Interstitial telomere sequences in chromosomes of Baikal planarians

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1. Introduction

Internal telomeric sequences (ITSs) are telomeric DNA located anywhere on the chromosome except the terminal regions (Meyne et al., 1990). They can arise for several reasons, including as a result of chromosomal rearrangements during genome evolution, repair of double-strand DNA breaks, and the introduction of extrachromosomal telomeric DNA into chromosomes and its amplification (Bolzán and Bianchi, 2006; Ruiz-Herrera et al., 2008; Bolzán, 2012). As a rule, ITSs are not associated with telomere functions, unless one takes into account their specific role in maintaining genome plasticity, since they are known to induce mutations and are localized at fragile sites where chromosome breaks occur (Moore et al., 2018; Lin and Yan, 2008).

There are four groups of ITSs: short ITSs, subtelomeric ITSs, fusion ITSs (resulting from the fusion of chromosomes) and heterochromatic ITSs (Bolzán, 2017). They differ in size, location and nucleotide environment, and also arise through different mechanisms. Short ITSs have a size of 100-120 bp and can be surrounded by both unique sequences and SINE, LINE, LTR retrotransposons. The length of subtelomeric ITSs is measured in hundreds of nucleotides and may include degenerate telomeric repeats. The ITSs fusions have a head-to-head orientation and are flanked by subtelomeric DNA, indicating instances of two chromosomes joining at telomeric regions. Their sizes can vary from several kilobases (kb) to several tens of kb. Heterochromatic ITSs are the largest sequences, up to several hundred kb in size. They are often found in pericentromeric regions, but can also occur on chromosome arms and also form microchromosomes (Bolzán, 2017).

The sensitivity of conventional Fluorescence in situ hybridization (FISH) allows the visualization of sequences 1000 bp or longer (Poon et al., 1999), i.e. it is difficult to detect short and subtelomeric ITSs using this method. However, this method is capable of recording the most significant chromosomal rearrangements in the evolution of the karyotype, which can affect telomeric regions. In the chromosomes of vertebrates, ITSs larger than 1000 bp are quite common and are located mainly near centromeres (Meyne et al., 1990; Bolzán et al., 2017; Vicari et al., 2022). Usually, this is a consequence of Robertsonian translocations (Slijepcevic, 1998), although such chromosomal mutations do not always lead to the appearance of ITSs (Souza et al., 2016). ITSs longer than 1000 bp are not so widespread among invertebrates (Vítková et al., 2005; Traut et al., 2007; Vicari et al., 2022). For example, among mollusks, such ITSs were found in only two of 23 species studied in this regard (Nomoto et al., 2001; Godwin et al., 2012). Among flatworms, FISH data with telomeric probes are known for 23 species, most of them parasites (Table 1), and ITSs were found only in the sex chromosomes of two trematode species (Hirai, 2014) and one cestode species (Špakulová et al., 2019). ITSs have not yet been identified in free-living flatworms.

 

Table 1. The analyzed species with/without ITSs and the sequenced 18S rRNA gene

Species	2n	ITSs (FISH)	Accession number 18S rRNA (GenBank)
TRICLADIDA
Baikalobia guttata Gerstfeldt, 1858	30	yes1	KY848668.1
B. variegata Korotneff, 1912	30	yes1	OR758633.1
Polycelis tenuis Ijima, 1884	14	no2	Z99949.1
Dugesia ryukyuensis Kawakatsu, 1976	14	no2	AF050433.1 (type II)
MONOGENEA
Paradiplozoon homoion Bychowsky et Nagibina, 1959	14	no3	KY640614.1
CESTODA
Caryophyllaeus laticeps Pallas, 1781	20	no4	AJ287488.1
Caryophyllaeides fennica Schneider, 1902	20	no4	KF990172.1
Nippotaenia mogurndae Yamaguti et Miyata, 1940	28	no4	AJ287545.1
Atractolytocestus huronensis Anthony, 1958	24 (3n)	yes9	OM972659.1
TREMATODA
Schistosoma mansoni Sambo, 1907	16	yes5	U65657.1
S. haematobium Bilharz, 1852	16	yes5	Z11976.1
S. japonicum Katsurada, 1904	16	no5	Z11590.1
S. sinensium Pao, 1959	16	no5	AY157225.1
Clonorchis sinensis Looss, 1907	14	no6	JF823988.1
Metorchis xanthosomus Creplin, 1846	14	no6	OK384552.1
M. bilis (Braun, 1790) Odening, 1962	14	no6	OK384551.1
M. orientalis Tanabe, 1920	-	-	JF314771.1
Opisthorchis viverrini (Poirier, 1886) Stiles & Hassal, 1896	14	no6	JF823987.1
O. felineus Rivolta, 1884 Blanchard, 1895	14	no6	MF077357.1
Bucephalus minimus (Stossich, 1887) Nicoll, 1914	14	no7	-
B. australis (Szidat, 1961) Yamaguti, 1971	14	no7	-
Monascus filiformis (Rudolphi, 1819) Looss, 1907	18	no7	-
Cercaria longicaudata Tang, 1990	16	no7	-
Bacciger bacciger (Rudolphi, 1819) Nicoll, 1914	12	no7	-
MACROSTOMORPHA
Macrostomum lignano Ladurner, Schärer, Salvenmoser, & Rieger, 2005	8	no8	FJ715306.1
ACOELOMORPHA
Hofstenia miamia Correa 1960	-	-	AM701817.1

Note: 1 - gene sequences and FISH results were gave by us; 2 - Joffe et al., 1996; 3 - Tasaka et al., 2013; 4 - Bombarová et al., 2009; 5 - Hirai et al., 2000, Hirai, 2014; 6 - Zadesenets et al., 2012; 7 - García-Souto and Pasantes, 2015; 8 - Zadesenets et al., 2016; 9 - Špakulová et al., 2019. Dash means absent of complete information.

 

We used FISH with telomeric TTAGGG probes to identify the localization of telomeric repeats in the chromosomes of two species of endemic planarians of the genus Baikalobia, an autochthonous group of Baikal Dendrocoelidae. ITSs were found in both worm species. Also in this work, we analyzed the phylogenetic relationships of flatworms taking into account ITSs in their chromosomes.

2. Materials and methods

Planarians were manually collected in the Listvyanichny Bay of Lake Baikal (51°52’02.4”N 104°49’55.2”E) in September 2011. The material was collected on the outside of stones at the depth of about 1 m. After that, worms were placed in a thermal container with Baikal water and taken to the laboratory, where they were kept until analysis. Tissues from two Baikalobia species were used for FISH: B. guttata Gerstfeldt, 1858 and B. variegata Korotneff, 1912 (3 individuals of each species). Species included in the phylogenetic analysis are presented in Table 1.

2.1. Fluorescence in situ hybridization (FISH)

Chromosome preparations were prepared from homogenized worm tissues. Tissues were placed in 0.56% KCl, minced and left at 37°C for 15 min. Then they were fixed with a mixture of methanol and acetic acid (3:1), kept at +5°C for 15 minutes, centrifuged, the supernatant was removed, and the procedure was repeated three times. A cell suspension was dropped onto glass slides cooled to -20°C over water vapor (70-80°C) and dried for 10 minutes. Before hybridization, the preparations were kept at room temperature for several days.

The telomeric probe was generated by template-free PCR (Ijdo et al., 1991) and labeled with Bio-11-dUTP by PCR with primers for telomeric regions. FISH of the telomere probe on the preparations was carried out in accordance with the protocol (Joffe et al., 1998) with some modifications. After washing in 1× PBS containing 50 mM Mg2+, the preparations were treated with 0.12% trypsin for 20 s. Next, the preparations were fixed in 0.5% formaldehyde and 1× PBS for 10 min, washed in 2× SSC, and dehydrogenated in ethanol. The hybridization mixture (20 μl) contained 50% formamide, 2xSSC and a telomeric probe. Before hybridization, the mixture was denatured for 5 min at 96°C, cooled in ice, and applied to the preparation. Hybridization proceeded overnight at 42°C. Detection of the biotinylated probe was carried out using fluorescently labeled streptavidin (Streptavidin-Cy3, Sigma, USA). The preparations were stained with DAPI fluorochrome (4,6-diamino-2-phenylindole, 0.5 μg/ml) in Vectashield medium (Vector laboratories, UK) and analyzed on an OIympus BX51 fluorescence microscope. Chromosomes were photographed at 100x magnification (DP70 camera, X-Cite 120Q light source).

2.2. Phylogenetic analysis

DNA extraction, PCR of the 18S rRNA gene and sequencing were carried out as described in article by Porfiriev and coauthors (Porfiriev et al., 2018). The alignment of the resulting nucleotide sequences was carried out in the ClustalW1.6 program (Thompson et al., 1994). The execution of phylogenetic reconstruction was done using MrBayes 3.2.7 (Huelsenbeck and Ronquist, 2001) in accordance the GTR+G model. The calculation of Markov chains (MCMC) involved 10 000 000 generations (4 chains in parallel) and the recording of parameters every 1000 generations. The likelihood method was stabilized by using the first 25% of generations, while the rest was used to estimate the posterior probability. The reliability criterion was a posterior probability exceeding 95%. MEGA7 (Kumar et al., 2016) was also used to reconstruct trees, which was accomplished by using both Neighbor-Joining and K2P models, which were tested in 1000 replications using a bootstrap test. The implementation of graphic editing of the tree was done in FigTree version 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) and MEGA7.

3. Results

3.1. FISH

B. guttata and B. variegata, two species of Baikal planarians, were the targets of this method’s using (Fig. 1). Their haploid set is the same and amounts to 15 chromosomes, that was shown by T. M. Umylina in the 70s (Umylina, 1973; 1976; 1977). Figure 1 shows that TTAGGG repeats are present not only at the ends, but also inner of chromosomes, which indicates the presence of ITSs in the analyzed species. Moreover, in the species B. guttata they are located on one chromosome (Fig. 1 c, d), and in the species B. variegata ITSs they are probably located on two different chromosomes (Fig. 1 h).

 

Fig.1. Telomere signals (pink) in meiotic chromosomes of Baikal planarians (chromosomes stained with DAPI, blue): a, e – appearance of worms, the scale is 1 cm; b, f – 15 pairs of chromosomes; c, d, g, h – FISH shows telomeric repeats TTAGGG in B. guttata and B. variegata. Arrows point at chromosomes with ITS. Yellow asterisks indicate ITS. A number of chromosome sets is shown in figure d.

 

3.3. Phylogenetic analysis and ITSs

To carry out phylogenetic analysis, nucleotide sequences of the 18S rRNA gene were obtained for B. guttata and B. variegata; for other species of flatworms, the sequences of this gene were taken from the GenBank database (Table 1). The length of the analyzed regions after the alignment was about 2000 bp. 655 informative sites were identified. Both MrBayes 3.2.7 and MEGA7 programs produced trees of similar topology. Figure 2 shows the phylogenetic tree obtained in MrBayes. In general, representatives of different orders cluster into separate clades with high statistical support; the representative of Macrostomorpha forms a separate branch, along with the outgroup Hofstenia miamia.

 

Fig.2. 18S rRNA gene phylogenetic tree (MrBayes 3.2.7). Species with ITSs are highlighted in pink. On the right are the flatworm order names. The nodes indicate the posterior probability values. The scale shows genetic distances.

 

4. Discussion and conclusions

4.1. ITSs in parasitic and free-living flatworms

In the evolution of some groups of animals, intra- or interchromosomal rearrangements, as well as genomic mutations, played a decisive role (Trifonov et al., 2012; 2016; Dehal and Boore, 2005). In this regard, tracking chromosomal mutation markers such as ITSs makes it possible to assess the contribution of chromosomal rearrangements to speciation.

During the evolution of flatworms, numerous karyotype transformations also occurred. As in the case of nematodes and other types of invertebrate animals (Stein et al., 2003; Ghedin et al., 2007; Dubinin et al., 1936), a significant contribution of intrachromosomal rearrangements was noted for flatworms (Swain et al., 2011), which is probably due to the presence of a large number of repeating sequences in their genome, including LTR retroelements (Grohme et al., 2018). ITSs data were obtained for a small number of representatives of different orders/classes (Table 1). Based on these data, we can conclude that ITSs are not typical for the chromosomes of these animals, as for other invertebrates. Previously, ITSs were found only in parasitic flatworms (Hirai, 2014; Špakulová et al., 2019). The detection of ITSs in the sex chromosomes of parasites, in this case schistosomes, is associated with several inversions and heterochromatization (Hirai et al., 2012; Hirai, 2014). The appearance of ITSs was associated with the spread of these parasites from Asia to Africa and with subsequent speciation (Hirai, 2012). It is worth noting that schistosomes have a unique sex determination system among hermaphroditic trematodes (ZZ male, ZW female), and the sex chromosomes have undergone significant reorganization during the evolution, as evidenced by ITSs. Baikal planarians, like most flatworms, are hermaphrodites and do not have separate gonosomes. Similarly, speciation in Baikalobia worms may have been accompanied by the emergence of ITSs (Fig. 1). Free-living flatworms often have genomic mutations associated with changes in the number of chromosomes. They can be random, as in the case of the macrostomorph Macrostemum lignano, which has a high percentage of aneuploids (Zadesenets et al., 2016). In the case of planarians, the adaptive nature of changes in the number of chromosomes was revealed: with increasing latitude, the number of chromosomes also increased (Lorch et al., 2016). At the same time, changes in the number of chromosomes during the evolution of planarians accompanied speciation, for example in the genus Bdellocephala, including among the Baikal representatives (Umylina, 1971; Kuznedelov et al., 2000; Novikova et al., 2006). Concurrently, changes occurred in the morphology of chromosomes, which indicates a significant reorganization of the genome. The endemic planarians of Lake Baikal have, as a rule, 30 chromosomes with a predominance of metacentrics and submetacentrics in the karyotype (Umylina, 1973, 1976, 1977). The stability of the number of chromosomes and the rarity of telocentric and acrocentric chromosomes in this morphologically and ecologically very diverse group of triclads may indicate the predominance of intrachromosomal changes during the evolution of their genomes, as in other flatworms. Unfortunately, we do not know whether the common ancestor of all Baikal triclads had ITSs in their chromosomes or whether they appeared only during the evolution of a separate branch of Baikalobia.

4.2. ITSs and flatworm phylogeny

Early in the evolution of flatworms, an important event occurred involving the loss of centrosomes (Azimzadeh et al., 2012). This accompanied the emergence of several groups of flatworms, which are now combined into a taxon Acentrosomata. It includes four orders Tricladida, Fecampiida, Prolecithophora, Bothrioplanida and three class of parasitic worms Monogenea, Cestoda and Trematoda (Egger et al., 2015; Collins, 2017). ITSs were identified in representatives of Tricladida, Trematoda and Cestoda, but were absent in the studied representatives of other Acentrosomata, as well as in the rather distant clade Macrostomorpha (Fig. 2), indicating the independence of the pathways leading to the appearance of ITSs in free-living and parasitic flatworms. Within the order Tricladida, representatives of the two families Planariidae and Dugesiidae lack ITSs, but they appear in the family Dendrocoelidae among Baikal endemics (Fig. 1). Representatives of only this family are found in Lake Baikal.

Further study of ITSs in representatives of the family Dendrocoelidae will help to understand at what stage of evolution the genome reorganization occurred, leading to the appearance of ITSs, and whether this event was a feature of the Baikal endemics or all dendrocoelids.

Acknowledgments

The work was carried out thanks to the State Project 0279-2021-0007 (121032300180-7) “Complex studies of the nearshore zone…” and 0279-2021-0005 (121032300224-8) “Environmental transformations of basins…, as well as was supported by Russian Foundation for Basic Research (RFBR) grants №51, №12-04-32052, №13-04-01270 and №18-34-00395.

Conflict of interest

The authors declare no conflict of interest.

About the authors

A. G. Koroleva

Limnological Institute Siberian Branch of the Russian Academy of Sciences

Author for correspondence.
Email: ankor-2015@yandex.ru
ORCID iD: 0000-0002-5255-0448
Russian Federation, Ulan-Batorskaya Str., 3, Irkutsk, 664033

E. V. Evtushenko

Institute of Molecular and Cellular Biology, Russian Academy of Sciences, Siberian Branch

Email: ankor-2015@yandex.ru
Russian Federation, 8/2 Lavrentiev Avenue, Novosibirsk, 630090

E. P. Zaytseva

Baikal Museum, Russian Academy of Sciences, Siberian Branch

Email: ankor-2015@yandex.ru
Russian Federation, 1 Akademicheskaya Str., Listvyanka, 664520

A. G. Porfiriev

Institute of Fundamental Medicine and Biology, Kazan Federal University

Email: ankor-2015@yandex.ru

Department of Zoology and General Biology

Russian Federation, 18 Kremlevskaya Str., Kazan, 420008

O A. Timoshkin

Limnological Institute Siberian Branch of the Russian Academy of Sciences

Email: ankor-2015@yandex.ru
ORCID iD: 0000-0001-6476-0074
Russian Federation, Ulan-Batorskaya Str., 3, Irkutsk, 664033

S. V. Kirilchik

Limnological Institute Siberian Branch of the Russian Academy of Sciences

Email: ankor-2015@yandex.ru
ORCID iD: 0000-0002-9997-6294
Russian Federation, Ulan-Batorskaya Str., 3, Irkutsk, 664033

References

Supplementary files