Discovery of deep-sea acoels from a chemosynthesis-based ecosystem

(a) Sampling

We collected more than 15 hofsteniid acoels (figure 1b) at a depth of 911 m at the off Hatsushima Island seep site in Sagami Bay, Japan (figure 1a) during the research cruise KM21-08 (R/V Kaimei, Japan Agency for Marine-Earth Science and Technology (JAMSTEC)). Bathymodiolus mussels were collected using a suction sampler installed on the remotely operated vehicle (ROV) KM-ROV. The samples from Bathymodiolus aggregations including the bottom sediments were gently stirred in cold seawater (4°C), and the supernatant was filtered through a 500 μm mesh plankton net. We found the acoels and other associated macro and meiobenthic animals including polychaetes, nemerteans, sea spiders and polyclad flatworms by sieving water and sediments collected while sampling Bathymodiolus mussels. Living specimens were photographed on board using a Nikon D5600 Digital SLR Camera (Nikon, Japan). The specimens studied in this study were deposited in the National Museum of Nature and Science, Tokyo (NSMT), Tsukuba, Japan.

(b) Internal morphology

For histological analysis, specimens were anaesthetized with 3.5% MgCl₂ in cold seawater, fixed in Bouin’s fluid for 24 h and later preserved in 70% ethanol. Paraffin samples were sectioned at 6 μm and stained using haematoxylin and eosin stains.

(c) DNA extraction, PCR amplification and sequencing

Total DNA was extracted from specimens preserved in 99% ethanol using the DNeasy Blood & Tissue Kit (Qiagen, Germany) according to the manufacturer’s protocol. To increase the number of OTUs, we additionally sequenced specimens of Hofstenia atroviridis [19] from Misaki (type locality) and an undescribed species from Sugashima, Hofstenia sp. Sugashima (figure 1b).

PCR amplification was performed using the following primers: for partial sequences of cytochrome oxidase c subunit I (COI), LCO1490 and HCO2190 [21]; for 28S rRNA (28S), U178, 1200F, U1846, L1642, R2176, L3449 [22], and the newly designed specific primers for the internal region between the region amplified with 1200F/L1642 and U1846/L3449, Hof_1747_F (5′-CACTGGGCTAAACCGTACCG-3′) and Hof_1845_R (5′-GATCGAGTTTGCCGACTTCC-3′) (present study); for 18S rRNA (18S), TIM A and TIM B [23]. The PCR protocol was as follows: preheating at 94°C for 2 min; 35 cycles of 94°C for 40 s, 52°C for 60−75 s and 72°C for 60 s; then a final extension at 72°C for 7 min. PCR products were purified using Exo-SAP-IT (Thermo Fisher Scientific, USA). Additional internal primers were used for sequencing of 18S: S30, 4FB, 5FK and 1806R [22–25]. After purification using Exo-SAP-IT (Thermo Fisher Scientific, USA), PCR products were sent to FASMAC sequencing service (FASMAC, Japan).

Alignment of COI datasets was performed with the Clustal Omega algorithm [26] implemented in Geneious Prime v. 1.0.18+10, following checking translation into amino acids for stop codons. For 28S and 18S, datasets were aligned by MAFFT v. 7 .490 [27] using the automatically selected strategy L-INS-i. Ambiguous sites were removed by Gblocks v. 0.91b [28] under a less stringent option; a 636 bp for COI (41.0%), a 1840 bp for 28S (36. 5%) and a 1306 bp for 18S (67.4%) remained. After tree topology was checked for each marker, partial sequences of COI, 28S and 18S were concatenated using Geneious Prime.

Phylogenetic analyses were performed using a concatenated dataset based on the partition model GTR + G + I in RAxML v. 8.2.12 [29] implemented in RAxML GUI v. 2.0 [30]. Branch supports were obtained based on 1000 bootstrap replicates. Maximum likelihood (ML) analyses were additionally performed using IQ-TREE software [31] based on partition model (GTR + F + I + G4 for all codon positions of COI and 28S; TIM2 + F + I + G4 for 18S) selected in the IQ-TREE web server [32]. Sequences newly obtained in this study were deposited in GenBank under the following accession numbers: for Hofstenia atroviridis, PP535705 (COI), PP536131 (18S) and PP536128 (28S); for Hofstenia sp. Sugashima, PP535704 (COI), PP536130 (18S) and PP536126 (28S); for Hoftherma hatsushimaensis gen. et sp. nov. PP535703 (COI), PP536129 (18S) and PP536127 (28S). Accession numbers for the other sequences used in our phylogenetic analyses are listed in electronic supplementary material, table S1 (136 OTUs in total).

This article has been registered under the ZooBank (https://zoobank.org/) LSID: urn:lsid:zoobank.org:pub:5CB4747A-9D20-4BEE-A022-788401666434.

(a) Evolution of xenacoelomorphs associated with chemosynthetic-based ecosystem

In our phylogenetic reconstruction based on one mitochondrial and two nuclear genes, we found the newly discovered acoels (figure 1b) nested within the shallow-water family, Hofsteniidae (figure 2, electronic supplementary material, figure S1). These findings prompt questions about the adaptation and evolution of Xenacoelomorpha in chemosynthetic-based ecosystems.

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Within Xenacoelomorpha, the genus Xenoturbella stands as the sole representative of chemosynthetic-based ecosystems, including hydrocarbon seeps, hydrothermal vents and whale-fall sites [14]. Consequently, our results suggested that colonization in reducing environments occurred independently at least twice within Xenacoelomorpha. Notably, most of the known species in Hofsteniidae are carnivorous and primarily feed on copepods, ostracods and flatworms [33]. Acoels presence in chemosynthetic ecosystems may be owing to the rich food resources present there, similar to species of Xenoturbella that feed on bivalves in hydrocarbon seeps [14,34].

Our phylogenetic tree indicated that Hofsteniidae forms a clade with Solenofilomorphidae [35], a family known to inhabit sulfide-rich environments in shallow-water sandy flats [36,37] (figure 2). This relationship suggests a hypothesis: the common ancestor of these two families may have possessed adaptations for life in sulfide-rich environments (figure 2). The shared traits of this ancestor could have facilitated the successful colonization of sulfide-rich ecosystems by the newly discovered species. However, we note that some solenofilomorphids live in sandy sediment that does not contain sulfide, and several Xenoturbella species inhabit oxygenated mud. These exceptions indicate potential parallelisms or reversals in this trait.

Our hypothesis gains further support from the presence of several hofsteniids in low-oxygen sulfide-rich habitats including relatively stable subtidal waters among dead and decaying leaves such as H. miamia and H. giselae (now synonymized with H. miamia) [33]. Apart from exceptions in intertidal species, such as H. atroviridis [20] living among Rhodophyta (red algae) and Hofsteniola pardii [38] among Posidonia seagrasses, where oxygen levels in the water are frequently replenished, similar habitats lend support to a common ancestor inhabited sulfide-rich ecosystems.

The uniform bright pink body coloration of our species distinguished it from other shallow-water hofsteniids, except for H. pardii from Mediterranean, which exhibits a bright-red body coloration but was found in non-hypoxic habitats [38]. The similar coloration in these species with different habitats suggests the need for a comparative study to unravel the potential functions of their pink or red pigmentation. Interestingly, some Xenoturbella species from the chemosynthetic environment are also known for their bright red, pink or purple colouration [14] although specific pigments and their functions in Xenoturbella remain unknown.

Red body coloration often corresponds to the presence of red blood cells (RBCs) containing haemoglobins (Hbs), implying a potential adaptation for oxygen transport crucial for survival in hypoxic environments [39–42]. For example, bivalves with RBCs thrive in sulfide-rich and hypoxic environments, ranging from mangrove swamps [40], burrows of endobenthic invertebrates [41] to deep-sea hydrothermal vents and hydrocarbon seeps [42]. Invertebrate Hbs are distributed in the RBCs and the cytoplasm of muscles, nerves, glial cells and gametes, and are also freely dissolved in vascular, coelomic and perienteric body fluids [43]. Given that acoels lack circulatory systems [15], if the observed body coloration in our species is attributed to Hbs, it is likely originating from the cytoplasm of internal organs or body fluids or parenchymal cells. It is further supported by previous transcriptome analyses revealing the presence of globin orthologues in acoels [43–45]. In-depth physiological and anatomical studies, particularly in comparison with H. pardii, incorporating measurements of oxygen concentration in the environment, are essential to unveiling the functional significance of the pink coloration for their adaptation to the chemosynthetic-based environments.

In conclusion, our findings shed light on the evolution of xenacoelomorphs in chemosynthetic-based ecosystems, suggesting multiple colonization events and potential ancestral adaptations. The distinctive pink coloration of the newly discovered species hints at intriguing physiological adaptations, warranting further research into its functional significance in hypoxic environments.

(b) Taxonomy

Order Acoela Uljanin, 1870 [8]

Family Hofsteniidae Bock, 1923 [20]

Genus Hoftherma gen. nov.

ZooBank LSID: urn:lsid:zoobank.org:act:EB5481E3-6E35-4B06-861F-150532A8D476

Diagnosis: Hofsteniidae with pharynx one-half the body length (figure 3c); pharynx musculature well developed (figure 3c); seminal vesicles enclosed in a thick muscular wall (figure 3f1,f2); dorsal body wall with two longitudinal furrows (figures 1b and 3a). Testes diffuse (figure 3g).

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Etymology: The generic name ‘Hoftherma’ is derived from elements of the Hofsteniidae family name and the Latin word ‘therma’, which means hot spring, from the Greek therme.

Hoftherma hatsushimaensis gen. et sp. nov.

[New Japanese name: Hatsushima-shiawase-uzumushi]

ZooBank LSID: urn:lsid:zoobank.org:act:BD9F1EF4-557F-4C46-8957-B0EFB88B0A90

Type material: Holotype: NMST-Xe−745, unsectioned complete specimen, fixed in Bouin’s fluid, collected on 24 October 2021 by N.H. and N.J., at a depth of 911 m, off Hatsushima (35°00.94′ N, 139°13.38′ E), Sagami Bay, Kanagawa, Japan (figure 1a). Paratypes: NMST-Xe-746, unsectioned complete specimen, fixed in Bouin’s fluid, collected on the same date and locality as the holotype. NMST-Xe-747-749, unsectioned complete specimen, fixed and preserved in 99% ethanol, collected on the same date and locality as the holotype. NMST-Xe-750, serial frontal sections, four slides. NMST-Xe-751, serial transverse sections, three slides. NMST-Xe-752, serial sagittal sections, three slides.

Description: Living specimens up to 4.8 mm long. Uniformly pink coloured without body pigmentation (figure 1b). Body shape anteriorly rounded and posteriorly thinner (figures 1b and 3a). Body shape frequently changes by contracting and elongating when alive. Mouth opening subterminal. Eye spots not observed. Statocyst present dorsoanteriorly (figure 3b). Body wall with epidermis, outer circular and inner longitudinal muscles, parenchyma and gastrodermis present (figure 3b,c). Pharynx musculature well developed (figure 3b,c). Male gonopore opening ventral to mouth (figure 3d1). Male copulatory organ located ventral to pharynx (figure 3d1–3), composed of male antrum (figure 3d1), eversible penis with a penis stylet (figure 3d2, e2, f1), vesicula granulorum and seminal vesicle (figure 3e1,d3). Seminal vesicle enclosed in a thick muscular wall (figure 3f1,f2); seminal vesicle muscular wall with dense radially developed muscle fibres (figure 3f2). Testes diffuse (figure 3e3). Female gonopore absent. Eggs are ventrolaterally situated on both sides of pharynx (figure 3g); oocytes 75 μm in the maximum diameter (figure 3g).

Etymology: The species name is derived from the type locality, off Hatsushima in Sagami Bay, Japan.

Type locality and distribution: The species is only known from the type locality, off Hatsushima, Sagami Bay, Kanagawa Prefecture, Japan, at a depth of 911 m (figure 1a).

Ecological notes: While sampling Bathymodiolus aggregations with a manipulator arm installed on an ROV, more than 15 specimens of H. hatsushimaensis gen. et sp. nov. were collected (some of the specimens were damaged during collection). Additionally, we also collected many polychaetes, nemerteans, sea spiders and polyclad flatworms found in the area.

Taxonomic remarks: Hoftherma hatsushimaensis gen. et sp. nov. shares several characteristics with hofsteniid species: the mouth opening subterminal; the muscular pharynx approximately half the length of its entire body (figure 3a,c); the male copulatory organ positioned anteriorly and ventral to the pharynx (figure 3d1–3); a female gonopore lacking. Our phylogenetic analysis robustly supports the phylogenetic position of H. hatsushimaensis gen. et sp. nov. within the Hofsteniidae clade (figure 2); the clade encompasses five species in two genera in the family, including the type species of Hofsteniidae, H. atroviridis, sequenced based on the topotype specimen from Misaki (figures 1b and 2).

Hoftherma hatsushimaensis gen. et sp. nov. is differentiated from two of the three hofsteniids genera, Hofstenia and Marcusiola, in having a seminal vesicle enclosed in muscular wall (figure 3f2), while in the latter two genera, a seminal vesicle is not enclosed in muscular wall (table 1). This new species is the most similar to the remaining genus, Hofsteniola ( = H. pardii), with its pink-coloured body and the seminal vesicle enclosed in muscular wall (figure 3f2). However, it can be clearly distinguished from H. pardii in having diffuse testes (figure 3e3); while testes are compact in H. pardii. This distinction is further supported by our ML analysis, where the two species do not form a clade, and the uncorrected p-distance based on a 549 bp COI sequence (19.2%) was higher than interspecific values in Acoela (e.g. [47]). Morphological differences between H. hatsushimaensis gen. et sp. nov. and other members in Hofsteniidae are summarized in table 1.

Given its phylogenetic position sister-related with all the other hofsteniids used in our analyses (figure 2), a reassessment of the generic identification of ‘Hofstenia’ sp. AW-2007b is required for taxonomic reappraisal involving Hofstenia, Hofsteniola and our new species. However, morphological data on ‘Hofstenia’ sp. AW-2007b are currently unavailable. Therefore, we propose the establishment of the new genus, Hoftherma gen. nov., based on discernible morphological differences from other species in Hofsteniidae, deferring the taxonomic reappraisal to future studies.