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A new hypotrich ciliate, Oxytricha xianica sp.nov., with notes on the morphology and phylogeny of a Chinese population of Oxytricha auripunctata Blatterer & Foissner, 1988(Ciliophora, Oxytrichidae)

  • Received Date: 2020-09-08
    Accepted Date: 2020-12-11
    Published online: 2021-03-02
  • Edited by Jiamei Li.
  • The morphology and infraciliature of the new hypotrichous ciliate, Oxytricha xianica sp.nov., and a Chinese population of Oxytricha auripunctata Blatterer & Foissner, 1988, are investigated using live observation and protargol staining.Oxytricha xianica sp.nov.is defined by the body size 85-130 μm × 30-50 μm in vivo, the typical oxytrichid 18 frontoventral-transverse cirri, cortical granules absent, undulating membranes reduced or absent, three caudal cirri, dorsal kineties in Oxytricha pattern, and two ellipsoidal macronuclear nodules.The Chinese population of O.auripunctata differs slightly from other populations in the arrangement and color of cortical granules and position of anterior end of dorsal kinety 4.Phylogenetic analyses based on SSU-rDNA sequences support the assertion that O.auripunctata belongs to Oxytricha and Oxytrichidae.
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A new hypotrich ciliate, Oxytricha xianica sp.nov., with notes on the morphology and phylogeny of a Chinese population of Oxytricha auripunctata Blatterer & Foissner, 1988(Ciliophora, Oxytrichidae)

  • 1. Laboratory of Protozoological Biodiversity and Evolution in Wetland, College of Life Sciences, Shaanxi Normal University, Xi'an 710119, China
  • 2. Insititute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China
  • 3. College of Life Science, Institute of Life Science and Green Development, Hebei University, Baoding 071002, China
  • 4. Department of Life Sciences, Natural History Museum, London SW7 5BD, UK
  • 5. Handan Foreign Language School of Hebei Province, Handan 056002, China

Abstract: The morphology and infraciliature of the new hypotrichous ciliate, Oxytricha xianica sp.nov., and a Chinese population of Oxytricha auripunctata Blatterer & Foissner, 1988, are investigated using live observation and protargol staining.Oxytricha xianica sp.nov.is defined by the body size 85-130 μm × 30-50 μm in vivo, the typical oxytrichid 18 frontoventral-transverse cirri, cortical granules absent, undulating membranes reduced or absent, three caudal cirri, dorsal kineties in Oxytricha pattern, and two ellipsoidal macronuclear nodules.The Chinese population of O.auripunctata differs slightly from other populations in the arrangement and color of cortical granules and position of anterior end of dorsal kinety 4.Phylogenetic analyses based on SSU-rDNA sequences support the assertion that O.auripunctata belongs to Oxytricha and Oxytrichidae.

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Introduction
Results

    Oxytricha xianica sp. nov. (Figs. 2f–i, 3a–f; Table 1)

  • Figure 2.  Oxytricha auripunctata (ae) and Oxytricha xianica sp. nov. (fi) from life (a, df) and after protargol staining (b, c, gi). a Ventral view, showing a representative individual. b, c Ventral and dorsal view, showing the infraciliature and nuclear apparatus. Arrowhead demonstrates the buccal cirrus; arrow marks the caudal cirri. d Dorsal views, showing the variability of body shape. e Arrangement of cortical granules. f Ventral view, showing a representative individual. g, h Ventral and dorsal views of holotype specimen, showing the ciliature and nuclear apparatus. Arrowhead denotes the buccal cirrus, arrow points to the caudal cirri, and circle indicates transverse cirri. i Ventral view of the anterior portion, to show the frontal cirri and frontoventral cirri. AZM adoral zone of membranelles, EM endoral membrane, FC frontal cirri, FVC frontoventral cirri, LMR left marginal row, Ma macronuclear nodules, Mi micronuclei, PM paroral membrane, RMR right marginal row, TC transverse cirri; 1–4 dorsal kineties 1–4; 5, 6, dorsomarginal kineties. Scale bars = 50 μm (ac, fh; 25 μm (i)

    Figure 3.  Photomicrographs of Oxytricha xianica sp. nov. from life (ac) and after protargol staining (df). a Ventral view of a representative individual. b Ventral view of adoral zone, showing the absence of undulating membranes, arrow indicates adoral zone of membranelles. c Ventral view of posterior region, showing the transverse cirri (red circles). d Ventral view, showing the infraciliature, red circle indicates transverse cirri. e Ventral view of anterior region, arrow indicating the vestigial undulating membranes. f Dorsal view of posterior portion, showing the caudal cirri (red circle). Ma macronuclear nodules. Scale bars = 50 μm

    Charactera Species HT Min Max Mean M SD CV n
    Length of body xia 131 87 150 113.4 116 19.8 17.4 19
    aur 97 163 136.3 135.9 15.4 11.3 21
    Width of body xia 48 29 53 38.6 40 7.2 18.5 19
    aur 30 77 49.7 50.2 13.3 26.8 21
    Length of adoral zone xia 45 34 51 40.2 41 5.2 13.0 19
    aur 34 50 41.6 41.1 4.5 10.9 21
    Number of adoral membranelles xia 28 24 32 28.2 28 2.4 8.6 19
    aur 26 34 30.8 31 2.0 6.5 21
    Number of buccal cirri xia 1 1 1 1 1 0 0 19
    aur 1 1 1 1 0 0 25
    Number of frontal cirri xia 3 3 3 3 3 0 0 19
    aur 3 3 3 3 0 0 25
    Number of frontoventral cirri xia 4 4 4 4 4 0 0 19
    aur 4 4 4 4 0 0 25
    Number of postoral ventral cirri xia 3 3 3 3 3 0 0 19
    aur 3 3 3 3 0 0 25
    Number of pretransverse cirri xia 2 2 2 2 2 0 0 19
    aur 2 2 2 2 0 0 25
    Number of transverse cirri xia 5 5 5 5 5 0 0 19
    aur 4 4 4 4 0 0 25
    Number of caudal cirri xia 3 3 3 3 3 0 0 8
    aur 3 3 3 3 0 0 10
    Number of left marginal cirri xia 17 15 21 17.9 18 1.6 8.6 19
    aur 18 24 21.4 22 1.8 8.5 25
    Number of right marginal cirri xia 18 17 21 19.3 19.5 1.3 6.9 19
    aur 18 24 20.8 20 1.7 8.0 25
    Number of dorsal kineties xia 6 6 6 6 6 0 0 9
    aur 6 6 6 6 0 0 9
    Number of basal bodies in dorsal kinety 1 xia 28 20 28 23.8 26 2.2 9.4 8
    aur 17 21 19.6 20 1.6 8.1 9
    Number of basal bodies in dorsal kinety 2 xia 23 20 28 24.5 24.5 2.3 9.5 8
    aur 17 22 19.4 20 1.4 7.3 9
    Number of basal bodies in dorsal kinety 3 xia 17 12 21 15.3 14.5 3.1 20.1 6
    aur 10 17 13.6 14 2.3 16.9 9
    Number of basal bodies in dorsal kinety 4 xia 20 18 24 19.7 18 2.4 12.3 7
    aur 6 15 9.1 8.5 2.8 30.1 8
    Number of basal bodies in dorsal kinety 5 xia 11 7 13 10 11 2.1 20.6 9
    aur 6 11 9.6 10 1.7 17.5 8
    Number of basal bodies in dorsal kinety 6 xia 6 5 8 6.9 7 0.9 13.5 9
    aur 2 7 4.6 4.5 1.5 32.6 8
    Total number of dorsal bristles xia 105 90 110 101 101.5 6.6 6.5 6
    aur 73 84 76.5 75 4.0 5.3 8
    Distance between cirrus Ⅲ/2 and undulating membranes xia / 8 15 11.6 11 2.6 22.5 5
    aur 4 10 5.9 5 1.6 27.5 17
    Distance between cirri Ⅲ/2 and Ⅳ/3 xia 4 4 7 5.4 5 0.8 14.4 18
    aur 3 5 4.1 4 0.6 13.7 17
    Distance between cirri Ⅴ/2 and Ⅴ/3 xia 22 13 34 21.7 20.5 6.0 27.7 16
    aur 60 135 83.4 81 18.5 22.1 16
    Distance between cirri Ⅴ/2 and Ⅵ/2 xia 12 9 20 14 13.5 3.2 23.0 16
    aur 3 6 4.4 4 1.1 25.6 17
    Distance between cirri Ⅴ/3 and Ⅴ/4 xia 15 11 31 18.8 15 6.9 37.0 16
    aur 7 17 9.9 10 2.4 24.5 15
    Number of macronuclear nodules xia 2 2 4 2.2 2 0.6 28.5 19
    aur 2 2 2 2 0 0 25
    Macronuclear nodules, length aur 17 10 25 15.9 15 3.4 21.7 24
    Macronuclear nodules, width aur 4 9 6.9 7 1.4 20.0 24
    Distance between two macronuclear nodules xia 7 5 9 7.4 7.5 1.4 18.7 10
    aur 15 21 17.7 7.5 1.9 10.7 10
    Number of micronuclei xia 3 2 5 3.3 3 1.0 30.0 9
    aur 1 3 1.8 1.5 0.9 50.7 8
    Diameter of micronuclei xia / 2 3 2.5 2.5 0.3 11.5 13
    CV coefficient of variation in %, HT holotype, M median, Max maximum, Mean arithmetic mean, Min minimum, n sample size, SD standard deviation, / data not available
    aAll data are based on protargol-stained specimens. All measurements in µm

    Table 1.  Morphometric characterization of Oxytricha xianica sp. nov. (xia) and the Chinese population of Oxytricha auripunctata (aur)

  • Diagnosis
  • Cell generally elongate-elliptical, body size in life 85–130 µm × 30–50 µm. Two ellipsoidal macronuclear nodules. Adoral zone consists of 24–32 membranelles, undulating membranes reduced or absent. Contractile vacuole about 40% down length of body. 18 frontoventral–transverse cirri arranged in typical oxytrichid pattern. Postoral ventral cirri well separated from rear end of adoral zone of membranelles. Left and right marginal rows composed of about 18 and 19 cirri, respectively. Three caudal cirri. Four dorsal kineties and two dorsomarginal kineties.

  • Type locality
  • Soil (salinity of soil percolate ca. 0) in campus of Shaanxi University of Science and Technology (34°22′45″N, 108°58′22″E), Xi'an, Shaanxi Province, China.

  • Type material
  • The holotype (registration number LY2015121502A) and two paratype slides (registration numbers LY2015121502B, C) were deposited in the Laboratory of Protozoological Biodiversity and Evolution in Wetland, Shaanxi Normal University, China. One paratype slide (registry no. LY2015121502D) with protargol-stained specimens is deposited in the Laboratory of Protozoology, Ocean University of China (OUC).

  • Etymology
  • The species-group name "xianica" refers to Xi'an, the type locality.

  • ZooBank registration
  • Zoobank number of present work:

    urn: lsid: zoobank.org: pub: 7468C65D-941A-4090-98DA-DE5F4EDBA26D.

    Zoobank number of Oxytricha xianica sp. nov.:

    urn: lsid: zoobank.org: act: 4495032B-C385-4FA9-99D1-07B1711CC3A8.

  • Description

  • Body size 85–130 µm × 30–50 µm in vivo, 87–150 µm × 29–53 µm after protargol staining, flexible, but not contractile, elongate and generally elliptical, right and left margins more or less convex, both ends rounded (Figs. 2f, 4a), ventral and dorsal sides flat when viewed laterally. Usually, two, rarely four, macronuclear nodules, each about 20–25 µm × 10–12 µm in vivo, positioned along cell mid-line; two-to-five globular micronuclei each closely associated with macronuclear nodules, 2–3 µm across (Fig. 2h). Cortical granules absent. One contractile vacuole near left margin, positioned about 40% down length of body, approximately 13 µm in diameter, contracts at intervals of about 10 s, conspicuous at low magnification. Cytoplasm colorless, no distinct content in food vacuoles recognizable, indicating that it feeds on bacteria. Locomotion by fast crawling on substrate.

    Figure 4.  Photomicrographs of Oxytricha auripunctata from life (ad) and after protargol staining (e, f). a, b Ventral views of a representative individual. c, d Ventral view to mark the distribution of cortical granules (arrows). e Ventral view, showing frontal cirri, buccal cirrus, and frontoventral cirri. f Dorsal view of posterior portion, showing the caudal cirri (red circle), arrow indicates dorsal kinety 1. Scale bars = 50 μm

    Adoral zone conspicuous, shaped like a question mark, consists of 24–32, on average 28, adoral membranelles, about 35% of body length with DE value of ca. 0.31 (n = 19), cilia of membranelles about 20 µm long in vivo (Figs. 2f, 4a). Undulating membranes usually absent. In 5 out of 19 cells, a vestigial endoral was observed, this being about 10–35 µm long, single-rowed composed of 12–30 kinetosomes, located at level of mid-region of adoral zone or near buccal vertex (Figs. 2i, 4e).

    Eighteen frontoventral–transverse cirri, including three enlarged frontal cirri, with cilia 20–25 µm long in vivo; one buccal cirrus, with cilia about 20 µm in vivo, located 40% down length of adoral zone, slightly right of mid-line; four frontoventral cirri in V-shaped pattern, cirrus Ⅲ/2 ahead of level of cirrus Ⅳ/3 and behind level of cirrus Ⅵ/3 (Fig. 2g, i); three postoral ventral cirri located behind buccal vertex and well separated from rear end of adoral zone of membranelles (average 16 µm, n = 5), with cilia 15–20 µm long in vivo, cirrus Ⅳ/2 arranged anterior to cirrus Ⅴ/4, distance between cirri Ⅴ/3 and Ⅴ/4 a little shorter than that between cirri Ⅴ/3 and Ⅴ/2 (Fig. 2g); two pretransverse cirri, five enlarged transverse cirri arranged in asymmetric "V" shape in two indistinct groups, cilia 25–30 µm long in vivo (Figs. 2g, 4a). One right and one left marginal row, composed of 17–21 and 15–21 cirri, respectively, cilia about 20 µm long in vivo. Left marginal row commences more or less at level of buccal vertex; marginal rows not confluent posteriorly; right marginal row starts right anterior to cirrus Ⅵ/4 (Figs. 2g, 4d).

    Dorsal ciliature in Oxytricha pattern, dorsal bristles about 3 µm long in vivo; dorsal kineties 1 and 2 bipolar, each comprising 20–28 dikinetids; dorsal kineties 3 and 4 almost bipolar, shortened posteriorly and anteriorly, composed of 12–21 and 18–24 dikinetids, respectively; dorsomarginal kineties starting near anterior end of cell, dorsal kinety 5 terminating about 35% down length of cell and composed of 7–13 dikinetids, dorsal kinety 6 terminating 25% down length of cell and composed of 5–8 dikinetids (Fig. 2h). Three caudal cirri with cilia about 15 µm long in vivo, one each at posterior ends of dorsal kineties 1, 2, and 4 (Figs. 2g, 4f).

Morphology of Chinese population of Oxytricha auripunctata Blatterer & Foissner, 1988 (Figs. 2a–e, 4a–f, Table 1)

    Voucher material

  • Three slides with protargol-stained specimens have been deposited in the collection of the Laboratory of Protozoological Biodiversity and Evolution in Wetland, College of Life Sciences, Shaanxi Normal University, China (accession numbers LY2015121504A–C).

  • Description

  • Body size 95–145 µm × 30–60 µm in vivo, 97–163 µm × 30–77 µm in protargol preparations. Body elongate-elliptical, very flexible but not contractile, anterior end broadly rounded, posterior end narrowly rounded resembling a short tail, dorso-ventrally flattened about 2:1 (Figs. 2a‒d, 3a, b). Two ellipsoidal macronuclear nodules positioned left of mid-line and about 35% and 65% down length of body, respectively, size 10–25 µm × 4–9 µm in protargol-stained specimens. One to three globular micronuclei closely associated with macronuclear nodules. Contractile vacuole approximately 12 µm in diameter, positioned about 40% down length of body near left margin, pulsing at intervals of about 30 s (Figs. 2a, 3a, b). Cortical granules arranged in numerous short, irregular, longitudinal rows or in small groups, citrine-yellow, about 1 µm across, (Figs. 2e, 3c, d), rendering cell yellowish even at low magnifications (Fig. 3a, b). Cytoplasm colorless, usually containing several food vacuoles (Fig. 2a). Locomotion by swimming while rotating about main cell axis or by crawling moderately quickly on substrate.

    Adoral zone formed like a question mark, about 25% of body length, composed of 26–34 adoral membranelles, (DE value ca. 0.24, n = 21). Cilia of adoral membranelles about 15 μm long in vivo. Endoral and paroral in Oxytricha pattern, curved and optically intersecting, almost equal in length; endoral comprises a single row of kinetosomes, starting about 35% down length of buccal field; paroral commences slightly ahead of endoral, kinetosomes forming a zig-zag pattern (Figs. 2b, 3e).

    Invariably 17 frontoventral–transverse cirri, including three frontal cirri, about 15 µm long in vivo. One buccal cirrus located slightly anterior to level of intersection of undulating membranes; four frontoventral cirri in V-shaped pattern with cirrus Ⅲ/2 behind level of cirrus Ⅵ/3; three postoral ventral cirri in a cluster behind buccal vertex with cirrus Ⅳ/2 in front of level of cirrus Ⅴ/4; four transverse cirri about 15 µm long in vivo, two pretransverse ventral cirri close to transverse cirri (Figs. 2a, b). One right and one left marginal row, each comprising 18–24 cirri, marginal cirri 12 µm long in vivo. Right marginal row starts at level of cirrus Ⅵ/3, left marginal row commences close to buccal vertex, posterior ends of marginal rows nearly confluent (Figs. 2b, 3e).

    Six dorsal kineties in Oxytricha pattern, dorsal cilia about 3 µm long in vivo: dorsal kineties 1 and 2 bipolar, comprising 17–21 and 17–22 dikinetids, respectively; dorsal kinety 3 commences near anterior end of cell and terminates ca. 80% down length of body, composed of 10–17 dikinetids; dorsal kinety 4 starts ca. 60% down length of body and extends to posterior end of body, comprises 6–15 dikinetids; dorsal kineties 5 and 6 are dorsomarginal kineties, kinety 5 composed of 6–11 dikinetids and terminates at midbody, dorsal kinety 6 composed of 2–7 dikinetids and terminates ca. 25% down length of body (Fig. 2c). Three caudal cirri, one each at posterior ends of dorsal kineties 1, 2, and 4, difficult to distinguish from marginal cirri (Figs. 2c, 3f). Cilia of caudal cirri about 13 µm long in vivo.

  • Phylogenetic analyses based on SSU-rDNA sequences

  • The SSU-rDNA sequence of Oxytricha auripunctata has been deposited in GenBank with the accession number MH844496. The length and GC content of the new sequence are 1761 bp and 45.88%, respectively.

    Phylogenetic trees inferred from SSU-rDNA sequences using ML and BI had similar topologies, so only the ML tree is presented here (Fig. 5). In both analyses, Oxytricha auripunctata has a close relationship with oxytrichids with a flexible body, and clusters near the Cyrtohymena citrina + Urosomoida spp. + Onychodromopsis flexilis + Paraurosomoida indiensis + Notohymena spp. + Neokeronopsis spp. + Austrolocirrus shii clade, albeit with low support (27% ML, 0.57 BI).

    Figure 5.  Maximum-likelihood (ML) tree based on SSU-rDNA sequence data. The newly sequenced O. auripunctata is in bold and marked by an arrow. Support values for nodes are for ML and BI (ML/BI), respectively. Disagreements in ML and BI tree topologies are indicated by "-". All branches are drawn to scale. The scale bar corresponds to 0.02 expected substitutions per site

Discussion

    Morphological comparison of Oxytricha xianica sp. nov. with similar species

  • In terms of the lack of cortical granules, elongate-elliptical body shape, two macronuclear nodules, more than one micronuclei, six dorsal kineties, and conspicuously long caudal cirri and dorsal bristles, three congeners shoud be compared with Oxytricha xianica sp. nov., namely O. similis Engelmann, 1862, O. parallela Engelmann, 1862 and O. proximata Shibuya, 1930.

    Oxytricha similis differs from O. xianica sp. nov. in having fewer caudal cirri (2 vs. 3), undulating membranes present (vs. reduced or absent) and conspicuously long (vs. not conspicuously long) caudal cirri (Berger 1999).

    Oxytricha parallela can be separated from O. xianica sp. nov. by having longer dorsal bristles (10 µm vs. 5 µm), more caudal cirri (4 vs. 3), conspicuously long (vs. not conspicuously long) caudal cirri, and the undulating membranes present (vs. reduced or absent) (Berger 1999).

    Oxytricha proximata Shibuya, 1930 can be distinguished from O. xianica sp. nov. by its obtusely pointed (vs. rounded) posterior end, undulating membranes present (vs. reduced or absent), and the number of transverse cirri (4 vs. 5) (Berger 1999).

  • Comparison of Chinese population of Oxytricha auripunctata with Austrian and Korean populations

  • Two populations of Oxytricha auripunctata have previously been described, one from Austria and the other from Korea (Blatterer and Foissner 1988; Kim and Min 2015). The Chinese population closely resembles both of these populations, the main differences being the color and the arrangement of the cortical granules (citrine-yellow, arranged in irregular short rows and small groups vs. orange–yellow, irregularly scattered and sometimes cluster in small groups, see Fig. 4d–g in Kim and Min 2015) and the position of the anterior end of dorsal kinety 4 (below vs. ahead of midbody). We believe that these differences are intraspecific variations and, therefore, not significant for species-level separation. Furthermore, all other key characters, i.e., body size, shape, nuclear features, arrangement of cirri, and morphometric data (Table 1; Berger 1999), are consistent with the original description. The identification of the Chinese population is therefore not in doubt.

  • Phylogenetic analyses

  • The new sequence obtained in this study provided the opportunity to infer the phylogenetic relationships of Oxytricha auripunctata. In the SSU-rDNA tree, O. auripunctata is most closely related to Urosomoida, Paraurosomoida, Notohymena, Cyrtohymena, Neokeronopsis, Australocirrus, and Onychodromopsis. The current topology, to some extent, also reflects the morphological similarity of these taxa, i.e., a flexible body and the possession of dorsomarginal kineties (Berger 1999; Kumar and Foissner 2015; Singh and Kamra 2013; Song and Wilbert 1989; Warren et al. 2002). In addition, this clade nests within the Dorsomarginalia, which is monophyletic in our phylogenetic tree. All analyses strongly support Berger's (2006) hypothesis that the dorsomarginal kineties are the main morphological apomorphy of the Dorsomarginalia.

Materials and methods

    Sampling, cultivation, and observation of morphology

  • Oxytricha xianica sp. nov. and O. auripunctata were isolated from a soil sample (soil percolate salinity 0‰, pH approximately 7) collected from the campus of Shaanxi University of Science and Technology (34°22′45″N, 108°58′22″E), Xi'an, China, on 15th December 2015 when the air temperature was 13 ℃ (Fig. 1a, b). At this location the mean annual temperature is 13.03–13.73 ℃ and annual precipitation is 522.4–719.5 mm. Ciliates were stimulated to excyst and emerge from the soil samples by employing the non-flooded Petri dish method (Foissner 1987). A clonal culture of O. auripunctata was established in the laboratory at room temperature (ca. 25 ℃) in a Petri dish containing spring water (Ningfu) and rice grains to enrich the growth of bacterial food organisms for the ciliates.

    Living cells were observed using bright field and differential interference contrast (DIC) microscopy (Olympus BX51). The protargol silver staining method of Wilbert (1975) was used to reveal the infraciliature and nuclear apparatus. The protargol reagent was made mainly according to Pan et al. (2013). Drawings of stained specimens were conducted with the help of drawing device. Terminology is mainly based on Berger (1999) and Foissner (2016).

  • DNA extraction, PCR amplification, and gene sequencing

  • The Genomic DNA extraction, PCR amplification, and gene sequencing were carried out according to Wang et al. (2020b).

  • Phylogenetic analyses

  • The SSU-rDNA sequence of Oxytricha auripunctata was aligned with sequences of 74 other hypotrich taxa downloaded from the GenBank database (for accession numbers, see Fig. 5). Four euplotids were used as the outgroup taxa. Phylogenetic analyses were carried out according to Wang et al. (2020a).

Acknowledgements
  • This study was supported by the Natural Science Foundation of China (project numbers: 32070428, 31872206).

Author contributions
  • JW and TZ collected the samples and performed the experiments. FL and YL assisted with the calculations, sequencing and phylogenetic analyses. AW revised and edited the manuscript. CS carried out the species identifcations and helped to revise the manuscript.

Compliance with ethical standards

    Conflict of interest

  • The authors declare that they have no confict of interest.

  • Animal and human rights statement

  • We declare that all applicable international, national, and/or institutional guidelines for sampling, care, and experimental use of organisms for the study have been followed and all necessary approvals have been obtained.

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