Diversity of Deep-Sea Scale-Worms (Annelida, Polynoidae) in the Clarion-Clipperton Fracture Zone

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ORIGINAL RESEARCH published: 25 August 2021 doi: 10.3389/fmars.2021.656899

Paulo Bonifácio1,2*, Lenka Neal3 and Lénaïck Menot2

1 Independent Researcher, Brest, France, 2 Ifremer, Centre Bretagne, REM EEP, Laboratoire Environnement Profond, Plouzané, France, 3 The Natural History Museum, London, United Kingdom

The polymetallic nodules lying on the seafloor of the Clarion-Clipperton Fracture Zone (CCFZ) represent over 30 billion metric tons of manganese. A single mining operation has potential to directly impact approximately 200 km2 of the seabed per year. Yet, the biodiversity and functioning of the bentho-demersal ecosystem in the CCFZ remain poorly understood. Recent studies indicate a high species diversity in a food-poor environment, although the area remains poorly sampled. Undersampling is aggravated by a combination of low densities of fauna and high habitat heterogeneity Edited by: Erica Goetze, at multiple spatial scales. This study examines the Polynoidae, a diverse family of University of Hawai‘i at Mânoa, mobile polychaetes. Sampling with an epibenthic sledge and a remotely operated United States vehicle was performed during the cruise SO239 within the eastern CCFZ. Five areas Reviewed by: Gustavo Fonseca, under the influence of a sea surface productivity gradient were visited. Specimens were Federal University of São Paulo, Brazil identified using morphology and DNA: (i) to provide a more comprehensive account Pat Hutchings, of polynoid diversity within the CCFZ, (ii) to infer factors potentially driving alpha and Australian Museum, Australia beta diversity, and (iii) to test the hypothesis that epibenthic polychaetes have low *Correspondence: Paulo Bonifácio species turnover and large species range. Patterns of species turnover across the [email protected] eastern CCFZ were correlated with organic carbon fluxes to the seafloor but there was also a differentiation in the composition of assemblages north and south of the Specialty section: This article was submitted to Clarion fracture. In contrast to the previous studies, patterns of alpha taxonomic and Deep-Sea Environments and Ecology, phylogenetic diversity both suggest that polynoid assemblages are the most diverse at a section of the journal Frontiers in Marine Science Area of Particular Environmental Interest no. 3, the most oligotrophic study site, located Received: 21 January 2021 north of the Clarion fracture. Without ruling out the possibility of sampling bias, the Accepted: 19 July 2021 main hypothesis explaining such high diversity is the diversification of polynoid subfamily Published: 25 August 2021 Macellicephalinae, in response to oligotrophy. We propose that macellicephalins evolved Citation: under extremely low food supply conditions through adoption of a semi-pelagic mode Bonifácio P, Neal L and Menot L (2021) Diversity of Deep-Sea of life, which enabled them to colonise new niches at the benthic boundary layer and Scale-Worms (Annelida, Polynoidae) foster their radiation at great depths. in the Clarion-Clipperton Fracture Zone. Front. Mar. Sci. 8:656899. Keywords: diversity and distribution, Clarion-Clipperton Fracture Zone, nodule province, scale-worms, doi: 10.3389/fmars.2021.656899 Polychaeta, diversification

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INTRODUCTION variable at a 100-km scale although the influence of productivity gradients is not as clear (Simon-Lledó et al., 2020). Polymetallic nodules are potato-shaped structures varying in Understanding the ecology of benthic communities in the size and mineral concentration, and patchily distributed on CCFZ is however still impaired by the incomplete diversity the seafloor (Morgan, 2000). They are mainly composed of assessment. For polychaetes, a species-rich group at abyssal manganese and iron, but also copper, nickel, and cobalt (Hein depths representing 36–55% of total macrofaunal abundances and Petersen, 2013). The Clarion-Clipperton Fracture Zone (Hessler and Jumars, 1974; Hecker and Paul, 1979; De Smet et al., (CCFZ), an area of ca. 6 million km2 of seabed in the central 2017; Chuar et al., 2020), the incomplete species inventories can Pacific, has attracted increasing commercial interest. This largest be attributed to undersampling, species lumping and sampling polymetallic nodule field in the world sits between 4000 and inaccuracy. Undersampling is visible at all scales. At local scale, 6000 m depth. The CCFZ potentially holds 34 billion metric tons the species rarefaction curve did not level off after extensive of manganese, representing at least 25 trillion USD (Morgan, sampling of 54 box-cores at Domes A in the western and most 2000; Volkmann et al., 2018). This area is managed by the oligotrophic site of the CCFZ (Wilson, 2017; Washburn et al., International Seabed Authority (ISA), which issues exploration 2021). In the eastern CCFZ, a total of 30 box-cores across mining contracts. To date, 18 such contracts have been signed four contract zones and one APEI yielded 275 species of which with the latest one in 2021 (International Seabed Authority, 49% were singletons (Bonifácio et al., 2020). Moreover, most 2021). When moving from exploration to exploitation, a single polychaete species remain undescribed (only 5–10% of collected mining operation could directly impact 182 km2 year−1 of polychaete species were identified to named species; Glover seafloor to achieve a production of 2 Mt annually, while et al., 2002) and the recent combination of morphological and sediment plume re-deposition might indirectly increase the molecular criteria to delineate species suggests that morphology footprint of mining by a factor of two to five (Oebius et al., significantly underestimates the magnitude of biodiversity 2001; Glover and Smith, 2003; Volkmann and Lehnen, 2018). (Janssen et al., 2015; Bonifácio and Menot, 2018). Sampling The ISA has approved a regional management plan that has needs to be more comprehensive because polychaetes encompass designated nine zones each measuring 400 × 400 km, known a large range of sizes and life modes, from minute infaunal as Areas of Particular Environmental Interest (APEIs). Such to large epibenthic and commensal species (Hutchings, 1998). areas are protected from the mining activities and expected to In the CCFZ, polychaete assemblages are sampled with a be representative of the full range of biodiversity, ecosystem box core, in accordance with the recommendations issued by structure, and habitats within the management area (Lodge the ISA (ISBA/25/LTC/6/Rev.1, International Seabed Authority, et al., 2014). These nine APEIs are located at the periphery of 2020). While macro-infaunal polychaetes are quantitatively and the CCFZ, however, their location is currently not completely accurately sampled with a box core (Hessler and Jumars, 1974), supported by the scientific data. Unsupervised classification of large epifaunal and commensal species are not. Such groups are benthic habitats based on derivatives of the GEBCO bathymetry, better targeted by trawls and epibenthic sledges (EBS). Among particulate organic carbon (POC) fluxes estimated from satellite the poorly sampled polychaetes, EBS samples showed that the data and nodule abundances derived from low-resolution kriging family Polynoidae is a highly diverse yet poorly studied group suggested that habitats in the network of APEIs are not fully at abyssal depths (Schüller et al., 2009; Guggolz et al., 2018; representative of habitats within mineable areas (McQuaid Bonifácio and Menot, 2018). et al., 2020). To provide quality knowledge for management Of all polychaetes, Polynoidae is one of the most diverse and conservation strategies, we need to better constrain families, both in the number of genera and species (868 valid habitat distribution models, which in turn requires enhanced species; Read and Fauchald, 2021). Polynoids belong to a group of comprehension of the factors determining biodiversity patterns. organisms called scale-worms (Aphroditiformia), distinguishable The CCFZ is a heterogeneous environment composed by their scale-like dorsal elytra. Of the eight subfamilies of of abundant hills (approximately 200 m high), numerous Polynoidae recognised by Bonifácio and Menot(2018) and seamounts, and nodule fields, which may explain why followed in this study, the subfamily Macellicephalinae appears biodiversity appears to be richer than previously thought to be restricted to the deep sea, the deep Antarctic shelf, and (Smith et al., 2008; Wedding et al., 2013; Glover et al., 2016). submarine caves (Pettibone, 1985b; Neal et al., 2018b; Bonifácio For example, a megafauna diversity assessment within APEI and Menot, 2018). In a census of deep-sea polychaete species, no. 6 found 129 morphospecies in a survey covering 15,840 m2 Paterson et al.(2009) counted 91 polynoid species (12% of of seabed. Changes in assemblage composition were associated total polychaete records) below 2000 m depth with 15 polynoid with variations in geomorphology and nodule abundance species below 4000 m depth (hadal depths), 13 of these belonging (Simon-Lledó et al., 2019). Beyond species restricted to hard to Macellicephalinae, including the deepest known polynoid substrates, the presence of nodules may also increase the diversity found at 10,190 m depth (Kirkegaard, 1956). According to of macro-infauna at a local scale (De Smet et al., 2017; Bonifácio Bonifácio and Menot(2018), the subfamily Macellicephalinae et al., 2020; Chuar et al., 2020). On a regional scale, northward forms a monophyletic group characterized by the loss of and westward gradients of decreasing primary productivity are the lateral antennae compared to other polynoid subfamilies, important drivers of variations in meiofaunal and macrofaunal which bear two lateral and one median antennae. Within the community structure (Hauquier et al., 2019; Bonifácio et al., Macellicephalinae, which currently contains 121 species (Read 2020). The structure of megafaunal assemblages is also highly and Fauchald, 2021), a monophyletic clade of 15 species, the

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so-called Anantennata clade, also lost the median antenna. of distance separates BGR and IOM areas whereas 1440 km Macellicephalins seem to have been particularly successful in separates BGR and Ifremer or APEI no. 3. colonizing and radiating in the deep sea (Uschakov, 1982; Levenstein, 1984; Bonifácio and Menot, 2018). Numerous Sampling Strategy genera are endemic to deep-sea chemosynthetic ecosystems The overarching aim of the sampling strategy was to cover the such as hydrothermal vents and cold seeps (Pettibone, 1983; whole range of biodiversity of benthic communities, crossing all Chevaldonné et al., 1998; Hatch et al., 2020), whereas others were faunal size groups (from meio- to megafauna) and habitats (from successful in colonizing pelagic deep-sea, nodule fields, abyssal soft-sediments with no nodules to basalt on seamounts). A variety depths, and even trenches (Pettibone, 1976, 1985a,b; Bonifácio of methods were used to collect biological samples from large and Menot, 2018). Predating the discovery of hydrothermal and qualitative EBS samples to smaller and quantitative box-core vents, Levenstein(1984) studied macellicephalin distribution samples and targeted samples with a ROV. around the world and pointed out that the Pacific Ocean hosts The EBS (Brenke, 2005) consists of a supra- and epibenthic a high rate of diversity with 21 of the 40 species (known at that net with cod ends of 300 µm each and an opening and time) and 15 endemic species. closing mechanism. A total of 12 EBS were recovered but From EBS and remotely operated vehicle (ROV) samples only eight were fully examined (Table 1). The ROV Kiel collected during the SO239 cruise across the eastern half of 6000 fitted with various sampling tools was also used to the CCFZ (Martínez Arbizu and Haeckel, 2015), Bonifácio and recover benthic macrofauna (Figure 1). One of the features Menot(2018) described 17 new polynoid species, of which 16 employed was the bio-box, a large box in which megafaunal were macellicephalins, with many remaining undescribed. In specimens collected with the manipulator arm were stored. the present study, we aim to provide a more comprehensive The United States Naval Electronics Laboratory (USNEL) account of polynoid diversity within the CCFZ and improve our spade box corer of 0.25 m2 (Hessler and Jumars, 1974) is understanding of macellicephalin species radiation in the deep proven to be an accurate and quantitative tool for benthic sea. Additionally, we aim to further test hypotheses regarding the biological studies. drivers of species turnover in the CCFZ. Based on quantitative Polynoids were recovered from box corer and EBS box-core sampling, Bonifácio et al.(2020) showed a high species deployments, as well as from the ROV bio-box. Polynoids turnover among infaunal polychaete assemblages across the were not intentionally sampled using the ROV but were most eastern CCFZ, attributing it to variations in trophic inputs and likely associated with the collected sponges or corals. Once on barriers to dispersal. In particular, the Clarion fracture was board, the megafauna specimens were sorted from the bio-box hypothesized to limit dispersal between the APEI no. 3 to the and the water was sieved through a 300 µm mesh in a cold room north and the core of the CCFZ to the south. Dispersal ability (full methods in Martínez Arbizu and Haeckel, 2015). Polynoids has also been advocated as a driver of differential distribution were sorted from the sieved residues. patterns between polychaete and isopods as well as among isopod Sieving and sorting were performed on board. The samples families in the CCFZ (Janssen et al., 2015, 2019; Brix et al., were maintained in cold seawater (4◦C) and sieved through 2020). By focusing on polynoids, we aspired to test whether a 300 µ mesh in a cold room. All specimens from ROV mobile epifaunal polychaetes would show lower species turnover sampling and some specimens from box corer and EBS sampling and greater species ranges than the more sedentary infaunal were sorted alive. The upper 10 cm of the box-core sample polychaete assemblages. was sliced into three layers (0–3, 3–5, and 5–10 cm), the first was sieved on board in the cold room with cold seawater (4◦C) whereas the deeper layers were fixed in formalin for MATERIALS AND METHODS 48–96 h, preserved in 96% ethanol and sorted back on land (for detailed processing of box corer sampling, see Bonifácio et al., Clarion-Clipperton Fracture Zone 2020). Sieving residues from the EBS samples were preserved Within the Equatorial Pacific Ocean, the CCFZ is bordered by in 96% ethanol at −20◦C. The ethanol was changed after the Clarion fracture to the north, the Clipperton Fracture to 24–48 h and the sieved residues were then sorted on board the south, the Kiribati islands to the west, and Mexico to the under ice. The collected polychaetes were fixed/preserved in cold east (Figure 1). As part of the JPI Oceans project “Ecological (−20◦C) 80% ethanol and stored at −20◦C. In the laboratory aspects of deep-sea mining,” the EcoResponse cruise SO239 on (on land), a few parapodia or small pieces of tissue were board the RV Sonne covered the eastern part of the CCFZ dissected, preserved in cold 96% ethanol, and stored at −20◦C from March 9 to April 30, 2015 (Martínez Arbizu and Haeckel, for molecular extraction. 2015). Sampling took place within four exploration contract areas and the APEI no. 3 at water depths ranging from 4000 to Molecular Methods 5000 m (Figure 1). While the ISA administers the APEIs, the Briefly, the DNA was extracted from sampled tissues using a exploration contracts were issued by ISA to the Federal Institute NucleoSpin Tissue kit (Macherey-Nagel). Two mitochondrial for Geosciences and Natural Resources of Germany (BGR); genes (i.e., COI, cytochrome c oxidase subunit I; and 16S) the InterOceanMetal Joint Organization (IOM); the G-TEC Sea and one nuclear gene (18S) were amplified using the following Mineral Resources NV (GSR); and the Institut Français de primers: polyLCO, polyHCO, LCO1490, and HCO2198 for COI Recherche pour l’Exploitation de la Mer (Ifremer). Only 243 km (Folmer et al., 1994; Carr et al., 2011); Ann16SF and 16SbrH

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FIGURE 1 | (A) Map of the nodule exploration contract areas, reserved areas and Areas of Particular Environmental Interest (APEIs) in the Clarion-Clipperton Fracture Zone (CCFZ) showing the sampled areas (in color). The background map shows the average particulate organic carbon (POC) ﬂux at the seaﬂoor during the 2002–2018 period estimated by Bonifácio et al.(2020). The sampled areas are enlarged in the following panels: BGR (B,C), IOM (D), GSR (E), Ifremer (F) and APEI no. 3 (G), with start positions in white and end positions in grey. Each has a detailed local hydro-acoustic map based on the multibeam system EM122 (Martínez Arbizu and Haeckel, 2015; Greinert, 2016) in the background.

for 16S (Palumbi, 1996; Sjölin et al., 2005); and 18SA, 18SB, (for 16S or 18S) or for 40 cycles (for COI)] – 72◦C/480 s. 620F, and 1324R for 18S (Medlin et al., 1988; Cohen et al., 1998; PCR products which resulted in bands of expected size after Nygren and Sundberg, 2003) for 18S. The polymerase chain electrophoresis on 1% agarose gel, were sent to the MacroGen reaction (PCR) mixtures were prepared as suggested for the Europe Laboratory in Amsterdam (Netherlands) to be sequenced Green GoTaq R by the manufacturers. The proﬁle of temperature with the same set of primers. was as follows: 95◦C/240 s – [94◦C/30 s – 52◦C/60 s – Overlapping sequence fragments (forward and reverse) were 72◦C/75 s (for COI and 16S) or 180 s (for 18S) for 35 cycles assembled into consensus sequences using Geneious Pro 8.1.7

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TABLE 1 | Details of sampling sites, total number of polynoid specimens (ind., individuals), and number of polynoid species collected from epibenthic sledges (EBS), box corer, and ROV deployments across the eastern Clarion-Clipperton Fracture Zone during the SO239 cruise.

Area – Station Date Depth (m) Sampling Sampling Sampling Sampling Trawling Number of Total species locality (dd/mm/yyyy) start start end end distance specimens (taxa gear−1) latitude longitude latitude longitude (m) (ind. gear−1)

EBS Per EBS Per EBS BGR-PA 20 21/03/2015 4144–4093 11.83717 −116.982 11.8385 −116.97967 2769 11 9 BGR-RA 50 26/03/2015 4360–4328 11.83117 −117.4915 11.83183 −117.4885 2469 1 1 BGR-RA 59 28/03/2015 4384–4307 11.8085 −117.4865 11.809 −117.48383 2469 2 2 IOM 81* 1/04/2015 4365–4346 11.07083 −119.60783 11.0715 −119.60483 2739 31 15 IOM 99* 4/04/2015 4398–4402 11.043 −119.661 11.04367 −119.6585 2529 23 16 GSR 117* 7/04/2015 4498–4521 13.879 −123.23317 13.87967 −123.2305 3129 54 15 GSR 133* 10/04/2015 4516–4427 13.8545 −123.2315 13.85517 −123.22883 2289 19 9 Ifremer 158* 15/04/2015 4946–4976 14.06283 −130.11083 14.0635 −130.108 3789 30 16 Ifremer 171* 17/04/2015 5024–5017 14.052 −130.07967 14.05333 −130.07683 2979 6 4 APEI no. 3 192* 21/04/2015 4821–4820 18.75417 −128.3425 18.755 −128.34017 2799 47 33 APEI no. 3 197* 22/04/2015 4805–4823 18.81717 −128.35767 18.818 −128.35467 2529 29 22 APEI no. 3 210 24/04/2015 4700–4740 18.8305 −128.40867 18.8315 −128.40617 3399 3 2 Total 256 89 ROV Per ROV dive Per ROV dive IOM 82 1–2/04/2015 4347 11.0575 −119.6315 11.061 −119.6275 – 1 1 GSR 131 9–10/04/2015 4478 13.87317 −123.2505 13.874 −123.248 – 1 1 GSR 135 10–11/04/2015 3593 13.97817 −123.149 13.98433 −123.144 – 2 2 APEI no. 3 189 20–21/04/2015 4931 18.79667 −128.30883 18.80217 −128.30333 – 10 3 APEI no. 3 200 22–23/04/2015 4672 18.82033 −128.42583 18.82667 −128.42467 – 1 1 APEI no. 3 212 24–25/04/2015 1844 18.54717 −128.748 18.54283 −128.74883 – 8 2 Total 23 6 Box corer Per box core Per box core GSR 138 11/04/2015 4503 13.84817 −123.23467 – – – 1 1

Asterisk indicates fully processed EBS samples.

2005–2015 (Biomatters Ltd.). For COI, the sequences were could not be morphologically discriminated, the principle of translated into amino-acid alignments and checked for stop the phylogenetic species concept was applied. Through this codons to avoid pseudogenes. approach, the genetic divergence among specimens belonging Newly assembled sequences were blasted in GenBank to check to the same species (intraspecific) is smaller than the divergence for contamination. Each set of genes was aligned separately using: among specimens from different species (interspecific) (Hebert MAAFT (Katoh et al., 2002) for 16S and 18S; and MUSCLE et al., 2003b). This creates a gap between intraspecific and (Edgar, 2004) for COI. All sequences obtained in this study have interspecific variations when plotted in a distribution of pairwise been deposited in BOLD1 (Ratnasingham and Hebert, 2007) or divergences among all sequences. When data were insufficient GenBank2. to define a barcode gap, molecular operational taxonomic units (MOTUs) were recognised using a threshold of 97 or Integrative Taxonomy 99% similarity between COI and 16S sequences, respectively The specimens were examined under a Leica M125 (Hebert et al., 2003a,b). Hereafter, for the sake of simplicity, stereomicroscope and a Nikon Eclipse E400 microscope. Only we use the term species to refer to the lowest taxonomic the specimens with heads were counted and morphologically resolution achieved by using this combination of morphospecies identified using deep-sea polynoid fauna bibliography (Pettibone, and MOTU concepts. 1976; Uschakov, 1982; Bonifácio and Menot, 2018), to the lowest taxonomic level possible (morphospecies). The naming of morphospecies is consistent with previous studies (Bonifácio Environmental Data and Menot, 2018; Bonifácio et al., 2020). Naming refers to The environmental data used are those compiled by Bonifácio the Ifremer code of the specimen, which served as a reference et al.(2020) from previous studies (Volz et al., 2018a; Hauquier for morphological characters defined in the diagnosis of the et al., 2019) which are publicly available (Hauquier et al., 2017; morphospecies (similar to type material). For specimens that Volz et al., 2018b,c,d,e,f,g). Sediment samples were recovered from the same areas as biological samples during the same cruise, 1http://www.boldsystems.org using a multi-corer or a gravity corer (see Martínez Arbizu 2https://www.ncbi.nlm.nih.gov/genbank/ and Haeckel, 2015 for details). Hauquier et al.(2019) reported

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data for clay fraction (<4 µm), silt fraction (4–63 µm), total between assemblages based on the unique branch length (branch nitrogen (TN in weight per cent), total organic carbon (TOC leading to another tip in the same sample) over the total in weight per cent), and chloroplastic pigment equivalents (CPE branch length observed among assemblages. UniFrac metric in µg ml−1). Volz et al.(2018a) reported POC flux (POC, ranges from 0 (i.e., no unique branch, all the terminals on mg C m−2 d−1) at the seafloor for all areas (eastern CCFZ). This the tree are shared among all assemblages) to 1 (i.e., only POC flux at the seafloor was used as a proxy for food supply to unique branches, the terminals leading to another tip are not benthic communities. shared between assemblages). Ordination of samples based on UniFrac distance metric was performed using Principal Data Analysis Coordinate Analysis (PCoA; Gower, 1966). PCoA, also known Phylogenetic Analyses as metric multidimensional scaling, is an ordination method Maximum likelihood and Bayesian phylogenetic analyses were similar to PCA but that can handle semimetric and non-metric run for two datasets. The first dataset included all 428 sequences dissimilarity measures (Borcard et al., 2018). Furthermore, a (COI, 16S, and 18S) from 238 specimens collected by all types of correlation between UniFrac distance and geographical distance gear from all study sites. Two sigalionids [Neoleanira tetragona was sought to test for a distance decay of phylogenetic similarity (Örsted, 1845) and Sthenelais boa (Johnston, 1833)] were chosen between polynoid assemblages. The UpSet plots were used to as outgroups. The phylogenetic analyses aimed at providing illustrate the distribution of rare, widely distributed and common a comprehensive account of known polynoid diversity in the species across the CCFZ. Haplotype networks were constructed eastern CCFZ. The second dataset was limited to specimens considering the infinite site model and a pairwise uncorrected collected from the fully processed EBS samples and included 156 distance between mitochondrial (COI or 16S genes) haplotypes, sequences (COI, 16S, and 18S) from 81 species. N. tetragona and the quantitative distribution of haplotypes within putative was used as an outgroup. The phylogenetic analyses were run to populations (sampling site). compute phylogenetic diversity indices (see below). The three genes were combined in a partitioned dataset with Alpha and Beta Taxonomic Diversity SequenceMatrix (Vaidya et al., 2011). The maximum likelihood Diversity patterns were analysed using rarefaction curves based analyses were carried out using Randomized Axelerated on the total number of individuals from fully examined EBS Maximum Likelihood (RAxML v.8.2.10; Stamatakis, 2014) on samples (Hurlbert, 1971; Gotelli and Colwell, 2001). Based on this XSEDE with rapid bootstrapping (1000 iterations). The Bayesian rarefied dataset, the expected number of species was calculated phylogenetic analyses were achieved using MrBayes v.3.2.6 on for 12 (ES12) and 35 (ES35) individuals for comparison XSEDE (Ronquist et al., 2012) with 60,000,000 generations with previous studies. Non-parametric and abundance-based in which every 1000 generation chain was sampled and 25% estimators included Chao1 and an abundance-based coverage discarded as burn-in. TRACER v.1.7.1 (Rambaut et al., 2018) was estimator (ACE; O’Hara, 2005; Chiu et al., 2014). used to check the convergence chain runs. Both phylogenetic A Hypergeometric Principal Component Analysis (H-PCA) analyses were computed in CIPRES Science Gateway (Miller was used to describe variations in assemblage composition et al., 2010). Node support is given as a maximum likelihood between fully examined EBS samples. The H-PCA relies on bootstrap and Bayesian posterior probability values. The tree Chord-Normalized Expected Species Shared (CNESS) distance files were plotted using RStudio environment or FigTree v.1.4.23. (Trueblood et al., 1994; Gallagher, 1999), which is computed from probabilities of species occurrence in random draws of m Alpha and Beta Phylogenetic Diversity (PD) individuals. The CNESS distance thus allows rarefying samples to Phylogenetic diversity was assessed using Faith’s PD (Faith, 1992). a similar number of individuals, limiting the bias due to different PD is the most widely used phylogenetic diversity measure sample sizes. Low values of m give high weight to dominant and is defined as the sum of branch lengths of a phylogenetic species whereas high values of m give high weight to rare species. tree connecting all species in a given assemblage. Similar to To choose the value of m, distance matrices are computed species richness, Faith’s PD is also dependent on sample size for all possible values of m, then Kendall’s τ correlations are and inventory completeness (Hsieh and Chao, 2017). We thus calculated between each of these matrices and both matrices for used sample-size-based rarefaction and extrapolation curves to m = 1 and m = m max (minimum sample total). The value compare PD between polynoid assemblages (Hsieh and Chao, of m used for calculation is the one that gives correlation with 2017). Extrapolations were computed for a sample twice the CNESS m = 1 which is roughly equivalent to its correlation with size of the empirical sample. The 95% confidence intervals were CNESS m = m max. The CNESS distance, which provides an computed using a bootstrap method with 200 replications. For objective trade-off between giving weight to either dominant or PD, the phylogenetic ultrametric tree was pruned to only reflect rare species was preferred over the Euclidean distance classically species with at least one sequence present in the entire dataset used in PCA, which gives high weight to abundant species, (i.e., without outgroup species or specimens without a sequence). and a Chi-square distance classically used in Correspondence Unweighted (presence/absence) UniFrac metric (Lozupone Analysis that gives high weight to rare species (Legendre and et al., 2006) was computed to assess beta phylogenetic diversity Gallagher, 2001). CNESS is also a metric distance, which contrary between assemblages. The metric measures the difference to semimetrics such as the Bray–Curtis dissimilarity, respects the relative distance between samples and can be plotted in 3http://tree.bio.ed.ac.uk/software/figtree/ the Euclidean space of a PCA. The influence of environmental

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variables on assemblage patterns was explored by fitting clay diverse with 65 specimens belonging to 42 species (Figure 2). fraction, silt fraction, TN, TOC, CPE, and POC flux at the Anantennata corresponded to 25.1% of the total number of seafloor onto the PCA ordination (envfit function in R library specimens and 45.6% of the total number of macellicephalin Vegan). The accuracy of fit of each variable was tested with species. Of the 11 genera identified, the following were the a permutation test (n = 999). This post hoc explanation of most abundant (>10% of the total number macellicephalin ordination axes was preferred over a constrained multivariate specimens) and/or diverse: Macellicephala with 77 specimens analysis such as a Redundancy Analysis (RDA). RDA involves (29.7%) and 11 species; Polaruschakov with 38 specimens multiple linear regressions of species abundance data, but our (14.7%) and 24 species; Macellicephaloides with 37 specimens data are not truly quantitative. For comparison with previous of Macellicephaloides moustachu; and Bathyfauvelia with 35 studies and to evaluate the distance decay of taxonomic similarity specimens (13.5%) and nine species. The most abundant species between assemblages, the New Normalized Expected Species was Macellicephala sp. 180 with 45 specimens (16.1% of the Shared (NNESS; Trueblood et al., 1994; Gallagher, 1999) was also total number of polynoid specimens). The identification of computed. NNESS is a similarity measure, which as for CNESS is 38 specimens (29 species) remained incomplete due to poor computed on rarefied samples (Trueblood et al., 1994; Gallagher, conservation or no fit within currently recognised genera. Eight 1999). specimens probably belong to new genera and 21 were identified All analyses were conducted with R language (R Core Team., at least as Anantennata. 2020) using RStudio (R Studio Team., 2020) and the following The UpSet plot (Figure 3) shows that 74 species were specific packages or functions: adespatial (Dray et al., 2020), restricted to only one area with 59 species represented by a single ade4 (Dray and Dufour, 2007), ape (Paradis and Schliep, 2019), specimen (Table 2). These singletons accounted for 62.1% of BiodiversityR (Kindt and Coe, 2005), Biostrings (Pagès et al., the total number of species and 21.1% of the total number of 2017), dplyr (Wickham et al., 2020), fossil (Vavrek, 2011), geiger polynoid specimens. Seventeen species were sampled at two or (Pennell et al., 2014), ggplot2 (Wickham, 2016), ggtree (Yu et al., three areas whereas only five species were recovered from four 2017), iNextPD (Hsieh and Chao, 2017), ness (Menot, 2019), areas. No species was common to all five studied areas. APEI pegas (Paradis, 2010), picante (Kembel et al., 2010), reshape2 no. 3 was the most species-rich zone, with 55 species in total, (Wickham, 2007), treeio (Wang et al., 2020), UpSetR (Conway of which 80% were unique to this site, a percentage that drops et al., 2017), and vegan (Oksanen et al., 2016). to a maximum of 52.7% for the other sites within the CCFZ. Interestingly, the two most abundant species Macellicephala sp. 180 and Macellicephaloides moustachu together representing 29% RESULTS of the total number of specimens were widely distributed in all areas except APEI no. 3. Diversity and Distribution The relationships among DNA sequences within putative A total of 280 polynoid specimens were sampled along the populations were explored for two relatively abundant and five areas studied within the eastern CCFZ, of which 256 widely distributed species. The haplotype networks for specimens were collected with the EBS, 23 with the ROV and Bathyfauvelia sp. 224 and Macellicephala sp. 180 based one from a box-core sample (Tables 1, 2). The combination of on sampled sites (putative populations) showed relatively morphological examination and DNA sequencing enabled the high numbers of haplotypes separated mostly by one or a identification of all but five poorly preserved specimens for which few mutational steps (Figure 4). The data did not show DNA sequencing was unsuccessful. The success rate of the DNA phylogeographic structure. sequencing varied according to the targeted genes. COI sequences were obtained from 136 specimens, 16S sequences from 217 Polynoidae Assemblages specimens, and 18S sequences from 68 specimens. Collectively, The structure of polynoid assemblages was analysed from 238 specimens were successfully sequenced for at least one of the eight fully processed EBS samples from IOM, GSR, Ifremer, targeted genes (Figure 2) while 38 specimens were sequenced for and APEI no. 3 areas totalling 239 specimens (Table 1). all three genes and 107 specimens were sequenced for at least two The proportion among subfamilies varied among the sites of the studied genes. (Figure 5). The subfamily Macellicephalinae was dominant The identified polynoids (275 specimens) accounted for 95 while the subfamily Polynoinae was represented by a few species belonging to the subfamilies Eulagiscinae, Polynoinae, specimens at IOM, GSR, and Ifremer areas. Within the subfamily and Macellicephalinae (Figure 2 and Table 2). Eulagiscinae was Macellicephalinae, the proportion of the Anantennata group represented by nine specimens belonging to the same species, (Macellicephalinae without median antenna) showed a two to Bathymoorea lucasi, which was found only at APEI no. 3. fivefold increase at APEI no. 3. Polynoinae was represented by 12 specimens belonging to two species (Harmothoe sp. 207 and Harmothoe sp. 414) found at Taxonomic Diversity BGR, IOM, GSR and Ifremer areas (Figure 2). Macellicephalinae Of the 239 specimens, 234 were identified to species (Table 3). was the most abundant and diverse group with 259 specimens In the case of five poorly preserved specimen, DNA sequencing (92.5% of total number of specimens) belonging to 92 species was not successful, preventing any identification. Based on an (Figure 2 and Table 2). Within Macellicephalinae, a clade integrative taxonomy, 84 species were recognised. The total called Anantennata was also abundant and surprisingly very number of species showed high variability between areas and

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TABLE 2 | Species list and total number of specimens per study area of Polynoidae sampled during the SO239 cruise in the eastern Clarion-Clipperton Fracture Zone.

BGR IOM GSR Ifremer APEI Total BGR IOM GSR Ifremer APEI Total no. 3 no. 3

Macellicephalinae 10 46 64 24 50 194 Anantennata 2 6 8 10 39 65 Hartman-Schröder, 1971 macellicephalins Abyssarya acus Bonifácio 4 4 Bathyedithia retierei 1 1 and Menot, 2018 Bonifácio and Menot, 2018 Bathyeliasona mariaae 1 1 1 1 4 Hodor anduril Bonifácio 2 2 Bonifácio and Menot, 2018 and Menot, 2018 Bathyfauvelia glacigena 1 4 1 6 Hodor hodor Bonifácio and 1 1 Bonifácio and Menot, 2018 Menot, 2018 Bathyfauvelia ignigena 1 1 3 5 Hodor sp. 666-2 1 3 4 Bonifácio and Menot, 2018 Bathyfauvelia sp. 224 3 2 2 3 10 Nu aakhu Bonifácio and 1 1 Menot, 2018 Bathyfauvelia sp. 225 1 3 2 6 Polaruschakov lamellae 1 2 3 Bonifácio and Menot, 2018 Bathyfauvelia sp. 626 1 1 Polaruschakov limaae 1 1 Bonifácio and Menot, 2018 Bathyfauvelia sp. 636-5-2 1 2 3 Polaruschakov omnesae 1 1 2 Bonifácio and Menot, 2018 Bathyfauvelia sp. 636-5-3 1 1 2 Polaruschakov sp. 211 1 1 Bathyfauvelia sp. 666-1-3 1 1 Polaruschakov sp. 219 1 1 Bathyfauvelia sp. 698 1 1 Polaruschakov sp. 315 1 1 2 Bathypolaria sp. 173 1 1 1 3 Polaruschakov sp. 343 2 2 Bathypolaria sp. 608 6 6 Polaruschakov sp. 514 1 1 Bruunilla nealae Bonifácio 1 1 Polaruschakov sp. 615 2 2 and Menot, 2018 Bruunilla sp. 651 1 1 Polaruschakov sp. 636-4-2 1 1 Bruunilla sp. 659-8 3 3 Polaruschakov sp. 636-4-3 1 1 Bruunilla sp. 664 1 1 Polaruschakov sp. 639-2 1 1 Bruunilla sp. 668-1 1 1 Polaruschakov sp. 642 1 1 Bruunilla sp. 668-2 1 1 Polaruschakov sp. 655-3-1 2 2 Bruunilla sp. 692 1 1 Polaruschakov sp. 655-3-4 1 1 Macellicephala clarionensis 4 4 Polaruschakov sp. 655-3-5 1 1 Bonifácio and Menot, 2018 Macellicephala parvafauces 1 1 2 Polaruschakov sp. 655-3-6 1 1 Bonifácio and Menot, 2018 Macellicephala sp. 180 1 12 29 3 45 Polaruschakov sp. 655-3-7 1 1 Macellicephala sp. 308 5 1 6 Polaruschakov sp. 659-1-2 1 1 Macellicephala sp. 320 1 1 Polaruschakov sp. 666-3-1 1 4 5 Macellicephala sp. 35 2 4 3 9 Polaruschakov sp. 666-3-2 1 1 Macellicephala sp. 437 1 1 Polaruschakov sp. 685 1 1 Macellicephala sp. 442b 2 2 Polaruschakov sp. 686 1 1 Macellicephala sp. 444 1 1 2 Polaruschakov sp. 690 1 1 Macellicephala sp. 464 1 1 1 3 Polaruschakov spp. 3 3 Macellicephala sp. 687 2 2 Polynoidae sp. 303 1 1 Macellicephaloides 2 7 15 13 37 Polynoidae sp. 450 1 1 moustachu Bonifácio and Menot, 2018 Polynoidae sp. 153 1 1 Polynoidae sp. 513 1 1 Polynoidae sp. 197 1 1 Polynoidae sp. 537-1-2 2 2 Polynoidae sp. 299 1 1 Polynoidae sp. 609 1 1 Polynoidae sp. 306 1 1 Polynoidae sp. 655-4 2 2 Polynoidae sp. 314 1 1 Polynoidae sp. 657-1-3 3 3 Polynoidae sp. 353 1 1 Polynoidae sp. 659-7 1 1 Polynoidae sp. 465a 1 1 Polynoidae sp. 670-2 1 1 Polynoidae sp. 521-4 1 1 Polynoidae sp. 679 1 1

(Continued)

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TABLE 2 | Continued

BGR IOM GSR Ifremer APEI Total BGR IOM GSR Ifremer APEI Total no. 3 no. 3

Polynoidae sp. 655-5 1 1 Polynoidae sp. 688 1 1 Polynoidae sp. 659-4 1 1 Polynoidae sp. 693 1 1 Polynoidae sp. 659-5 1 1 Polynoidae sp. 697 1 1 Polynoidae sp. 666-5 1 1 Polynoidae spp. 1 1 Polynoidae sp. 666-6 1 1 Eulagiscinae Pettibone, 9 9 1997 Polynoidae sp. 673 1 1 Bathymoorea lucasi 9 9 Bonifácio and Menot, 2018 Polynoidae sp. 691 1 1 Polynoinae Kinberg, 2 3 5 2 12 1856 Polynoidae sp. 696 1 1 Harmothoe sp. 207 1 2 2 2 7 Polynoidae spp. 1 1 Harmothoe sp. 414 1 1 3 5 Yodanoe desbruyeresi 1 1 Bonifácio and Menot, 2018 Yodanoe sp. 659-3 1 1 Total 14 55 77 36 98 280

Subfamilies and species’ grouping are marked in bold. Values represent a sum of specimens belonging to the given taxon or grouping of species.

no clear trend. A total of 24 species were identified at IOM pairwise comparisons can be highlighted. The three pairwise area from 54 individuals, 19 species at GSR area from 73 comparisons between exploration contracts and APEI no. 3 individuals, 19 species at Ifremer from 35 individuals, and 49 consistently show the lowest values of similarity, irrespective species identified at APEI no. 3 from 72 individuals (Table 3). of distance. For pairwise comparison among the three Species rarefaction curves (individual-based) did not reach an exploration contracts, similarity decreases with distance but asymptote at any sampled area and suggested higher diversity the correlation is not statistically significant (R2 = 0.84, p = 0.26; at APEI no. 3 (Figure 6A). The results also suggest that the Supplementary Figure 1C). diversity at Ifremer and IOM areas is similar and possibly higher than at the GSR area. The non-parametric estimation Phylogenetic Diversity of species richness followed the same patterns as rarefaction Out of the 239 specimens recovered from fully examined EBS, curves, showing the highest values at APEI no. 3 (123 species 230 were sequenced and identified to one of the 80 species with Chao1; Table 3). The richness estimates computed by Chao1 represented in the phylogenetic tree. Maximum likelihood and and ACE for APEI no. 3 are four to five times higher than Bayesian inference resulted in very similar phylogenetic trees for the GSR area, which presented a similar sample size. When (Supplementary Figure 2). For phylogenetic diversity analyses, data from the four areas were pooled, the rarefaction curve the distance between species in the Bayesian inference tree did not level-off (Figure 6C). The non-parametric estimation has been considered. The rarefaction curves of the Faith of species richness at this regional scale yielded estimates phylogenetic diversity showed significantly higher diversity in ranging from 176 to 202 polynoid species for Chao1 and ACE APEI no. 3 than Ifremer, GSR, and IOM areas (Figure 6B). estimators respectively. The same pattern was observed for the estimated asymptotes The ordination of EBS samples in the two first axes of an where APEI no. 3 was expected to have two to three times H-PCA based on polynoid assemblage structure is illustrated in higher diversity (Table 4). Pooling of samples from the four Figure 7A. The first two axes explained 61% of total variance areas did not result in rarefaction curve levelling off either in the composition of polynoid assemblage (Figure 7A). The (Figure 6D). first axis explained 40% of total variance and discriminated The PCoA based on phylogenetic distance showed similar eastern areas (IOM and GSR) from APEI no. 3. The second patterns to the H-PCA ordination (Figure 7B). APEI no. 3 was axis, explaining 21% of total variance, discriminated the Ifremer discriminated from southern areas, and Ifremer separated from area, and particularly one EBS sample. TOC was identified GSR and IOM, which are closely related. (post hoc) as the environmental variable most significantly related The phylogenetic distance showed no relationship with (p < 0.01) to the first axis (Figure 7A). POC, silt and clay geographic distance (R2 = 0.10, p = 0.54; Supplementary were also significantly related to the first axis (p < 0.05). The Figure 1B). Two groups of pairwise comparison were evident, as ordination of species (Figure 7A) showed that Macellicephala previously found with taxonomic similarity. Pairwise comparison sp. 180 and Macellicephaloides moustachu were the species most with the APEI no. 3 had the highest phylogenetic distances characteristic in eastern areas (particularly in IOM and GSR for irrespective of geographic distances while pairwise comparison the first, and in GSR and Ifremer for the second). among exploration contracts showed a pattern of increasing The relationship between distance and assemblage phylogenetic distance as a function of geographic distance. This similarity showed no significant correlation (R2 = 0.12, pattern was however not statistically significant (R2 = 0.84, p = 0.49; Supplementary Figure 1A) but two groups of p = 0.26; Supplementary Figure 1D).

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C Bat u chu hu C 709 P s 5 C C ZP8 ru i a a m hu u 1 lar 53 as n o st a c C F 7 6 d e p. 537 − 6 CCFZ FZP 1 P726 i CCF Z |P oly c hu C CF 9 C FZ P s 5 n stac h CC C 64 a B C | a C Z −19| s C athypo C 7 so st a c P8 8 |P o u e 9 8 c liaso o u PCFZ p. c 2 F m 2 − C t a C P − C ZP7 −19| 9 B C 6 no s − e ia mous F ZP 1 ol lyno s ha −8 s o − − FZ 1 hyel s 9 8 − 19 c 51 1−2 1 9| Z Z P p y mou ta F ou F ZP72 t P FZ ZP 65 1 − . 696 6− ha kov 9 8 − el 8 u 6 19| a CCF Z − lias s i ko . 1 h m Z P 3 3 P ola d 800 de ust 2 es p Z r 3 59 1− 1 5 26 1 | o 6 . i u P7 7 8 o 6 Ba . 659−5 9−1 − P i a P810− 7 u P at s o P5 19 da v s P 8 9

i C ac 1 l d 6 6 llic l c 1 − 19 elli 9 l h d u M l ac a − 2 a 6− e − ha u 1 − 6 r CC − M c −19| a 6−2 nal nea 9| | 6− o e i 2 c 2 r 5 M 8 s k l 8 s 9 873− 3 1 r i 2 Mace p. k sp o l 1 3 p 1 8 P Ma s o 808 v Sampled areas 8 P2 0 . Ma P − FZP77 Z p . | v P

P8 664 Z P6 0 19| P 6 a P s 424− 19| 1 Z 6 9 F . F 19| Z P8 11−19| s P F 6 e p. Z 659−

− CC C p 5 60 FZ 8 F 0− . 6 7

− C 6 CC CFZ CC CF ZP210− P CFZ C 6−1 CFZ 6 C − 8

1 P475−19| C C F Z 7 BGR CFZ 4 C C 85 C 1 6 C C F 7 7 P − CC P 3 Subfamily CC Z FZ CCFZ F C C FZP638 CC Polynoinae CC IOM Eulagiscinae GSR Macellicephalinae with median antenna Ifremer Macellicephalinae without median antenna APEI no. 3

FIGURE 2 | Maximum likelihood inference of polynoid phylogeny based on concatenated gene data (COI, 16S, and 18S) showing distribution of each sequence within the eastern CCFZ. Some species and respective DNA data were already published by Bonifácio and Menot(2018). Dataset includes all sequences of specimens from ROV, EBS, and box corer. Colors indicate subfamilies or sampled area. Circles on branches represent bootstrap supports ≥90%. Bayesian inference has not converged to the stationary distribution (not presented).

DISCUSSION these patterns were food inputs, sediment grain size, and a barrier to dispersal. Causes of Polynoid Species Turnover in the Clarion-Clipperton Fracture Zone Conflicting Patterns of Community Structure Along a Taxonomic and phylogenetic beta diversity patterns were Gradient of Surface Primary Productivity similar for polynoid assemblages across the eastern CCFZ. The Sea surface primary productivity decreases from south-east to composition of polynoid assemblages discriminated the eastern north-west across the eastern CCFZ with POC fluxes ranging (i.e., IOM and GSR) from the western areas (i.e., Ifremer and from 1.54 mg C m−2 d−1 at IOM to 1.07 mg C m−2 d−1 at APEI APEI no. 3). It was also possible to notice clear differences no. 3 (Volz et al., 2018a). In previous studies, beyond species between the Ifremer and APEI no. 3. These patterns in species turnover, the influence of food inputs on community structure turnover resemble those already reported among the same was supported by positive correlations between POC fluxes and sampling sites for infaunal polychaetes (Bonifácio et al., 2020), the abundance or taxonomic richness of infauna (Błazewicz˙ et al., tanaids (Błazewicz˙ et al., 2019), and nematodes (Hauquier et al., 2019; Hauquier et al., 2019; Bonifácio et al., 2020). However, 2019). So far, the three main processes evoked to explain here, the highest species richness of polynoids was found at

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50 sp. 464

44 sp. 636-5-3 sp. 414 sp. 173 sp. 180 40 sp. 444 Harmothoe Bathyfauvelia Macellicephala Polaruschakov omnesae 30 Bathypolaria and Macellicephala Macellicephala sp. 666-3-1 sp. 315 sp. 35 and sp. 308, sp. 225, 20 sp. 636-5-2, sp. 224 sp. 207, sp. 666-2 and Number of species Macellicephaloides moustachu Polaruschakov 10 Polaruschakov 10 9 Macellicephala 7 Bathyfauvelia and and Bathyfauvelia Harmothoe and Macellicephala Hodor Bathyfauvelia ignigena Bathyfauvelia glacigena Polaruschakov lamellae Bathyfauvelia Bathyeliasona mariaae 333 Macellicephala parvafauces 4 3 2 11 2 1 11 0 55 APEI no. 3

19 Ifremer

23 GSR

25 IOM

12 BGR

60 40 20 0 Number of species

FIGURE 3 | UpSet plot showing the intersection of species distribution along sampled areas within the eastern CCFZ. Horizontal (colored) bars present total number of species for corresponding areas with vertical (black) bars presenting restricted (dots) or shared species (linked dots). Shared species are indicated.

A Bathyfauvelia sp. 224 (COI) B Macellicephala sp. 180 (16S) IX

VIII VII VII VIII

12 VI

IV I VI 4 II V 1

III I III IOM GSR Ifremer APEI no. 3 V

II IV XI X

FIGURE 4 | Haplotype networks for Bathyfauvelia sp. 224 (10 sequences) based on COI gene (A) and for Macellicephala sp. 180 (36 sequences) based on 16S gene (B). Each roman number indicates one haplotype with colors indicating sampling areas and dashes indicating mutational steps between haplotypes. Circle size is proportional to the number of samples observed for a speciﬁc haplotype.

APEI no. 3, the most oligotrophic site. The EBS used in this content and lower porosity (Volz et al., 2018a; Hauquier et al., study, while very efficient in sampling the poorly known vagile 2019). Higher contents of finer sediments have been postulated epifauna (Brandt and Schnack, 1999), is a qualitative sampler to increase sediment shear strength making it more difficult for contrary to the box corer used to sample infauna. Diversity data fauna to burrow (Trueman et al., 1966). Chuar et al.(2020) extrapolated from EBS trawls should thus be interpreted with pointed out that sediment shear strength may impact negatively caution. Yet, without ruling out the sample bias, a high number of infaunal abundance in the OMS area located at the south-eastern specimens and species of polynoids at APEI no. 3, driven mainly end of the CCFZ. Together with low food input, inhospitable by Macellicephalinae, might also be explained by adaptations to sediments may thus have contributed to the low abundance oligotrophy (see below). of infaunal polychaetes, tanaids, and nematodes at APEI no. 3 (Błazewicz˙ et al., 2019; Hauquier et al., 2019; Bonifácio et al., The Influence of Sediment Grain Size – Fact or 2020). In turn, the low infaunal standing stock may increase the Artefact? relative availability of resources to epifaunal communities. Like In addition to low POC flux, the sediments at APEI no. 3 polynoids, isopods from EBS samples also showed a number of were characterised by a lower average grain size, higher clay specimens and species similar to or even higher at APEI no.

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that the Clarion fracture is a biogeographic barrier between the 1.00 northern APEI no. 3 and the southern exploration contract areas (Ifremer, GSR, IOM, and BGR). This fracture represents a long and narrow submarine mountain range displaying peak-and- trough patterns with up to 1800 m of difference in elevation 0.75 (Hall and Gurnis, 2005). Ridges and fractures can work as physiographic barriers affecting the dispersal of different taxa to a lesser or greater

0.50 degree. For example, the Mid-Atlantic Ridge (MAR) allows the dispersal of nematodes of the genus Acantholaimus (Lins Percentage (%) Percentage et al., 2018), copepods of the genus Mesocletodes (Menzel et al., 2011) and isopods of the family Munnopsidae (Bober 0.25 et al., 2018), but is mostly impermeable to isopods of the families Macrostylidae, Desmosomatidae, and Nannoniscidae (Bober et al., 2018). Polychaetes did not show a clear pattern in the permeability of the MAR as a barrier (Guggolz et al., 2018). 0.00 Guggolz et al.(2018) examined the distribution of polychaetes IOM GSR Ifremer APEI no. 3 and species composition of spionids and polynoids along the

Macellicephalinae Macellicephalinae Vema Fracture Zone across the MAR. They observed signiﬁcant Polynoinae with median antenna without median antenna changes in species composition across the MAR and suggested them to be the result of limited dispersal potential and diﬀerent FIGURE 5 | Bar chart of the relative abundance of two polynoid subfamilies – habitat characteristics. Only six of 32 polynoid species crossed the Polynoinae and Macellicephalinae, and within Macellicephalinae with median MAR (Guggolz et al., 2018). antenna only or no antennae (i.e., the “Anantennata clade”). Comparisons of Based on our CCFZ samples, 11 of 96 polynoid species data from fully processed EBS samples from the IOM, GSR, Ifremer, and APEI no. 3 study areas. (10%) were found on both sides of the Clarion Fracture Zone, which is a much higher proportion of faunal sharing than for infaunal polychaetes (1%; Bonifácio et al., 2020) or infaunal TABLE 3 | Observed species richness (Sobs) and individual-based estimators of tanaidaceans (0%; Błazewicz˙ et al., 2019), in the same order of polynoid species richness for each sampled area and for the eastern CCFZ (pooled areas) from the fully processed EBS samples. magnitude as isopods sampled with an EBS (5%; Brix et al., 2020), and still lower than scavenging amphipods (90%; Patel Assemblage Sobs Sample size n Chao1 ACE ES12 ES35 et al., 2020). For isopods, variations in species ranges across

IOM 24 54 64 ± 29 62 ± 4 9 ± 1 18 ± 2 the CCFZ were attributed to variable swimming habits, and GSR 19 73 25 ± 5 27 ± 3 6 ± 1 13 ± 2 thus dispersal abilities (Brix et al., 2020; Janssen et al., 2015). Ifremer 19 35 54 ± 26 68 ± 2 8 ± 1 19 ± 0 Among the most abundant species of polynoids, Bathyfauvelia APEI no. 3 49 72 123 ± 36 114 ± 4 11 ± 1 28 ± 2 sp. 224 (10 specimens) remarkably occurs on both sides of the Eastern CCFZ 84 234 176 ± 36 202 ± 9 9 ± 1 22 ± 2 fracture while Macellicephaloides moustachu (37 specimens), and Macellicephala sp. 180 (45 specimens) were clearly restricted to ES indicates the expected number of species for a given number of individuals “n.” south of the fracture. The subfamily Macellicephalinae, which “±” indicates the standard error. is dominant among deep-sea polynoids, shows morphological characters that facilitate a benthopelagic lifestyle (see below). 3 than at the southern areas (Ifremer, GSR, IOM, and BGR; However, M. moustachu has been described as having very thin Brix et al., 2020). Contrasting patterns in community structure neurochaetae which would evidently affect its ability to swim, and between infaunal and epifaunal assemblages at APEI no. 3 may have contributed to limit its distribution south of the Clarion are thus consistent for different taxonomic groups. However, fracture. This species also has morphological structures attached sampling bias cannot be ruled out, as sediment heterogeneity to the body that may potentially be related to reproduction may influence the sampling efficiency of an EBS (Guggolz (Bonifácio and Menot, 2018). et al., 2018). At APEI no. 3, the higher clay content might The life cycle of polynoids is mostly known from shallow have facilitated sediment flushing through the nets, limiting water species, which have generally a planktotrophic larval sediment clogging and increasing the effective sampling time, development (Giangrande, 1997). A few species in the contrary to southern sites where the EBS mesh would have deep sea are however assumed to undergo lecithotrophic filled up faster. development (Glover et al., 2005), a more suitable condition in oligotrophic waters (Tyler and Young, 1999). External brooding Clarion Fracture, a True Barrier to Dispersal? of eggs under the dorsal elytra has also been observed in The pairwise comparisons of taxonomic and phylogenetic polynoids of the Antarctic shelf (Gambi et al., 2001). Species composition show that the polynoid assemblage from APEI no. from the CCFZ also showed swollen or sac-like structures 3 is systematically the most different from all other assemblages, on the dorsal side, which may be linked to reproduction regardless of geographic distance between sites. This may suggest (Bonifácio and Menot, 2018).

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A B 50

30 20

20 Number of species Phylogenetic diversity Phylogenetic 10

0 0 0 20 40 60 0 50 100 150 Number of individuals Number of individuals rarefaction extrapolation IOM GSR Ifremer APEI no. 3

C D 40 80

30 60

20 40 Number of species Phylogenetic diversity Phylogenetic

20 10

0 0

0 50 100 150 200 0 100 200 300 400 Number of individuals Number of individuals rarefaction extrapolation Eastern CCFZ

FIGURE 6 | Individual-based rarefaction curves of fully processed EBS samples based on species richness for each sampled area (A) and for the eastern CCFZ (pooled areas, C); and based on phylogenetic diversity for each sampled area (B), and for the eastern CCFZ (pooled areas, D).

Overall, the benthopelagic lifestyle of some deep-sea polynoids Enhanced Diversity at Great Depths combined with the planktotrophic larval development inherited Potentially Related to Low Food Input from their shallow-water relatives might explain the relatively large geographic ranges of Polynoidae at community scale in the and Mode of Life CCFZ, with 10% of species shared between north and south of the In a global census of abyssal polychaetes (Paterson et al., 2009), Clarion fracture. However, this is still a low sharing proportion, polynoids represented the most species-rich family with 91 considering that the purpose of APEI no. 3 is to preserve species out of a total of 768 polychaete species occurring below representative biodiversity of benthic communities within the 2000 m depth. In addition, 13 out of the 15 species occurring CCFZ (Wedding et al., 2013). Further, while some polynoids below 4000 m depth belonged to the subfamily Macellicephalinae. radiated in the deep sea, much remains to be learned about their Interrogation of the Ocean Biodiversity Information System reproduction and mode of life. yielded 125 valid species of polynoids below 2000 m depth (OBIS,

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A B

171 1

M acelli cep hala 81 Clay * TOC ** sp. 180133 197 99 192 117

POC * achu 158

Axis 2 Silt * moust des Dimension 2

icephaloi

Macell

171 133 117 99 158 81 197 −2 −1 0 −0.2 0.0 0.2 0.4 0.6 192

−2 −1 0 1 2 −0.2 0.0 0.2 0.4 0.6 Axis 1 Dimension 1 IOM GSR Ifremer APEI no. 3

FIGURE 7 | Ordination plot of polynoid assemblages from fully processed EBS samples. (A) H-PCA biplot based on the Chord-Normalized Expected Species Shared (CNESS with m = 3) distance of species (scaling 2) with variables significantly correlated to the projected points (post hoc envfit permutation test) and showing species contributing most to two first axes (A, scaling 2). Significance codes: **p < 0.01 and *p < 0.05. (B) PCoA plot based on UniFrac phylogenetic distances.

TABLE 4 | Individual-based phylogenetic diversity (PD) of polynoid assemblages.

Assemblage Sobs Sample size n Observed PD Sample coverage (%) Estimated asymptote PD 95% conﬁdence interval

IOM 53 23 9.9 72 15 ± 4 9.9, 23.2 GSR 72 18 9.6 89 12 ± 1 9.6, 14.3 Ifremer 35 19 9.5 58 17 ± 5 9.5, 25.8 APEI no. 3 70 47 18.7 53 47 ± 10 26.8, 67.9 Eastern CCFZ 80 230 27 79 43 ± 6 31.7, 54.7

Dataset includes all sequenced specimens from fully examined EBS. Sobs indicates observed species richness. Eastern CCFZ comprises IOM, GSR, Ifremer, and APEI no. 3 data pooled. “±” indicates the standard error. The 95% conﬁdence intervals were calculated by a bootstrap method based on 200 replications.

2020). The Macellicephalinae (sensu Bonifácio and Menot, 2018) Guggolz et al.(2018) were able to identify eight Anantennata is the most species-rich deep-sea subfamily with 70 species, species among the 31 Macellicephalinae species recognised. followed by Polynoinae with 41 species. The depth ranges of OBIS Macellicephalins are thus polychaetes thriving in cold and records differ for these two subfamilies. For 311 valid species of dark environments that have successfully colonised deep-sea Polynoinae, the depth range varies from 0 to 5400 m depth, with habitats such as chemosynthetic ecosystems (hydrothermal vents a median at 21 m; while for 89 valid species of Macellicephalinae and cold seeps; Pettibone, 1983; Chevaldonné et al., 1998; the depth range varies from 298 to 10,180 m, with a median Hatch et al., 2020), trenches (Levenstein, 1971; Pettibone, at 2451 m. In total, 121 species are currently described within 1976), manganese nodules (Bonifácio and Menot, 2018), and Macellicephalinae (36 genera) including only 15 Anantennata the deep Antarctic shelf (Neal et al., 2018a,b) but also (Read and Fauchald, 2021), with the recent additions of 37 new analogous shallow water habitats such as submarine caves species and four new genera (Bonifácio and Menot, 2018; Jimi (Pettibone, 1985b). et al., 2018; Neal et al., 2018a; Zhang et al., 2018; Zhou et al., 2018; Macellicephalins differ from other polynoid subfamilies in Lindgren et al., 2019; Wu et al., 2019; Hatch et al., 2020; Kolbasova having lost their antennae. Polynoids from shallow waters et al., 2020). typically possess two lateral and one median antennae, whereas Our results (including 16 named species from Bonifácio macellicephalins have either only a median antenna or no and Menot, 2018) indicate the presence of at least 92 antennae at all. Macellicephalins without antennae form a Macellicephalinae species with 42 being Anantennata in the monophyletic group (i.e., the “Anantennata clade”; Bonifácio eastern CCFZ, in the north-east Pacific. In the Atlantic Ocean, and Menot, 2018). Macellicephalinae probably originated from

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short-body polynoids such as Bathymoorea (Eulagiscinae), bottom and feeding on small pelagic crustaceans or scavenging a morphology that is reminiscent of macellicephalins from dead planktonic organisms on the bottom (Pettibone, 1993). chemosynthetic habitats (a robust body, thick elytra and short Evolution of similar behavior may have enabled macellicephalins body appendages; Desbruyères and Hourdez, 2000) and is to explore benthic and pelagic niches in the deep sea, particularly likely associated with a basal position within Macellicephalinae within hadal depths where they are the most characteristic (Bonifácio and Menot, 2018). and diverse polychaetes (Paterson et al., 2009; Jamieson, Deep-sea, polar and cave-endemic macellicephalins not living 2015). in chemosynthetic habitats share mostly distinct morphological characters such as a soft body, delicate elytra, loss of eyes, relatively thin, flattened and long chaetae, elongated parapodia, CONCLUSION and sometimes extremely long dorsal cirri or reduction of jaws (Uschakov, 1977, 1982; Bonifácio and Menot, 2018; Gonzalez Variations in epibenthic polynoid assemblages across the CCFZ et al., 2018). Pettibone(1985a) also observed some of these show similarities with other faunal groups in that species morphological characters specific for pelagic life in Natopolynoe turnover covaries with POC flux and thus food supply. A major kensmithi Pettibone, 1985a which was described to be abundant difference from the infaunal pattern (Bonifácio et al., 2020) is not only on the seafloor but also swimming up to 10 m that species richness was similar to or even higher at APEI no. 3, above it. Some Macellicephalinae seem to be benthopelagic, the most oligotrophic site located north of the Clarion fracture, swimming in near-bottom water in search of food (Knox, in comparison to other exploration mining areas to the south. 1959; Pettibone, 1976; Uschakov, 1977, 1982). Other studies, This unexpected pattern may result from sampling bias, but could examining megafauna, have also recorded macellicephalins also be due to: (i) higher shear strength of APEI no. 3 sediments swimming in the water column (Smith and Hamilton, 1983; making them less hospitable to infauna to the benefit of epifauna Rybakova et al., 2019). Some macellicephalins also have elongated and (ii) evolutionary adaptations of macellicephalins towards a papillae on their pharynx, assumed to be helpful in rapid benthopelagic life strategy under oligotrophic conditions in the capture of small suspended particles, a character also shared deep sea. The difference in species composition and community with the pelagic family Alciopidae (Pettibone, 1976; Uschakov, structure at APEI no. 3 brings into question the key principles of 1982). Evidence presented so far supports the hypothesis of the APEI network, as this area appears not representative of the Gonzalez et al.(2018) of a secondary pelagic mode of life biogeography and habitat of the broader region (Wedding et al., as a deep-sea adaptation in polynoids. These authors also 2013). However, such a conclusion is tentative, given the limited suggested that scale worms living in aphotic environments (i.e., sampling within this APEI. submarine caves and the deep-sea) are subjected to the “darkness The polynoids in the CCFZ are highly diverse, with most syndrome” promoting morphological and behavioral changes diversity confined to the subfamily Macellicephalinae, which has such as loss of eyes, elongation of appendages and shifting to particularly radiated in the deep sea. Together with the results of swimming behavior. Bonifácio and Menot(2018) from the same areas in the CCFZ, Elongated appendages could provide an evolutionary our results increase the number of known Macellicephalinae advantage in two ways for deep-sea polynoids. Firstly, cirri species worldwide. We have newly identified 42 Anantennata elongation increases the surface area thus contributing to species whereas only eight species have been described worldwide attainment of neutral buoyancy (Gonzalez et al., 2018). prior to our visit in 2015. This number indicates, how Secondly, cirri elongation could increase their sensitivity underestimated macellicephalin diversity currently is. Other to prey detection. By removing the sensory appendages in questions remain unanswered as well: Do they have a pattern of Harmothoe species, Daly(1972) showed that the ability to vertical movement? Is the elongation of appendages driven by locate a source of vibrations is an important factor in feeding swimming behavior or prey detection? How do they interact with behavior. His experiments further suggested that palps provide other species? How do they reproduce and disperse? the worm with contact exploration of the object and chemical information (e.g., if suitable as prey or not) whereas dorsal DATA AVAILABILITY STATEMENT cirri were responsible for relaying chemical information and vibration source location (Daly, 1972). Therefore, the elongation DNA sequences are available in BOLD (https://dx.doi.org/ of appendages in deep-sea polynoids could represent an 10.5883/DS-POLYNOID; Bonifácio, 2021) and GenBank. The adaptation of the subfamily Macellicephalinae to food limitation total number of specimens and species data examined in the by increasing the access to food (invasion of new niches) present study with respective BOLD IDs and GenBank accession and prey detection. numbers are available in the PANGAEA database (https://doi. The swimming behavior in Macellicephalinae was likely org/10.1594/PANGAEA.926674; Bonifácio et al., 2021). the key to exploitation of new trophic resources, unavailable to worms with a benthic lifestyle. This rare semi-pelagic mode of life within polynoids has been well-documented for AUTHOR CONTRIBUTIONS a Polynoinae species, Bylgides sarsi (Kinberg in Malmgren, 1866), which rises above the bottom to mid-water during LM was responsible for the project planning and sampling design. the night, escaping the poor oxygen conditions close to the All authors carried out the sampling and the subsequent sample

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processing on board. PB was responsible for the identiﬁcation Special thanks to Helena Wiklund and Thomas Dahlgren of Polynoidae and data analysis. All authors involved in data who initiated PB in phylogenetic and connectivity studies. interpretation and preparation of the manuscript. We are thankful to Alison Chalm for the English editing. Finally, we extend our sincere thanks to Erica Goetze and the two referees for their critical reviews and helpful comments FUNDING on the manuscript.

This study received funding from the Ifremer programme “Ressources Minérales Marines” (REMIMA), the JPI Oceans SUPPLEMENTARY MATERIAL pilot action “Ecological Aspects of Deep-Sea Mining,” and the European Union Seventh Framework Programme (FP7/2007– The Supplementary Material for this article can be found 2013) under the MIDAS project, grant agreement no. online at: https://www.frontiersin.org/articles/10.3389/fmars. 603418. 2021.656899/full#supplementary-material

Supplementary Figure 1 | Distance decay of New Normalized Expected Species ACKNOWLEDGMENTS Shared (NNESS) between IOM, GSR, Ifremer, and APEI no. 3 (A) and the same areas excluding APEI no. 3 (C). Distance decay of UniFrac phylogenetic distance between IOM, GSR, Ifremer, and APEI no. 3 (B) and the same areas excluding We are grateful to Pedro Martínez, the crew of the RV APEI no. 3 (D). Sonne, the team of the ROV Kiel 6000 and all people who participated in the ﬁeld sampling and sample processing during Supplementary Figure 2 | Molecular phylogenetic relationship among sampled polynoid species based on concatenated genes (COI, 16S, and 18S) and the SO239 cruise. Many thanks for sample processing to Stefanie performed with maximum-likelihood (A) and Bayesian inference (B) analyses. Kaiser, Sarah Schnurr and Ana Hilário; and to Emmanuelle Outgroup (sigalionid species) colored in gray. Node values indicate the Omnes for assistance with DNA extraction and ampliﬁcation. maximum-likelihood bootstrap (A) and Bayesian posterior probabilities (B).

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Total organic carbon contents of sediment cores retrieved Publisher’s Note: All claims expressed in this article are solely those of the authors during R/V SONNE cruise SO239 in the BGR area. Pangaea 2018:894854. and do not necessarily represent those of their affiliated organizations, or those of doi: 10.1594/PANGAEA.894854 the publisher, the editors and the reviewers. Any product that may be evaluated in Volz, J. B., Mogollón, J. M., Geibert, W., Martinez Arbizu, P., Koschinsky, A., and this article, or claim that may be made by its manufacturer, is not guaranteed or Kasten, S. (2018c). Total organic carbon contents of sediment cores retrieved endorsed by the publisher. during R/V SONNE cruise SO239 in the IOM area. Pangaea 2018:894858. doi: 10.1594/PANGAEA.894858 Copyright © 2021 Bonifácio, Neal and Menot. This is an open-access article Volz, J. B., Mogollón, J. M., Geibert, W., Martinez Arbizu, P., Koschinsky, A., and distributed under the terms of the Creative Commons Attribution License (CC BY). Kasten, S. (2018d). Total organic carbon contents of sediment cores retrieved The use, distribution or reproduction in other forums is permitted, provided the during R/V SONNE cruise SO239 in the GSR area. Pangaea 2018:894855. original author(s) and the copyright owner(s) are credited and that the original doi: 10.1594/PANGAEA.894855 publication in this journal is cited, in accordance with accepted academic practice. Volz, J. B., Mogollón, J. M., Geibert, W., Martinez Arbizu, P., Koschinsky, A., No use, distribution or reproduction is permitted which does not comply with and Kasten, S. (2018e). Total organic carbon contents of sediment cores these terms.

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