Research Article |
Corresponding author: Romain Gastineau ( romain.gastineau@usz.edu.pl ) Academic editor: David Gibson
© 2022 Romain Gastineau, Leigh Winsor, Jean-Lou Justine.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Gastineau R, Winsor L, Justine J-L (2022) The complete mitogenome of the potentially invasive flatworm Australopacifica atrata (Platyhelminthes, Geoplanidae) displays unusual features common to other Rhynchodeminae. ZooKeys 1110: 121-133. https://doi.org/10.3897/zookeys.1110.83228
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We sequenced the complete mitochondrial genome of the flatworm Australopacifica atrata. The species, originally described from New South Wales, Australia, has been found in various locations in the British Isles, New Zealand and in the United States of America; it is thus potentially invasive. The genome is 16513 bp long, encodes for 12 protein coding genes, two ribosomal RNA genes and 20 tRNA genes, and is completely colinear with the other two available Rhynchodeminae. In addition, it shares with them some unusual characters discriminating them from members of the other subfamilies of Geoplanidae, the most noticeable being the extra length of its cox2 gene. The data allow a reliable multigene phylogeny to be derived, and also provide a means of accurate biomonitoring of possible invasiveness by A. atrata.
Biomonitoring, Continenticola, cox2 gene, multigene phylogeny, next generation sequencing, Parakontikia ventrolineata, Tricladida
In the recent years, an increasing number of reports have emerged from Europe and abroad concerning invasive terrestrial flatworms of the family Geoplanidae Stimpson, 1857 (
Two species, smaller than the aforementioned large ones and with a mostly scavenging behaviour, are also potentially invasive (
In recent years, several complete mitochondrial genomes from different species of terrestrial invasive flatworms have been sequenced (
Here we report the complete mitochondrial genome of a morphologically identified specimen of A. atrata and compare it with other species. We provide further evidence concerning the specific features of the mitogenomes of Rhynchodeminae compared to land flatworms in other subfamilies. We present a multigene phylogeny demonstrating its proximity with Pa. ventrolineata. Finally, we discuss the next steps that should take place in the investigation of invasive terrestrial flatworms by means of next generation sequencing.
All specimens were collected from a native plant nursery in Maffra, Victoria, Australia (37°57'S, 146°59'E), from November 2019 to February 2020 and identified on the basis of external morphology and colour pattern (
A tissue sample in 95% ethanol was sent to the Beijing Genomics Institute (BGI) in Shenzhen, China, where DNA extraction and sequencing took place. A total of ca 40 million of 150 base pair clean paired-end reads was obtained on a DNBseq platform. The reads were assembled using SPAdes 3.14.0 (
A phylogeny was inferred with the amino-acid sequences of the conserved mitochondrial proteins, following a protocol adapted from
The mitogenome was retrieved among the other contigs from the assembly file, in the form of a contig containing all conserved coding parts, with redundant extremities which allowed us to circularize it. The genome has been deposited on GenBank with accession number OM456243.
The mitogenome is 16513 bp long. Its composition is 4810 A, 1248 C, 2461 G and 7994 T. It encodes for 12 protein coding genes, 2 ribosomal RNA genes and 20 t RNA genes (Fig.
The maximum likelihood phylogenetic tree (Fig.
Maximum Likelihood phylogenetic tree obtained from concatenated amino-acid sequences of the mitochondrial proteins of Australopacifica atrata (Steel, 1897) and other flatworms. The tree with the best likelihood is shown (-75607.404300). Subfamilies of Geoplanidae are indicated on the right.
The mitogenome of A. atrata is shorter than those of the two other Rhynchodeminae Pl. manokwari (19959 bp, MT081580) and Pa. ventrolineata (17210 bp, MT081960), but larger than the largest recorded for a Bipaliinae, Bipalium vagum Jones & Sterrer, 2005 (17149 bp, MZ561468) or the Geoplaninae, O. nungara (14909 bp, KP208777) and Amaga expatria (14962 bp, MT527191). However, the presence of repeated sequences in the mitogenome of Geoplanidae has been strongly suggested (
So far, the Rhynchodeminae can be differentiated from Bipaliinae and Geoplaninae by the position of the t RNA-Cys. For A. atrata, Pl. manokwari and Pa. ventrolineata, it is located between the protein coding genes cox3 and atp6, clustering with t RNA-Ile, t RNA-Gln and t RNA-Lys, as the first t RNA of this cluster. For B. kewense as well as O. nungara and A. expatria, it is located between the protein coding gene ND2 and the 12S rRNA gene, clustering with t RNA-Met and t RNA-His, located after these two t RNA. Another difference regarding t RNA is the apparent lack of a tRNA-Thr among all Rhynchodeminae. Among the other species, t RNA-Thr has been found between the 16S rRNA gene and the protein-coding cob gene, clustering with t RNA-Leu and t RNA-Asn. It is worth emphasising here that there is a difference between the Geoplaninae, in which the order of this cluster is 16S, t RNA-Thr, t RNA-Leu, t RNA-Asn, cob and B. kewense, in which t RNA-Leu and t RNA-Thr exchange their positions. However, it might be noted that for the recently described species of Bipaliinae, Humbertium covidum Justine, Gastineau, Gros, Gey, Ruzzier, Charles & Winsor, 2022 (
Several studies failed to find a start codon for various mitochondrial genes of terrestrial flatworms such as O. nungara, A. expatria, Diversibipalium multilineatum Makino & Shirasawa, 1983 and B. vagum (
For all three Rhynchodeminae, the ND5 gene has an early termination because of the presence of tRNA-Ser immediately following the last T residue of the gene, for which we suspect that the functional TAA stop codon is obtained by being completed by the addition of 3’ A residues to the mRNA, while a canonical stop codon was found for all other species. Also, it is interesting to note that there is an overlap between the ND4L and the ND4 genes, and that the size of this overlap is always 32 bp. This character, which is common to Pl. manokwari and Pa. ventrolineata, has been also reported in O. nungara and C. alpina. A rapid investigation of the mitogenomes of all available Bipaliinae shows that this overlap is totally lacking. Trying to simulate this overlap by extending the open reading frame at its N terminal ending leads to a fictious polypeptide with no initial methionine but that may comprise several stop codons in its early part.
As was observed in both the other Rhynchodeminae, the cox2 gene has an important extra length. This extra length does not result from a missing stop codon, as it is located in the middle of the gene, and not on the 3’ extremity. The size of the cox2 putative protein of A. atrata is nearly identical to those of Pa. ventrolineata (434 and 433 amino acids respectively) (Table
Sizes in amino acids (AA) of the cox2 proteins encoded by the available mitogenomes of Continenticola (Tricladida).
Name | Family | GenBank accession number | Size of the putative cox2 protein (in AA) | Specific features |
---|---|---|---|---|
Schmidtea mediterranea | Dugesiidae | JX398125 | 292 | Start codon not determined |
Girardia sp. | Dugesiidae | KP090061 | 389 | Start codon not determined |
Girardia tigrina | Dugesiidae | MW972220 | 389 | Start codon not determined |
Dugesia japonica | Dugesiidae | AB618487 | 227 | NA |
Dugesia ryukyuensis | Dugesiidae | AB618488 | 230 | TAA stop codon completed by the addition of 3’ A residues to the mRNA |
Crenobia alpina | Planariidae | KP208776 | 239 | NA |
Phagocata gracilis | Planariidae | KP090060 | 297 | NA |
Obama nungara | Geoplanidae | KP208777 | 259 | Start codon not determined |
Amaga expatria | Geoplanidae | MT527191 | 260 | Start codon not determined |
Bipalium kewense | Geoplanidae | MK455837 | 225 | NA |
Bipalium vagum | Geoplanidae | MZ561468 | 229 | NA |
Bipalium adventitium | Geoplanidae | MZ561467 | 227 | NA |
Diversibipalium multilineatum | Geoplanidae | MZ561469 | 228 | NA |
Diversibipalium mayottensis | Geoplanidae | MZ561470 | 246 | NA |
Humbertium covidum | Geoplanidae | MZ561471, MZ561472 | 248 | NA |
Platydemus manokwari | Geoplanidae | MT081580 | 452 | NA |
Parakontikia ventrolineata | Geoplanidae | MT081960 | 433 | NA |
Australopacifica atrata | Geoplanidae | OM456243 | 434 | NA |
Following this work, we see three major paths of investigation. The first one will continue to focus on Rhynchodeminae. It will be interesting to compare all the characters considered here (colinearity, composition in t RNA, type of termination for the ND5 gene, overlap between ND4L and ND4 and of course the extra length of the cox2 gene) with more species. Of the five tribes of Rhynchodeminae, we have now sequenced representatives of two: the Rhynchodemini (i.e. Pl. manokwari) and the Caenoplanini (Pa. ventrolineata and A. atrata). However, living examples of many of the other species assigned to the remaining tribes will be difficult to find considering their origin and repartition, but at least one of them, Anzoplanini Marionfyfea adventor Jones & Sluys, is present in Europe (
We are most grateful to Mitch Findlater-Smith for collecting specimens of A. atrata, and to Reiner Richter for kindly allowing us permission to use his photographs of A. atrata supplied as ARR05976.JPG and ARR05978.JPG under the CC-BY 4.0 license. ProSciTech Pty. Ltd., Townsville, Queensland, is thanked for generously donating consumables for specimen collection. We also thank Prof. Hugh D. Jones and Dr Marta Álvarez-Presas for kindly sharing with us their molecular barcoding results.