrangements of the phytoplankton community in the pelagic zone of the lake (Bondarenko et al. 2019) and changes in the structure of bottom phytocenoses of the coastal zone (Kravtsova et al. 2014) have been detected. Moreover, the propagation of Palearctic species of invertebrates such as Lymnaea mollusks (Gastropoda, Lymnaeidae) and the caddis fly Apatania majuscula McLachlan, 1872 (Insecta, Trichoptera, Apataniidae) have been observed in Lake Baikal (Rozhkova et al. 2018). In addition, the mass death of the endemic species of Porifera sponges (Denikina et al. 2016;Khanaev et al. 2018) has also been recently observed. On the contrary, representatives of Hydra have begun to play a significant role along the open coasts of Lake Baikal. Previously, representatives of hydras were found in small numbers in the coastal zone of Lake Baikal. The first record of the mass development of Hydra oligactis Pallas, 1766 was in the 2000s (Stepanyants et al. 2003). Now, Hydra is abundant along the open coasts of all three basins of Lake Baikal (Peretolchina et al. 2018a).
Of the 80 Hydra species names described in the world only 12 to 15 are valid (Jankowski et al. 2008). The uncertainty of the position of individual hydra taxa results from the complexity of their identification, due to their primitive structure and limited number of reliable diagnostic features. The species diversity of Hydra within the morphological boundaries of Lake Baikal is not high; there are four species of Hydra reported from the Baikal Region: H. oligactis Pallas, 1766 and Hydra baikalensis Swarczewsky 1923, as well as the rare Hydra circumcincta Schulze, 1914and Hydra oxycnida Schulze, 1914(Peretolchina et al. 2018b. Hydra baikalensis, H. oligactis and H. oxycnida belong to the 'oligactis group' (Hemmrich et al. 2007;Martínez et al. 2010;Schwentner and Bosh 2015). However, the morphological descriptions of H. baikalensis and H. oligactis are very similar in both polyp morphology and holotrichous isorhiza structure (Swarczewsky 1923;Anokhin 2002).
The aim of our study was to verify the identity of H. oligactis and H. baikalensis using both morphological and molecular data.
Morphological studies of the cnidome (stenoteles, desmonemes, holotrichous and atrichous isorhizas) were carried out using an Olympus CX22 microscope with 1000fold magnification under oil immersion. Morphometric measurements of holotrichous isorhizas were made with the Image-Pro program. Hydra specimens were identified according to keys provided by Schuchert (2010Schuchert ( , 2018 and Anokhin (2002).
DNA was extracted from a single live or fixed individual as described by Doyle and Dickson (1987). Gene fragments of mitochondrial cytochrome c oxidase subunit I (COI) were amplified using standard Folmer primers for invertebrates (Folmer et al. 1994), and the internal transcribed spacer 1, 5.8S ribosomal DNA and internal transcribed spacer 2 (ITS1-5.8S-ITS2) were amplified using primers published in White et al. (1990).
All PCR reactions were performed in a final volume of 15 μL using 2-Red PCR mix (10 X PCR buffer, 50 mM MgCI2 and 0.02 unit/ μL Taq DNA polymerase). The PCR amplification conditions were as follows: denaturation at 94 °C for 5 min, 30 cycles at 94 °C for 30 sec, 50 °C for 45 sec, 72 °C for 2 min, and a final elongation step at 72 °C for 10 min. Direct sequencing of forward sequences was performed using an ABI 3130 automated sequencer (Research and Production Company "SYNTOL", Moscow, Russia).
The DNA sequences obtained were aligned using default settings in CLUSTAL W (Thompson et al. 1994), implemented in BIOEDIT v.7.2.5 (Hall 1999) and optimized by eye. The resulting COI alignment was translated to check for the absence of stop codons. To confirm species identity, we compared our sequence dataset to published orthologous sequences from other members of Hydra and analyzed genetic distances using MEGA v.6 (Tamura et al. 2013).
Phylogenetic analyses were performed using MRBAYES v.3.2 (Ronquist and Huelsenbeck 2003). The datasets for the COI and ITS1-5.8S-ITS2 fragments (Table 1) consisted of unique haplotypes of Baikal hydras produced for this study and specimens (three species from the 'oligactis group': Hydra canadensis Rowan, 1930, H. oligactis, H. oxycnida, and a representative of H. circumcincta, as outgroup) retrieved from GenBank. To estimate the posterior probabilities of the phylogenetic tree, we used 20,000,000 generations of Metropolis-coupled Markov chain Monte Carlo simulation (two runs with four chains). We used the JMODELTEST v.2.1 (Darriba et al. 2012) to determine the substitution models for the two genes separately. The best-fit model for phylogenetic analysis in the case of COI was HKY, and in the case of ITS1-5.8S-ITS2 it was GTR+I+G. We constructed a majority-rule (50%) consensus tree following 25% burn-in of all sampled trees.

Molecular analysis
In total, we produced 30 COI sequences (603 bp long) and 30 ITS1-5.8S-ITS2 sequences (up to 710 bp long). Inspection of the Baikal hydra sequences revealed nine unique haplotypes for the COI gene fragment (shared haplotypes were found in all sampling localities) and three unique haplotypes for the ITS1-5.8S-ITS2 sequences.
The consensus tree topology based on COI (27 unique haplotypes) indicated that Baikal H. oligactis and H. baikalensis did not form separate clades, but instead were clustered together with representatives of Hydra robusta Itô, 1947 from Japan and China and H. oligactis from Japan and Europe, forming a neighboring clade with the majority of hydras from Western Europe and North America (Fig. 1A). The consensus phylogenetic tree based on ITS1-5.8S-ITS2 (14 unique haplotypes) showed that differentiation between hydras from Europe + North America and Asia was not evident (Fig. 1B).
Mean pairwise p-distances based on COI sequences of Hydra species are given in Table 2. Intraspecific genetic distances of Hydra varied from 0.010 to 0.040, whereas interspecific distances were about 0.100. The mean genetic distance between Baikal   (Table 2).

Morphological analysis
In our collections, hydras with a length/width ratio (L:W) of holotrichous isorizas > 2 were identified as H. oligactis and hydras with L:W ≤ 2 were identified as H. baikalensis, following Schuchert (2010Schuchert ( , 2018 and Anokhin (2002). However, morphological analysis of polyps did not reveal specific features between Baikal hydras and H. oligac-tis, and the main diagnostic trait (L:W of holotrichous isorizas) varied from 1.85 to 2.46 (Table 3), depending on the environment, in different sites of Lake Baikal. Hydra specimens with a L:W of holotrichous isorizas ≤ 2.0 were collected at a depth of 15-18 m, where the water temperature was low (6-7 °C, near the bottom). Hydra specimens with holotrichous isorizas ≥ 2.0 were collected in shallow water from 1-5 m, where the water temperature reaches 16-24 °C in summer (Kozhov 1962).

Discussion
According to Schwentner and Bosch (2015), the 'oligactis group' includes H. oxycnida, H. canadensis, H. oligactis and H. robusta (possibly synonymous to H. oligactis). On the one hand, low genetic distance between the latter two species could be a consequence of shared ancestral polymorphism. On the other hand, the results may be due to population genetic variability of H. oligactis (= H. robusta). It is known that genetic polymorphism of species with wide geographic distributions is higher than that of species with restricted ranges, where genetic diversity decreases from the center of distribution to range boundaries (Brown 1984;Schuchert 2014). We suppose that H. oligactis is a widely distributed species with genetic (population) structure ( Figure 1A, B). The "Baikal" clade includes not only regional haplotypes but also haplotypes of H. oligactis from Europe, China and Japan (Fig. 1A). We believe that this level of genetic diversity corresponds to population differences, not to interspecific ones. Thus, according to our molecular phylogenetic study, all the haplotypes belong to the same species, H. oligactis (H. oligactis = H. robusta = H. baikalensis).
In Lake Baikal, the type locality of the endemic H. baikalensis is Chivyrkuyskiy Bay (Swarczewskiy 1923). This species has also been reported from Listvennichniy Bay, the littoral zone near Bolshoy Ushkaniy Island, Bolshie Koty Bay, and in small lakes along the Bolshie Koty River. Hydra oligactis occurs in the same areas and was also reported from Dagarskaya Bay, Mukhor Bay (Stepanyants et al. 2006), and Barguzinskiy Bay (Peretolchina et al. 2018a).
Previous researchers (Schulze 1927;Anokhin 2002; Jankowski and Anokhin 2019) distinguished H. oligactis from H. baikalensis mostly based on differences in the proportions of their holotrichous isorhizas. However, the differences in the length/width ratio of holotrichous isorhizas in H. baikalensis and H. oligactis are within the range of intraspecific variability (Table 3). Therefore, this morphological feature cannot be used as a diagnostic criterion for H. baikalensis. It is known that proportions and relative sizes of the nematocysts vary considerably depending on the stage of development of the nematocysts (Weill 1934) and on environmental factors (Östman 1987;Itô 1951). In our case, the size of the hydras and the proportions of the holotrichous isorhizas varied depending on microhabitat and environmental conditions. That is, specimens sampled in the open littoral of Lake Baikal were larger and their holotrichous isorhizas were shorter and slightly thicker (8-9 μm, length/width ratio ≤ 2), whereas hydras from shallow depths were smaller but their holotrichous isorhizas were longer and thinner (9-10 μm, length/width ratio > 2) ( Table 3). In addition, the packing of stinging threads in the holotrichous isorhizas of both Baikal H. baikalensis and H. oligactis was the same. Moreover, the species H. baikalensis and H. oligactis are similar in the number and morphology of chromosomes, in the symmetrical structure of the karyotype, and in C-heterochromatin localization. They only differ in the length ratio of the 1 st and 15 th pairs of chromosomes (Anokhin 2002). We suppose that the length ratio of chromosomes is a distinctive feature but may be not sufficient to distinguish H. baikalensis as an independent species.
In addition, molecular phylogenetic analyses did not reveal any differences between H. baikalensis and H. oligactis specimens. Genetic distances among all Baikal hydras (0.006) were less than the interspecific distances of other hydra (Table 2). Moreover, we found shared haplotypes among representatives of Baikal H. baikalensis and H. oligactis. The sympatric occurrence of H. oligactis and H. baikalensis and the low level of their genetic distances (revealed using both mitochondrial and nuclear markers) do not allow us to consider these two groups as separate species.
Thus, the morphological characteristics, patterns of haplotype diversity, and the results of phylogenetic analyses lead us to consider H. baikalensis as a synonym of H. oligactis.  Swarczewsky, 1923 Diagnosis. The Baikal Hydra is large, typically with 5-7 long tentacles, and a more or less distinct peduncle. The cnidome includes four types of nematocysts: stenoteles, holotrichous isorhizas, atrichous isorhizas and desmonemes (Figs. 2, 3). Holotrichous isorhizas are elongated, with a length/width ratio of about 2, and with thread-forming longitudinal, irregular coils inside the capsule. Other types of nematocysts are of a size and shape typical of other hydras. Holotrichous isorhiza measurements are summarized in Table 3, with photographs in Fig. 2. Distribution. Baikal region: Port Baikal, Ulanovo, Listvennichny Bay, Bolshie Koty, Varnachka, Sobolev Cape, Turali Cape, Elokhin Cape, Mukhor Bay, Posolsky Sor Dagarskaya Bay, littoral zone near Bolshoy Ushkaniy Island, small lakes along the Bolshie Koty River, and Lake Kuzmikhinskoye (Artificial reservoir near the Angara River). This species is also widespread and common on the entire European continent, including the British Isles and Iceland as well as Russia and North America (Hyman 1930;Holstein 1995;Stepanyants et al. 2006).