Amoenothamnion planktonicum

West, John A., 2023, Amoenothamnion planktonicum (Heterothamnieae Ceramiaceae, Rhodophyta), an annual drift event along sandy beaches of southern Australia and its affinities, Notulae Algarum 271, pp. 1-7 : 1-4

Amoenothamnion planktonicum is now known from Dongara, Western Australia to Gabo I., Victoria, and south-eastern Tasmania to Twofold Bay, New South Wales ( Wollaston 1968; Womersley 1998).

Athanasiadis (1996: 184) established a new genus Leptoklonion Athanasiadis based upon L. elongatum (E.M.Wollaston) Athanasiadis , later referred by Womersley (1998: 190) to Leptoklonion fastigiatum (Harvey) Womersley (type from Georgetown, Tasmania).

Amoenothamnion was referred to the tribe Heterothamnieae E.M.Wollaston by Wollaston (1968: 407) and by Womersley & Wollaston in Womersley (1996: 156). The tribe Heterothamnieae currently includes Acrothamniopsis Athanasiadis & Kraft , Amoenothamnion , Elisiella Womersley , Heterothamnion J.Agardh (the type), Tetrathamnion E.M.Wollaston and Trithamnion E.M. Wollaston ( Guiry & Guiry 2023) . However, no molecular information is currently available for the types of any of these genera.

Samples of Amoenothamnion planktonicum were collected on 10 January 2021 by John Miller, at Bridgewater Bay, Victoria. The samples were collected in waist-deep water about 10 m from the shore using a small, hand-held dip-net. Collecting in the water rather than beach-washed specimens ensured that they were fresh, intact, and largely free of sand and other debris. The specimens were then transferred to a small seawater-filled dish on the beach where the samples were further sorted and cleaned of any obvious detritus, and prepared in four ways for transport:

1. Some specimens were added to vials with 5% Formalin in seawater for morphological examination.

2. Some were pat-dried with tissues and placed in small plastic clip-lock bags containing silica crystals.

3. Some specimens were dried and mounted as voucher specimens.

4. Some specimens were added to small vials containing just seawater from the site.

All specimens were processed and packaged the same day and mailed to J.A. West. Living specimens were observed and photographed on the stereomicroscope and compound scope. Silicadried specimens were prepared for molecular analyses.

DNA was extracted from the dried Amoenothamnion sample in silica using a CTAB method described by Cremen & al. (2016). An Illumina sequencing library was prepared with the VAHTS Universal DNA kit and ca. 3 gigabases of sequencing data was generated on the Novaseq platform (paired-end, 150 bp) by Azenta. Raw sequencing data were assembled with megahit v.1.2.9 ( Li & al. 2016). Plastid genome contigs were identified and gene predictions were made as described elsewhere ( Costa & al. 2016; Marcelino & al. 2016). The rbc L gene was extracted from the contig and used in downstream analysis. The SSU gene was extracted from the same dataset.

For phylogenetic analyses, sequences of the rbc L and SSU were added to sequences downloaded from GenBank and aligned using MAFFT in Geneious Prime ® 2019.2.3. Phylogenetic analyses were performed using the maximum likelihood (ML) method, the best-fit model of each gene sequence was determined with IQTREE2 ( Minh & al. 2020) and ModelFinder ( Kalyaanamoorthy & al. 2017). The rbc L was partitioned by codon position and separate DNA substitution models were chosen for each codon. The SSU was not partitioned. Support for individual internal branches was determined by non-parametric bootstrap (500 replicates) ( Felsenstein 1985).

Bayesian inference analysis also partitioned rbc L by codon position and implemented in MrBayes3 ( Ronquist & Huelsenbeck 2003). The model partitions were unlinked, with variable rates, and six rate categories. Two parallel runs of four Markov chain Monte Carlo were performed for 3,000,000 generations, sampling every 1,000 generations. Estimated samples size, split frequencies, and stationarity were checked after each run. Post-analysis, 10% of generations were removed as a burn-in and posterior probabilities visualized. Figtree v1.1.4 ( Rambaut 2009) and Canvas X Draw (Canvas GFX, Inc., Boston, MA, USA) were used to manipulate trees for presentation. Species of Rhodomelaceae were used as outgroups (e.g. Melanothamnus ) for rbc L and Schimmelmanniaceae (e.g. Schimmelmannia ) for the SSU datasets.

With our rbc L dataset (1467 bp alignment, model used: 1 st codon–GTR+F+I+G4; 2 nd codon: TIM+I+G4; 3 rd codon: TIM +F+I+G4), the sample of Amoenothamnion planktonicum from Bridgewater Bay groups with members of the Ceramiaceae (e.g. Antithamnion , Ceramium , Pterothamnion ; 74% ML BS, 1.0 PP). Its relationships within this clade are unsupported with this dataset. It is excluded, for example, from the clade containing Antithamnion and Hollenbergia . Many of the generic relationships seen in this dataset indicate that further taxonomic work is needed as many genera are not monophyletic ( Ceramium , Hollenbergia, Griffithsia ).

Our SSU dataset (1819 bp alignment, model used: TN+F+I+G4), again shows that Amoenothamnion planktonicum does not group with any particular genus with any support. It is in an unsupported polytomy with Tetrathamnion sp. GWS464 (and Ceramium and Centroceras ), but is excluded from the clade containing Heterothamnion sp. GWS460 (and Antithamnionella spp. ). Both Heterothamnion and Tetrathamnion are currently assigned to the tribe Heterothamnieae ( Guiry & Guiry 2023) , along with Amoenothamnion . The monophyly of this tribe is thus questionable and further work is needed, as several genera have not been sequenced, plus many backbone relationships within the Ceramiaceae are unsupported with the SSU data set, as they are with rbc L. Nevertheless there are indications in Figs 2 and 3 that the tribe Heterothamnieae is sister to the tribe Ceramieae .

Funding was provided by the Australian Biological Resources Council (Activity ID 4-G046WSD to HV) and computational work was carried out on the Melbourne Research Cloud (Research Computing Services, University of Melbourne). The cost of field observations and collections was supported with personal funds of Joan Powling and John Miller. Microscopy and the preparation of photographic plates were supported with personal funds of John West and Susan de Goër.

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Figures

Fig. 2. Phylogenetic relationships based on ML analysis of the rbcL data set (1467 base pairs) of Amoenothamnion planktonicum from Bridgewater Bay, Victoria (in bold).

Fig. 3 Phylogenetic relationships based on ML analysis of the SSU data set (1819 base pairs) of Amoenothamnion planktonicum (in bold).