Dugesia sp.

Stocchino, Giacinta Angela, Sluys, Ronald, Solà, Eduard, Riutort, Marta & Manconi, Renata, 2024, The long-eared freshwater planarians of Madagascar form a separate phylogenetic clade within the genus Dugesia (Platyhelminthes: Tricladida), with the description of two new species, Zoological Journal of the Linnean Society (zlae 143) 202 (4), pp. 1-29 : 21-22

Dugesia sp. GS34-Dmad4

Material examined

CGAS Pla 28. 1 (GS34-1), Central High Plateau , Western Slope , Iandratsay River , upper Tsiribihina basin, under bridge crossing Route 34, 4 km from Betafo (19°50 ʹ 24″S 46°51 ʹ 18″E), 1300 m a.s.l., Madagascar, September 2011, coll. R. Manconi, one set of sagittal sections on four slides of a sexually immature specimen ; CGAS Pla 28. 2 (GS 34-2), one sexually immature specimen embedded in paraffin ; CGAS Pla 28. 3 (GS34-3), one sexually immature specimen fixed in Bouin’s fluid and, subsequently, preserved in 70% ethanol .

Other material: Three individuals from the same sample were preserved in absolute ethanol; one of these was used for DNA extraction and sequencing bySolà et al. (2022). GenBank accession numbers of the obtained sequences are given in Table 2 .

Habitat

Planarians were found under volcanic pebbles and boulders in shallow turbid water.

Reproduction and behaviour

At collection, the specimens turned out to be asexual ( Fig. 14 ). During the following 5 years of rearing under laboratory conditions, the three living worms underwent post-pharyngeal fission. This resulted in an increase in the number of animals in the culture (up to a maximum of 10 specimens), without, however, showing any case of sexualization. As in D. insolita , we observed in these animals also the same autotomy-like process, as well as the same type of locomotion (see above).

Karyology

Analysis of metaphasic plates revealed that specimens constantly showed a set of 10 chromosomes. In all of the 13 analysed plates it was always possible to obtain five groups of elements. However, notwithstanding its numerical stability, the karyotype is highly variable and characterized by conspicuous heteromorphism. Arrangement of the chromosomes in pairs was possible in eight out of 13 metaphasic plates examined, in which chromosomes could be arranged, according to their length, into five groups of two chromosomes with a diploid chromosome set of 2n = 10; n = 5 ( Fig. 15 ). In the remaining plates, the chromosome arrangement resulted in pairs, triplets, and single elements.

The karyometric data indicated that the diploid chromosome complement is characterized by metacentric heterobrachial chromosomes, with the exceptions of chromosome 4, which is submetacentric, and chromosome 5, which is metacentric, bordering on metacentric isobrachial ( Fig. 15 ; Table 5 ). In general, the dimensional difference between the first two pairs was clear, while from the third to the fourth pair of chromosomes it was so little that contiguous chromosomes could well be overlapping. The fifth pair of chromosomes was the smallest.

In all of the eight diploid metaphase plates examined, chromosome length decreased gradually with low standard deviation values; the same applied to the elements within each of the pairs of chromosomes. In contrast, centromeric indices showed great variation; the standard deviation was particularly high for some elements ( Fig. 15 ; Table 5 ).

Comparative discussion

In all phylogenetic approaches, specimen Dugesia sp. GS34- Dmad4 closely groups with a specimen of a different population, Dugesia sp. GS46C-Dmad10, which probably belongs to the same species. These two individuals present a well-supported sister-group relationship to the clade Dugesia sp. GS75-Dmad15 + Dugesia sp. GS15 ( Fig. 2 ). In its turn, this clade of four taxa is sister to Dugesia sp. GS21. Although the clade containing all of these specimens mentioned above is fully supported, its precise relationship to the rest of the long-eared Madagascan Dugesia is not well resolved.

The genus Dugesia encompasses ~110 nominal species. Unfortunately, karyological information is available for only 46 species ( Table 4 ). Until now, their haploid chromosome sets were classified into three karyological groups: n = 7, n = 8, and n = 9. Four species are known from the n = 7 group: D. hepta from Sardinia Island (Western Palaearctic Region), D. batuensis Ball, 1970 from Malaysia (Indian subregion), D. pendula Chen & Dong, 2024 ( Wang et al. 2024) from China, and D. ryukyuensis Kawakatsu, 1976 from the south-western Far East. The second group with n = 8 chromosomes includes the majority of Dugesia species from Europe, Africa, central and south-west Asia, and the Far East, while the basic number n = 9 is shared by seven species, including D. insolita , from the Mediterranean region, Yemen, and the Afrotropical region (cf. Stocchino et al. 2004, 2012, 2014, Harrath et al. 2013, Wang et al. 2021a, b, Chen et al. 2022). Therefore, the basic number of n = 5 of the Madagascan population of Dugesia sp. GS34-Dmad4 represents a new, as well as the lowest, chromosome number presently known for the genus ( Fig. 16 ; Table 4 ). It is noteworthy that, notwithstanding the numerical stability of the chromosome set, the new karyotype is characterized by a high polymorphism. Usually, in the genus Dugesia fissiparous, populations are characterized by great chromosomal variability, with aneuploid, polyploid, and/or mixoploid conditions, as well as occurrence of supernumerary chromosomes.Besides its exclusive karyotype, Dugesia sp. GS34- Dmad4 appears also molecularly well distinct from both D. insolita , as well as the other populations of long-eared planarians analysed in the present study, excepting specimen Dugesia sp. GS46C-Dmad10 with which it forms a sister-group relationship, thus suggesting conspecific affinity (see above). Unfortunately, for the moment we cannot compare it with the two other known species of long-eared planarians of Madagascar, i.e. D. milloti and D. crassimentula , since for these species we do presently lack molecular data.

Figures

Figure 2. Maximum likelihood molecular phylogenetic tree obtained from the concatenated dataset (COI, ITS1, 18S, and 28S) using the program IQ-tree 2.2.2.6. Supports on each node correspond with the supports of bootstrap (BS) of the maximum likelihood-based RaxML and IQ-tree programs, and supports of posterior probability (PP) of the Bayesian inference-based program MrBayes, respectively. Dashes (–) indicate those nodes not recovered in the corresponding approaches. Black squares on some nodes show full support for all three approaches. Scale bar: substitutions per position.

Figure 14. Dugesia sp. GS34-Dmad4.External features of a preserved fissiparous specimen.

Figure 15. Dugesia sp. GS34-Dmad4. A, metaphasic plate; B, karyogram; C, idiogram.

Figure 16. Worldwide distribution of the four karyological groups in the genus Dugesia.