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Evidence for Karyotype Polymorphism in the Free-Living Flatworm, Macrostomum Lignano, a Model Organism for Evolutionary and Developmental Biology

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Evidence for Karyotype Polymorphism in the Free-Living Flatworm, Macrostomum Lignano, a Model Organism for Evolutionary and Developmental Biology

Kira S Zadesenets et al. PLoS One.

Abstract

Over the past decade, the free-living flatworm Macrostomum lignano has been successfully used in many areas of biology, including embryology, stem cells, sexual selection, bioadhesion and aging. The increased use of this powerful laboratory model, including the establishment of genomic resources and tools, makes it essential to have a detailed description of the chromosome organization of this species, previously suggested to have a karyotype with 2n = 8 and one pair of large and three pairs of small metacentric chromosomes. We performed cytogenetic analyses for chromosomes of one commonly used inbred line of M. lignano (called DV1) and uncovered unexpected chromosome number variation in the form of aneuploidies of the largest chromosomes. These results prompted us to perform karyotypic studies in individual specimens of this and other lines of M. lignano reared under laboratory conditions, as well as in freshly field-collected specimens from different natural populations. Our analyses revealed a high frequency of aneuploids and in some cases other numerical and structural chromosome abnormalities in laboratory-reared lines of M. lignano, and some cases of aneuploidy were also found in freshly field-collected specimens. Moreover, karyological analyses were performed in specimens of three further species: Macrostomum sp. 8 (a close relative of M. lignano), M. spirale and M. hystrix. Macrostomum sp. 8 showed a karyotype that was similar to that of M. lignano, with tetrasomy for its largest chromosome being the most common karyotype, while the other two species showed a simpler karyotype that is more typical of the genus Macrostomum. These findings suggest that M. lignano and Macrostomum sp. 8 can be used as new models for studying processes of partial genome duplication in genome evolution.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Map of the collection sites for the different Macrostomum species and specimens.
The sites mentioned in Table 1 are identified by number: UV in Bibione (1), P1 in Lignano Sabbiadoro (2), and PS on Isola di Martignano (3), Vourvourou (4), Porto Koufo (5), Étang de Biguglia (6), Palavas-les-Flots (7), and San Rossore Regional Park (8).
Fig 2
Fig 2. Karyotype variation among Macrostomum species.
(a) the 'normal' chromosome set of Macrostomum lignano, 2n = 8, (b) the 'normal' chromosome set of Macrostomum sp. 8, 2n = 10, and the invariant chromosome sets of (c) M. spirale, 2n = 6, and (d) M. hystrix, 2n = 6. DAPI-staining (inverted image). Scale bar 10 μm.
Fig 3
Fig 3. Karyotype diversity among individually karyotyped specimens of Macrostomum lignano and Macrostomum sp. 8, based on ≥10 chromosome plates per specimen.
Karyotype diversity of Macrostomum lignano (a-i). (a) 'normal' 2n = 8 (two large and six small metacentrics); (b) 2n = 9 (three large and six small metacentrics); (c) 2n = 10 (four large and six small metacentrics); (d) 'abnormal' 2n = 8 (three large and five small metacentrics); (e) 'abnormal' 2n = 9 (four large and five small metacentrics); (f) 'abnormal' 2n = 9 (two large and seven small metacentrics); (g) 'abnormal' 2n = 16 (four large and twelve small metacentrics); (h) 'abnormal' 2n = 9 (two large, six small metacentrics and one extra small chromosome); (i) 'abnormal' 2n = 8 (one large metacentric, three medium-sized submetacentrics, and four small metacentrics). Karyotype diversity of Macrostomum sp. 8 (j-l). (j) 'normal' 2n = 10 (four large and six small metacentrics); (k) 2n = 9 (three large and six small metacentrics); (l) 2n = 11 (five large and six small metacentrics). Chromosome rearrangements are marked with arrows. DAPI-staining (inverted image). Scale bar 10 μm.
Fig 4
Fig 4. Chromosomes of Macrostomum lignano at different condensation levels, stained with DAPI (inverted image).
(a-c) mitotic metaphase chromosomes; (d) pachytene chromosomes. AT-positive bands are marked with arrows. Scale bar 10 μm.
Fig 5
Fig 5
Chromosomes of Macrostomum lignano (a, b) and Macrostomum sp. 8 (c, d) stained with both DAPI and CMA (inverted images). Intensively stained chromosomal material is marked with arrows.
Fig 6
Fig 6
Localization of clusters of 28S rDNA (green) and telomeric (red) repeats in chromosomes of M. lignano (a-c) and Macrostomum sp. 8 (d), using fluorescence in situ hybridization (FISH). The chromosomes were stained with DAPI (blue colour).
Fig 7
Fig 7
Fluorescence in situ hybridization (FISH) using microdissected DNA probes obtained from the large chromosome (Mli1—green) and the small chromosomes (Mlism–red) on either highly condensed (a) and less-highly condensed (b) chromosomes of M. lignano. The chromosomes were stained with DAPI (blue colour).

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Grant support

The reported study was supported by: (1) the Russian Foundation for Basic Research (RFBR), http://www.rfbr.ru/rffi/eng, Project grant no. 14-04-32007 (KZ); (2) Swiss National Science Foundation (SNSF), http://www.snf.ch/en/Pages/default.aspx, Grant no. 143732 (LS); (3) European Research Council (ERC), https://erc.europa.eu/, Starting Grant no. 310765 (EB); (4) Federal Agency for Science and Innovation, http://fano.gov.ru/en/, grant 0324-2015-0003, Russian Academy of Sciences, http://www.ras.ru/index.aspx (NBR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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