Morphological diversity and molecular phylogeny of five Paramecium bursaria (Alveolata, Ciliophora, Oligohymenophorea) syngens and the identification of their green algal endosymbionts

Molecular Phylogeny of Paramecium bursaria and Identification of its Endosymbionts

The SSU and ITS rDNA of the nuclear ribosomal operon were sequenced to infer the genetic variability of the investigated strains. The SSU and ITS rDNA sequences were aligned according to their secondary structure (examples are presented for the strain SAG 27.96; Fig. 1 and Supplementary Fig. 1). Additional sequences acquired from GenBank were incorporated into a dataset, which included all syngens also from references known for P. bursaria. The phylogenetic analyses revealed five highly supported lineages among the P. bursaria strains, which corresponded to their syngen assignment. As demonstrated in Fig. 2, all investigated strains belonging to the syngens R1, R2 and R5 originated from Europe, whereas the others of the syngens R3-R4 showed a worldwide distribution. The three known green algal endosymbionts, i.e., Chlorella variabilis (Cvar), Chlorella vulgaris (Cvul) and Micractinium conductrix (Mcon) showed no or only little affiliation to specific syngens.

Synapomorphies of the Paramecium bursaria Syngens

As demonstrated in Fig. 2, the subdivision of the P. bursaria strains into syngens is supported by the phylogenetic analyses of the SSU and ITS rDNA sequences. To figure out if these splits were also supported by characteristic molecular signatures, we studied the secondary structures of both SSU and ITS of all available sequences. We discovered 30, respectively 23 variable positions among the SSU and ITS sequences (numbers of these positions in the respective alignments are given in Fig. 3). All syngens showed characteristic patterns among the SSU and ITS. Only the syngens R1 and R2 could not be distinguished using the SSU only, however, in combination with the ITS, each syngen is characterized by unique synapomorphies as highlighted in yellow (Fig. 3). In addition, few variable base positions within syngens (marked in blue in Fig. 3) have been recognized in the ITS regions. For comparison with literature data, we also analyzed all available sequences of the mitochondrial COI gene to find synapomorphies for the five syngens. Within this gene, only 18 variable positions at the amino acid level could be discovered of which 13 are diagnostic for the five syngens (Fig. 3).

The synapomorphies discovered above were used to get insights into the geographical distribution of each P. bursaria syngen. Despite the complete SSU and ITS rDNA sequences included in the phylogeny presented in Fig. 2, records of the partial SSU or ITS rDNA sequences are available in GenBank (BLASTn search; 100% identity;¹³). Considering the metadata of our investigated strains and of the entries in GenBank (Supplementary Table 1), we constructed three haplotype networks using the Templeton-Crandall-Sing (TCS) approach. The SSU haplotype network (Fig. 4) containing 84 records showed that the syngens R1, R2 and R5 were only found in Europe, whereas the other three syngens have been discovered around the world. A similar distribution pattern occurred when using the ITS (101 entries in GenBank). Records of syngens R1 and R5 have only been found in Europe, whereas all other syngens were distributed around the world. The 132 COI records found in GenBank by the BLASTn search were used for the haplotype network, which also showed the similar pattern (Fig. 4).

Ciliate Taxonomy

Considering all our findings, P. bursaria is morphologically highly variable, and obviously represents a cryptic species complex (Figs. 5, 6; Supplementary Table 2). The known five syngens most likely represent biological species according to Mayr¹⁴ and can be attributed to the cryptic species described by Greczek-Stachura et al.¹¹. As mentioned above, the assignments of these cryptic species by Greczek-Stachura et al.¹¹ have not been validly described according to the ICZN. In addition, the naming using a mixture of Latin prefix and Greek suffix is also not appropriate (the epithet bursa derived from the Greek word byrsa). Therefore, we describe the five syngens as new species as follows. The general morphological features of these species are summarized in Table 1.

Paramecium protobursaria sp. nov.

Synonym: Paramecium primabursaria nom. inval.

Description: The strains SAG 27.96 and PB-25 belong to syngen R1 according to Greczek-Stachura et al.^10,11 and differ from other syngens by their SSU and ITS rDNA sequences (MT231333). From morphology, the cells are ellipsoidal to broadly ellipsoidal and dorso-ventrally flattened in vivo. The cells measure 70–164 × 44–65 µm; the single macronucleus is located around mid-cell and measures 25–38 × 11–22 µm; the adjacent single compact micronucleus measures 11–20 × 5–8 µm; the usually two (rarely one) contractile vacuoles, one in the anterior and one in the posterior cell portion have radial collecting channels and 1–3 excretory pores each; the number of ciliary rows/20 µm is 14–22; the length of the caudal cilia is 9–19 µm; the numerous trichocysts located in the cell cortex are 4–6 µm in length. The symbiotic algae belong to M. conductrix; the larger algae measure 4–7 × 4–7 µm; the smaller algal cells measure 2–5 × 2–5 µm.

Geographic distribution: The investigated strains of syngen R1 were found in Europe: Göttingen, Germany; Lake Mondsee, Austria. In addition, this species has been reported from different places in Europe, Asia and North America (see details in Supplementary Table 1).

Reference material: Strain SAG 27.96 and the clonal strain SAG 2645 derived from SAG 27.96 are available at the Culture Collection of Algae (SAG), University of Göttingen, Germany.

Holotype: Two slides (one holotype, one paratype) with protargol-impregnated specimens from the clonal culture SAG 2645, which derived from the reference material SAG 27.96, isolated from the pond of the Old Botanical Garden of the University of Göttingen (Germany), have been deposited in the Oberösterreichisches Landesmuseum at Linz (LI, Austria).

Zoobank Registration LSID: AFD967ED-BC2A-43FD-847E-5DF588BB025C.

Paramecium deuterobursaria sp. nov.

Synonym: Paramecium bibursaria nom. inval.

Description: The strains CCAP 1660/34 and CCAP 1660/36 belong to syngen R2 according to Greczek-Stachura et al.^10,11 and differ from other syngens by their SSU and ITS rDNA sequences (OK318487). From morphology, the cells are ellipsoidal to broadly ellipsoidal and dorso-ventrally flattened in vivo. The cells measure 81–167 × 35–83 µm; the single macronucleus is located around mid-cell and measures 24–46 × 10–32 µm; the adjacent single compact micronucleus measures 10–18 × 5–9 µm, no micronucleus seen in live cells of strain CCAP 1660/34; the usually two (rarely one or three) contractile vacuoles, one in the anterior and one in the posterior cell portion have radial collecting channels and 1–3 excretory pores each; the number of ciliary rows/20 µm is 13–22; the length of the caudal cilia is 11–20 µm; the numerous trichocysts located in the cell cortex are 4–6 µm in length. The symbiotic algae belong to M. conductrix; the larger algae measure 5–7 × 4–7 µm; the smaller algal cells measure 3–5 × 2–5 µm.

Geographic distribution: The investigated strains of syngen R2 were found in Europe: Zurich, Switzerland; Lake Piburg, Austria. In addition, this species has been reported from different places in Europe, Asia and Australia (see details in Supplementary Table 1).

Reference material: Strain CCAP 1660/36 is available at the Culture Collection of Algae and Protozoa (CCAP) at the Scottish Association for Marine Science, Oban, Scotland.

Holotype: Two slides (one holotype, one paratype) with protargol-impregnated specimens from the reference material CCAP 1660/36, isolated from Lake Piburg (Tyrol, Austria), have been deposited in the Oberösterreichisches Landesmuseum at Linz (LI, Austria).

Zoobank Registration LSID: D1C20BE6-9A15-4A3D-A7E5-DFC31FF04679.

Paramecium tritobursaria sp. nov.

Synonym: Paramecium tribursaria nom. inval.

Description: The strains CCAP 1660/26, CCAP 1660/28 and CCAP 1660/31 belong to syngen R3 according to Greczek-Stachura et al.^10,11 and differ from other syngens by their SSU and ITS rDNA sequences (MT231339). From morphology, the cells are ellipsoidal to broadly ellipsoidal and dorso-ventrally flattened in vivo. The cells measure 80–153 × 49–73 µm; the single macronucleus is located around mid-cell and measures 21–53 × 12–31 µm; the adjacent single compact micronucleus measures 9–17 × 3–6 µm; no micronucleus seen in live cells of strain CCAP 1660/28; the usually two (rarely one or three) contractile vacuoles, one in the anterior and one in the posterior cell portion have radial collecting channels and 1–3 excretory pores each; the number of ciliary rows/20 µm is 12–20; the length of the caudal cilia is 8–19 µm; the numerous trichocysts located in the cell cortex are 4–6 µm in length. The symbiotic algae belong to C. variabilis; the larger algae measure 4–7 × 3–6 µm; the smaller algal cells measure 3–5 × 2–4 µm.

Geographic distribution: The investigated strains of syngen R3 were found in Europe and Asia: Lake Piburg, Austria; Tokyo, Japan; Khabarovsk region, Amur River, Russia. In addition, this species has been reported from different places in Europe, Asia, North and South America as well as in Australia (see details in Supplementary Table 1).

Reference material: Strain CCAP 1660/26 is available at the Culture Collection of Algae and Protozoa (CCAP) at the Scottish Association for Marine Science, Oban, Scotland.

Holotype: Two slides (one holotype, one paratype) with protargol-impregnated specimens from the reference material CCAP 1660/26, isolated from Japan, have been deposited in the Oberösterreichisches Landesmuseum at Linz (LI, Austria).

Zoobank Registration LSID: CC0FBA7E-9E3A-4C37-B424-C9BFF2018EC0.

Paramecium tetratobursaria sp. nov.

Synonym: Paramecium tetrabursaria nom. inval.

Description: The strains CCAP 1660/25 and CCAP 1660/33 belong to syngen R4 according to Greczek-Stachura et al.^10,11 and differ from other syngens by their SSU and ITS rDNA sequences (MT231347). From morphology, the cells are ellipsoidal to broadly ellipsoidal and dorso-ventrally flattened in vivo. The cells measure 65–179 × 37–79 µm; the single macronucleus is located around mid-cell and measures 18–53 × 10–29 µm; the adjacent single compact micronucleus measures 8–18 × 4–10 µm; the usually two (rarely one or three) contractile vacuoles, one in the anterior and one in the posterior cell portion have radial collecting channels and 1–3 excretory pores each; the number of ciliary rows/20 µm is 14–19; the length of the caudal cilia is 12–20 µm; the numerous trichocysts located in the cell cortex are 4–7 µm in length. The symbiotic algae belong to C. variabilis (CCAP 1660/25) and M. conductrix (CCAP 1660/33); the larger algae measure 3–6 × 3–6 µm; the smaller algal cells measure 2–5 × 1–4 µm.

Geographic distribution: The investigated strains of syngen R4 are found in North- and South America: Burlington, North Carolina, USA; San Pedro de la Paz, Laguna Grande, Chile. In addition, this species has been reported from Europe (see details in Supplementary Table 1).

Reference material: Strain CCAP 1660/25 is available at the Culture Collection of Algae and Protozoa (CCAP) at the Scottish Association for Marine Science, Oban, Scotland.

Holotype: Two slides (one holotype, one paratype) with protargol-impregnated specimens from the reference material CCAP 1660/25, isolated from a pond in Burlington (North Carolina, USA), have been deposited in the Oberösterreichisches Landesmuseum at Linz (LI, Austria).

Zoobank Registration LSID: 78BA9923-07A9-4918-AD7C-9E5E15CC9CDB.

Paramecium pentobursaria sp. nov.

Synonym: Paramecium pentabursaria nom. inval.

Description: The strain CCAP 1660/30 belongs to syngen R5 according to Greczek-Stachura et al.^10,11 and differs from other syngens by their SSU and ITS rDNA sequences (MT231348). From morphology, the cells are ellipsoidal to broadly ellipsoidal and dorso-ventrally flattened in vivo. The cells measure 161–194 × 76–99 µm; the single macronucleus is located around mid-cell and measures 24–47 × 19–31 µm; the adjacent single compact micronucleus measures 13–20 × 4–9 µm; the usually two (rarely one or three) contractile vacuoles, one in the anterior and one in the posterior cell portion have radial collecting channels and 1–4 excretory pores each; the number of ciliary rows/20 µm is 13–19; the length of the caudal cilia is 14–25 µm; the numerous trichocysts located in the cell cortex are 5–7 µm in length. The symbiotic algae belong to C. variabilis; the larger algae measure 5–6 × 5–6 µm; the smaller algal cells measure 4–5 × 3–4 µm.

Geographic distribution: The investigated strain of Syngen R5 was found in Europe: Astrakhan Nature Reserve, Russia.

Reference material: Strain CCAP 1660/30 is available at the Culture Collection of Algae and Protozoa (CCAP) at the Scottish Association for Marine Science, Oban, Scotland.

Holotype: Two slides (one holotype, one paratype) with protargol-impregnated specimens from the reference material CCAP 1660/30, isolated from Astrakhan Nature Reserve (Russia), have been deposited in the Oberösterreichisches Landesmuseum at Linz (LI, Austria).

Zoobank Registration LSID: 6629FA71-E00F-48C6-83AB-61C0CA4823B6.

Syngen Affiliation related to Ciliate Morphology, Endosymbionts and Geographic Distribution

Pearson-correlations of morphometric, syngen-specific and endosymbiont datasets of the P. bursaria strains revealed four significant positive correlations (p < 0.05 and − 0.75 > r > 0.75) between ciliate cell length (BLEN) and width (BWID), BWID and macronucleus width (MACWID), as well as length and width of large symbiotic algae (LSALEN and LSAWID; Fig. 7).

The results of the principal component analysis (PCA) are summarized in the ordination diagram in Fig. 8. The first two axes explain 44.4% of the total variation in the investigated features. Only the first five components (out of 18) had eigenvalues > 1, accounting for 73.1% variation in total (Supplementary Table 3). Principal component axis 1 (PC1) appears to be most negatively weighted by syngen (SYN) and width of the macronucleus (MACWID), separating CCAP 1660/30 and CCAP 1660/33 from the other strains. Principal component axis 2 (PC2) is primarily positively influenced by symbiotic algae characteristics (LSALEN, LSAWID, small symbiotic algal length (SSALEN) and width (SSAWID)) and, ciliate cell length (BLEN) and width (BWID; Supplementary Table 4), partitioning strain PB-25, CCAP 1660/26 and CCAP 1660/36 from CCAP 1660/31 and SAG 27.96 (Fig. 8).

The redundancy analysis (RDA; Fig. 9) revealed a large difference between morphometric features and the tested set of explanatory variables (i.e., algal species (ALSPEC), LSAWID, SSALEN, SYN and GEO) as only 26.9% of the total variation could be explained.

Source: Ecology - nature.com