Rediscovery of Bipalium admarginatum de Beauchamp, 1933 (Platyhelminthes, Tricladida, Geoplanidae) in Malaysia, with molecular characterisation including the mitogenome

Abstract

We present here the first observation of Bipalium admarginatum de Beauchamp, 1933 since its original description 90 years ago. Three specimens were found on Perhentian Kecil Island, off Terengganu State, Malaysia and photographed in the field, and two were collected. This report thus includes the first colour photographs published for this species, from a locality close to the type-locality, Tioman Island (which is ca. 200 km south of the locality in this study, on the east coast of Peninsula Malaysia). We describe the external morphology and colour pattern of the species, which correspond well to the original description, itself based only on two preserved specimens. We performed an in-depth molecular characterisation of the species, including its complete mitochondrial genome, the 18S sequence and elongation 1-alpha (EF1-α) sequence. In addition, EF1-α sequences were also retrieved for 5 additional geoplanid species. No tRNA-Thr could be detected in the mitogenome of B. admarginatum, a lack already reported in several species of geoplanids, but we found a 13 bp sequence that contains the anticodon loop and seems to be conserved among geoplanids and might thus possibly represent a non-canonical undetected tRNA. We discuss the difficulties encountered in trying to reconstruct the cluster of nuclear ribosomal genes, a problem already mentioned for other Triclads. Three phylogenies, based respectively on all mitochondrial proteins, 18S, and EF1-α, were computed; the position of B. admarginatum within the Bipaliinae was confirmed in each tree, as sister-group to various bipaliine species according to the sequences available for each tree. In the mitochondrial proteins tree, which had high support, B. admarginatum was sister to Bipalium kewense and Diversibipalium multilineatum.

The invasive land flatworm Arthurdendyus triangulatus has repeated sequences in the mitogenome, extra-long cox2 gene and paralogous nuclear rRNA clusters

“…Using long-reads sequencing, it was possible to obtain the complete sequences of two paralogous nuclear rRNA gene clusters for A. triangulatus . Similarly to what was already suspected for Bipalium admarginatum de Beauchamp, 1933 54 , these clusters showed different coverages after assembly (Table 2 ), suggesting that their numbers of copies in the nuclear genome are noticeably different. When submitted to a Megablast analysis, the 18S rRNA gene version from the ‘high coverage’ cluster (HCC, OR797297) was found to correspond to type II (99.77% identity with AF033044), while the ‘low coverage’ cluster (LCC, OR797296) corresponded to type I (99.44% identity with AF033038).…”

“…For this species, a D-Loop missing tRNA was found at a position congruent with other species in which this tRNA was found. As in Soo et al 54 , it was possible to find the completely conserved TGT anticodon of a putative tRNA-Thr between the 16S rRNA gene and cytochrome b genes in the other species. However such tRNA would once again have a poorly conserved structure (no cloverleaf shape and missing D- and T-loops) and therefore was not annotated as such for any of these species.…”

“…The dataset previously used to infer a phylogeny of the Geoplanidae 31 , 54 based on 21 mitogenome-encoded proteins was appended with the five new species examined here plus the recently described Dugesia constrictiva Chen & Dong, sp. nov. 95 .…”

“…Another peculiarity not restricted to Rhynchodeminae, but also observed in two families of the superfamily Geoplanoidea (Table 1 ), is the presence of two paralogous clusters encoding the nuclear rRNA genes 40 , 54 – 57 . Aside from representing a biological trait that deserves deeper studies, the existence of these divergent nuclear rRNA gene clusters may be problematic for molecular barcoding and phylogenetic analyses based on nuclear rRNA genes.…”

“…Since the pioneering work by Solà et al 48 , the first to include the description of the complete mitogenome of a geoplanid, i.e., that of Obama nungara (Carbayo, Álvarez-Presas, Jones & Riutort, 2016), several other species have been similarly investigated phylogenomically 31 , 49 – 54 . Amongst these results, one peculiar feature that was noticed was the presence of an unusually long cox2 gene among the three species of Rhynchodeminae studied 50 – 52 , namely Platydemus manokwari de Beauchamp, 1963, Parakontikia ventrolineata (Dendy, 1892) and Australopacifica atrata (Steel, 1897).…”