Hungry scale worms: Phylogenetics of Peinaleopolynoe (Polynoidae, Annelida), with four new species

Abstract Polynoidae Kinberg, 1856 has five branchiate genera: Branchipolynoe Pettibone, 1984, Branchinotogluma Pettibone, 1985, Branchiplicatus Pettibone, 1985, Peinaleopolynoe Desbruyères & Laubier, 1988, and Thermopolynoe Miura, 1994, all native to deep-sea, chemosynthetic-based habitats. Of these, Peinaleopolynoe has two accepted species; Peinaleopolynoe sillardi Desbruyères & Laubier, 1988 (Atlantic Ocean) and Peinaleopolynoe santacatalina Pettibone, 1993 (East Pacific Ocean). The goal of this study was to assess the phylogenetic position of Peinaleopolynoe, utilizing DNA sequences from a broad sampling of deep-sea polynoids. Representatives from all five branchiate genera were included, several species of which were sampled from near the type localities; Branchinotogluma sandersi Pettibone, 1985 from the Galápagos Rift (E/V “Nautilus”); Peinaleopolynoe sillardi from organic remains in the Atlantic Ocean; Peinaleopolynoe santacatalina from a whalefall off southern California (R/V “Western Flyer”) and Thermopolynoe branchiata Miura, 1994 from Lau Back-Arc Basin in the western Pacific (R/V “Melville”). Phylogenetic analyses were conducted using mitochondrial (COI, 16S rRNA, and CytB) and nuclear (18S rRNA, 28S rRNA, and H3) genes. The analyses revealed four new Peinaleopolynoe species from the Pacific Ocean that are formally described here: Peinaleopolynoe orphanae Hatch & Rouse, sp. nov., type locality Pescadero Basin in the Gulf of California, Mexico (R/V “Western Flyer”); Peinaleopolynoe elvisi Hatch & Rouse, sp. nov. and Peinaleopolynoe goffrediae Hatch & Rouse, sp. nov., both with a type locality in Monterey Canyon off California (R/V “Western Flyer”) and Peinaleopolynoe mineoi Hatch & Rouse, sp. nov. from Costa Rica methane seeps (R/V “Falkor”). In addition to DNA sequence data, the monophyly of Peinaleopolynoe is supported by the presence of ventral papillae on segments 12–15. The results also demonstrated the paraphyly of Branchinotogluma and Lepidonotopodium Pettibone, 1983 and taxonomic revision of these genera is required. We apply the subfamily name Lepidonotopodinae Pettibone 1983, for the clade comprised of Branchipolynoe, Branchinotogluma, Bathykurila, Branchiplicatus, Lepidonotopodium, Levensteiniella Pettibone, 1985, Thermopolynoe, and Peinaleopolynoe.

al. (2016), which was subsequently described as Branchinotogluma bipapillata Zhou, Wang, Zhang & Wang, 2018. Due to the previous lack of DNA data from the type species Peinaleopolynoe sillardi, the phylogenetic position of Peinaleopolynoe was not examined until this study.
We present new DNA sequence data for a series of known and new branchiate scale worm specimens including some from nearby the type localities for P. sillardi, P. santacatalina, B. sandersi, and T. branchiata. We sequenced DNA for the following loci: mitochondrial cytochrome c oxidase subunit I (COI), 16S rRNA (16S), and cytochrome b (CytB), as well as nuclear 18S rRNA (18S), 28S rRNA (28S), and histone h3 (H3). We reassess branchiate scale worm phylogeny and the phylogenetic placement of Peinaleopolynoe by including representatives from all five branchiate genera. Additionally, we construct the first molecular phylogeny of Peinaleopolynoe by including DNA sequence data from both previously accepted Peinaleopolynoe spp. and describe four new Peinaleopolynoe spp. The morphology supporting the monophyly of the genus is examined and paraphyly of Branchinotogluma and Lepidonotopodium Pettibone, 1983 is explored.

Sample collection and morphology
Most new samples represent a range of polynoids collected on cruises using ROVs or the HOV "Alvin" in the eastern Pacific from 2004-2019. The majority of samples were collected via Monterey Bay Aquarium Research Institute's R/V "Western Flyer" and ROVs "Tiburon" and "Doc Ricketts". Other eastern Pacific samples were obtained with the R/V "Falkor" and ROV "SuBastian" and the R/V "Atlantis" and HOV "Alvin". Specimens of B. sandersi were collected by the E/V "Nautilus" and ROV "Hercules" from vents near the type locality for this species (Galápagos Rift vents). A specimen of T. branchiata was collected by the R/V "Melville" and ROV "Jason II" from a Lau Back Arc Basin hydrothermal vent, a few hundred kilometers from the type locality of vents in the North Fiji Basin in the western Pacific. A specimen of P. sillardi was collected from the central Atlantic at 3900 m and identified by the third author (SH). Tables 1, 2 provide further details of collection localities, deposition of types and vouchers, and GenBank accession numbers. The holotypes, most paratypes, and vouchers are deposited at the Scripps Institution of Oceanography, Benthic Invertebrate Collection (SIO-BIC), La Jolla, California, USA. Some paratypes are also deposited at the Museo de Zoología (Universidad de Costa Rica), San José, Costa Rica (MZUCR) and the Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autónoma de México (UNAM-ICML-EMU), Mazatlán, Mexico. The voucher for P. sillardi is deposited at the Muséum national d'Histoire naturelle (MNHN-IA), Paris, France and those of B. sandersi are at Harvard University's Museum of Comparative Zoology (MCZ), Cambridge, Massachusetts, USA.     Prior to preservation, whole specimens were generally relaxed with 7% MgCl 2 in fresh water and photographed alive using Leica MZ8 or MZ9.5 stereomicroscopes with a Canon EOS Rebel T6i attachment. They were then fixed in either 95% ethanol for DNA extraction or 10% formaldehyde in seawater for morphological work. For those fixed in formalin, some elytra were also fixed in 95% ethanol. After a day, specimens preserved in formalin were rinsed and transferred to 50% ethanol. Post-preservation, specimens of the new species Peinaleopolynoe orphanae sp. nov., Peinaleopolynoe elvisi sp. nov., Peinaleopolynoe goffrediae sp. nov., and Peinaleopolynoe mineoi sp. nov. were examined (Table 2) and photographed using the Leica S8 APO, DMR HC, and/ or Leica MZ9.5 microscopes with a Canon EOS Rebel T6i attachment.

DNA extraction, amplification, and sequencing
DNA from samples fixed and preserved in 95% ethanol was extracted using the Zymo Research DNA-Tissue Miniprep kit, following the manufacturer's protocol. Partial mitochondrial cytochrome c oxidase subunit I (COI) DNA sequences were obtained for these specimens for 'species' delimitation (Table 1). Representatives from each 'species' within the combined COI data set from this study and terminals that had only been sequenced for COI in Goffredi et al. (2017) were then sequenced for mitochondrial (16S rRNA (16S) and cytochrome b (CytB)) and nuclear (18S rRNA (18S), 28S rRNA (28S), and histone h3 (H3)) genes. All sequences obtained are deposited in GenBank (Table 1). Amplification was carried out using a PCR mixture of 12.5µl Apex 2.0× Taq Red DNA Polymerase Master Mix (Genesee Scientific), 1µl each of the appropriate forward and reverse primers (10µM), 8.5µl of ddH 2 O, and 2µl of eluted DNA, or when amplification using this mixture failed, 12.5µl Apex 2.0× Taq Red DNA Polymerase Master Mix (Genesee Scientific) was substituted with 12.5µl Conquest PCR 2.0× Master Mix 1 (Lamda Biotech). DNA sequencing was completed with the following PCR primers (Table 3) and temperature profiles, performed in a thermal cycler (Eppendorf ). Final PCR products were purified with ExoSAP-IT (USB Affimetrix, Ohio, USA), and Sanger sequencing was performed by Eurofins Genomics

Phylogenetic analyses
Consensus sequences were created via De Novo Assembly on Geneious v.11.0.5 (Kearse et al. 2012) with default settings. Alignments of the newly generated sequences, along with data for the six genes logged on GenBank from several different studies (Table 1), were performed for each gene using MAFFT v.7 server (Katoh and Standley 2013) with the G-INS-1 progressive method. In this study, we referred to the Branchinotogluma sandersi sequences JN852923, JN852889, JN852821, and JN852851, sourced from Norlinder et al. (2012), as Branchinotogluma cf. sandersi, because the specimen was collected from Juan de Fuca hydrothermal vents in the northeast Pacific, as opposed to from the type locality along the Galápagos Rift. We have included B. sandersi sequences from the type locality and the Gulf of California (Table 1). Aligned sequences for COI, 16S, 18S, 28S, H3, and CytB were concatenated using SequenceMatrix v.1.8 (Vaidya et al. 2011). A maximum likelihood (ML) analysis was performed using RAxML v.8.1.22 (Stamatakis 2014) on the concatenated data set partitioned by gene, using the model GTR+G. Node support was assessed via the thorough bootstrapping option (with 1000 pseudoreplicates). The deep-sea polynoids Austropolaria magnicirrata Neal, Barnich, Wiklund & Glover, 2012, Gesiella jameensis (Hartmann-Schröder, 1974), and Pelagomacellicephala cf. iliffei were chosen as the most appropriate outgroup based on previous phylogenetic results (Gonzalez et al. 2017b). A Bayesian inference (BI) analysis of the concatenated data partitioned by gene was also conducted using Mr. Bayes v.3.2.6 (Ronquist et al. 2012). Best-fit models for these partitions were selected using the Akaike information criterion (AIC) in jModelTest 2.1.10 v.20160303 (Guindon and Gascuel 2003;Darriba et al. 2012). COI, 16S, 18S, 28S, and CytB were assigned the GTR+I+G model; H3 was assigned the HKY+G model. A maximum parsimony (MP) analysis was conducted using PAUP* v.4.0a165 (Swofford 2002), using heuristic searches with the tree-bisection-reconnection branch-swapping algorithm and 100 random addition replicates. Support values were determined using 100 jackknife replicates each with 100 random addition searches and heuristic search with tree-bisection-reconnection. The ML tree of the combined analysis of COI, 16S, 18S, 28S, H3, and CytB was annotated with ML bootstrap percentages from RAxML, BI posterior probability, and MP jackknife support values. Minimum uncorrected interspecific pairwise distances and maximum uncorrected intraspecific distances were calculated for the Peinaleopolynoe COI dataset with PAUP* v.4.0a165 (Swofford 2002).

Character transformations
A cutdown ML molecular phylogeny of Peinaleopolynoe with its sister group, a clade composed of Branchinotogluma sp. nov. 1 and B. bipapillata, was generated using the same data (realigned) and with the same parameters with RAxML. Character transformations for two morphological features were then mapped onto this tree using Mesquite v.3.6 (Maddison and Maddison 2018). The Mk1 likelihood model was used for the transformations, because this incorporates branch length information into the transformation. The morphological characters and states used were: 1 Ventral segmental papillae and/or lamellae: State 0, Males with two pairs of papillae on segments 12-13 and four pairs of lamellae on segments 14-17, and females with five pairs of papillae on segments 11-15; State 1, Four pairs of papillae on segments 12-15. 2 Elytral number: State 0, 10 pairs of elytra; State 1, 9 pairs of elytra.

Phylogeny and species delimitation
All analyses ( Fig. 1) recovered B. cupreus as the sister taxon to a well-supported clade of the remaining ingroup taxa, a clade that comprised the remaining four branchiate genera, as well as the non-branchiate scale worms Levensteiniella spp., Lepidonotopodium spp., and Bathykurila guaymasensis Pettibone, 1989. In the ML and MP analyses, the branchiate T. branchiata was recovered as the sister taxon to Lepidonotopodium fimbriatum Pettibone, 1983 and nested among other non-branchiates: Lepidonotopodium spp., Levensteiniella spp. and B. guaymasensis. Lepidonotopodium, with L. fimbriatum as the type, was found to be paraphyletic (Fig. 1).
The ML, BI, and MP analyses ( Fig. 1) of the concatenated data set were not congruent at deeper nodes and this is reflected in the low support for some of these nodes. However, all analyses showed the same topology for relationships within Branchinotogluma at shallower nodes (3, 4, 5, 6 and 7 discussed below). The BI placement of Branchinotogluma segonzaci (Miura & Desbruyères, 1995) and Branchinotogluma trifurcus (Miura & Desbruyères, 1995) (Suppl. material 1: Fig. S1) was congruent with the ML analysis (1 and 2 discussed below), but these taxa had unresolved placement (collapsed nodes) in the MP analysis (Suppl. material 1: Fig. S2). Although differing at some nodes with regards to the clade composed of Levensteiniella spp., Lepidonotopodium spp., B. guaymasensis, and T. branchiata, both the BI (Suppl. material 1: Fig. S1) and the MP (Suppl. material 1: Fig. S2) analyses recovered Lepidonotopodium as non-monophyletic. The ML, BI, and MP analyses all supported the monophyly of Branchipolynoe and recovered the same relationships among the nine Branchipolynoe spp., which were the same as reported in Lindgren et al. (2019).
The ML, BI, and MP analyses also recovered Branchinotogluma as non-monophyletic, with the genus scattered across the ingroup (Fig. 1, see numbers 1-7):  The focus in this study, Peinaleopolynoe, was a well-supported clade in all analyses ( Fig. 1) and the relationships within Peinaleopolynoe were congruent in the ML and BI analyses. Peinaleopolynoe mineoi sp. nov., P. santacatalina, P. elvisi sp. nov., and P. sillardi formed a grade with respect to a P. goffrediae sp. nov. and P. orphanae sp. nov. clade. The MP topology (Suppl. material 1: Fig. S2) differed in showing P. elvisi sp. nov. and P. sillardi as a clade (as opposed to a grade) that was sister group to the P. goffrediae sp. nov. and P. orphanae sp. nov. clade.

Haplotype networks
The 17 specimens of P. orphanae sp. nov. that were collected with elytra remaining on the dorsum were coded for elytral color in the COI haplotype network ( Fig. 2A), which displayed eleven distinct haplotypes. There was no correlation between elytral color and haplotype for the specimens with pink, blue, and white elytra; these speci- mens were spread amongst nine different haplotypes. Although the two specimens with red elytra and black elytra respectively had their own distinct haplotypes, there were only 17 total specimens analyzed, so it is unlikely this represents the full diversity of haplotypes. When the specimens of P. orphanae sp. nov. that had lost their elytra were included (N = 24 in total), there were eleven haplotypes (Fig. 2B) from the type locality of the Pescadero Basin. The single specimen from Monterey Canyon shared a haplotype with a Pescadero Basin specimen. For the four specimens of P. santacatalina, there were four haplotypes (Fig. 3A). For the four specimens of P. mineoi sp. nov., there were four haplotypes that differed from each other by only one base (Fig. 3B). Lastly, for the five specimens of P. elvisi sp. nov., there were five haplotypes across the three localities from Costa Rica to California (Fig. 3C).

Character transformations
The state for the B. sp. nov. 1 and B. bipapillata clade was males with two pairs of papillae on segments 12-13 and four pairs of lamellae on segments 14-17, and females with five pairs of papillae on segments 11-15. The ancestral state of ventral segmental papillae and/or lamellae was inferred to be four pairs of papillae present on segments 12-15 for Peinaleopolynoe, which is arguably also an apomorphy for the clade (Fig. 4A).
The ancestral state of elytra was unclear for Peinaleopolynoe, but there was a slightly greater likelihood of possessing nine pairs of elytra (Fig. 4B). A most parsimonious transformation under this scenario would imply a reversal to the outgroup state of ten pairs of elytra for P. santacatalina (Fig. 4B). The other equally parsimonious alternative would be for nine pairs of elytra to have evolved independently in P. mineoi sp. nov. and the clade comprised of P. elvisi sp. nov., P. sillardi, P. goffrediae sp. nov. and P. orphanae sp. nov.
Etymology. Peinaleopolynoe orphanae sp. nov. is named after Dr. Victoria J. Orphan, not only for her invaluable research on deep-sea microorganisms, but also for her exploration of deep-sea chemosynthetic ecosystems and her love of the animals that thrive there.
Ecology. Peinaleopolynoe orphanae sp. nov. is unusual among Peinaleopolynoe in that most specimens were associated with bacterial mats adjacent to hydrothermal vents in the Pescadero Basin at ~3700 m depth. One specimen (SIO-BIC A10926) was found at a cold seep with abundant vesicomyid clams suggesting that P. orphanae sp. nov. may be more of a habitat generalist than its close relatives.
Superior neurochaetae (supra-acicular) with double rows of spines (Fig. 13D). Inferior neurochaetae (sub-acicular) with double rows of teeth from the mid swelling to the hooked tips; smooth beneath the mid swelling (Fig. 13E). Inferior neurochaetae teeth are less prominent than the superior neurochaetae spines. Hooked jaws with small teeth on inner borders (Fig. 10B).
Remarks. Peinaleopolynoe elvisi sp. nov. is unique from the remaining Peinaleopolynoe taxa in having six pairs of border papillae on the pharynx (Table 5). Additionally, P. elvisi sp. nov. differs from its closest relatives P. santacatalina and P. sillardi in having branchiae start on segment 3, as opposed to on segment 2. Finally, the posterior margin of the elytra displays a single macrotubercle compared to the few found in the other species.
Etymology. Peinaleopolynoe elvisi sp. nov. is named after the legendary King of Rock and Roll, Elvis Presley; the iridescent golden/pink elytra are reminiscent of the sparkly, sequined costumes he favored in his late career.
Remarks. Peinaleopolynoe goffrediae sp. nov.'s closest relative is P. orphanae sp. nov. (Fig. 1). Peinaleopolynoe goffrediae sp. nov. can be distinguished from P. orphanae sp. nov. by the segmental range of branchiae, the former present on segments 2-17 and the latter on segments 3-18 (Table 5). Additionally, the four pairs of ventral papillae on P. goffrediae sp. nov. are long, tapered, and curved laterally, distinguishing it from the small, rounded, cylindrical papillae of P. orphanae sp. nov. Peinaleopolynoe goffrediae sp. nov. is unique among Peinaleopolynoe taxa in that the angle formed by the ventral part on the neuroacicular lobe is clearly diagonal while it is nearly horizontal in the other species.
Etymology. Peinaleopolynoe goffrediae sp. nov. is named after Dr. Shana K. Goffredi for her notable contribution to the exploration and research of deep-sea chemosynthetic ecosystems (especially whalefalls), focusing on symbiotic relationships between bacteria and marine invertebrates.
Ecology. Peinaleopolynoe goffrediae sp. nov. was only found associated with a whalefall (Table 5). Fig. 6H shows the holotype observed in situ on a whale carcass before collection.  : 1 mm (A, B, D); 0.5 mm (C, E-J).
Etymology. Peinaleopolynoe mineoi sp. nov. is named after Ronald M. Mineo, MD, in recognition of support from the Mineo family, their interest in the deep sea, and support for our research.
Ecology. Peinaleopolynoe mineoi sp. nov. was found associated with bones and wood (Table 5). Like P. santacatalina and P. orphanae sp. nov., it may be more of a habitat generalist than other Peinaleopolynoe.

Discussion
In this study we provided new data for five loci (16S, CytB, 18S, 28S and H3) for a series of specimens that had previously been documented for only COI in Goffredi et al. (2017). That study was a biodiversity inventory of vents in the southern Gulf of California and included COI data for previously described polynoids such as B. hessleri (the type species for genus), B. sandersi, B. cupreus (a monotypic genus), L. fimbriatum (the type species for genus), Lepidonotopodium williamsae Pettibone, 1984, an undescribed Lepidonotopodium sp., and a previously undescribed Peinaleopolynoe (here described as P. orphanae sp. nov.). We also have added here a new 16S sequence for B. guaymasensis to supplement the previous 18S and COI data from Glover et al. (2005). Seven COI sequences were generated for B. sandersi from the type locality (Galápagos) and these matched the B. sandersi data from the Gulf of California, confirming the identification in Goffredi et al. (2017). We also provided the first DNA data for the only branchiate genus for which such data was lacking, T. branchiata. This new data, combined with the new data for Peinaleopolynoe spp. allowed for a further assessment of the relationship among deep-sea polynoids associated with vents, seeps and food falls (see below).
The prime focus of this study was Peinaleopolynoe and we generated DNA data for the two described species P. sillardi and P. santacatalina, and a series of undescribed species in addition to the Peinaleopolynoe reported in Goffredi et al. (2017). The monophyly of Peinaleopolynoe was supported here by the phylogenetic analysis of DNA data (Fig. 1), as well as by the presence of ventral papillae on segments 12-15 (Table 5, Fig. 4A). In addition to the phylogenetic results, species delimitation is supported by morphology (Table 5) and the marked difference in the uncorrected COI distance analysis; the intraspecific COI distances range from 0-1.46%, while the interspecific COI distances range from 12.65-19.64% (Table 4). These distances are in excess of what has often been used to delineate species level taxa in annelids (see review by Nygren 2014). We also were able to find apomorphic features for all four new species (Table 5): the branchiae of P. orphanae sp. nov. terminate on segment 18 and the four pairs of ventral papillae are small, cylindrical, and rounded; P. elvisi sp. nov. has six pairs of border papillae on the pharynx and a single macrotubercle on the elytra; the ventral part of the neuroacicular lobe is diagonal in P. goffrediae sp. nov.; and P. mineoi sp. nov. has large, rounded, protruding teeth on the inner borders of their jaws. The discovery of these four new species takes the number of Peinaleopolynoe spp. to six. The Peinaleopolynoe clade was recovered as sister group (Fig. 1) to what we refer to here as Branchinotogluma clade 5 (B. bipapillata and B. sp. nov. 1). Although the state of elytral number for the common ancestor between Peinaleopolynoe and Branchinotogluma clade 5 was unresolved (Fig. 4B), we conclude that it is likely ten pairs of elytra. The sister group to the combined Branchinotogluma clade 5 with Peinaleopolynoe is B. japonicus with Branchipolynoe (Fig. 1), which all possess ten pairs of elytra (Miura and Hashimoto 1991;Zhang et al. 2018a;Lindgren et al. 2019). The most likely ancestral state for Peinaleopolynoe is nine pairs of elytra, supporting a reversal in P. santacatalina, which is the only Peinaleopolynoe species that possesses ten elytra in the clade (Fig. 4B).
The first two Peinaleopolynoe spp., P. sillardi and P. santacatalina, were described from organic falls, and Desbruyères and Laubier (1988) highlighted this preference in the genus name, which includes a reference to hunger or being famished. This preferred habitat is indeed unusual among the deep-sea polynoids to which Peinaleopolynoe is closely related, where hydrothermal vents and methane seeps are the normal habitat. An exception is B. guaymasensis, which is known from whalefalls and hydrothermal vents (Pettibone 1989;Glover et al. 2005). Table 5 summarizes the habitats for the six Peinaleopolynoe now known and it is notable that with the exception of P. orphanae sp. nov., all of these 'hungry' scale worms have been found on organic remains such as whalefalls, deployed bones and wood. Unusually, P. santacatalina was also found at a seep, and the derived position of P. orphanae sp. nov. within Peinaleopolynoe suggests its occurrence at seeps and vents may be a secondary colonization.
Our addition of DNA data for new taxa and additional loci for previously published specimens (Table 1) has allowed for an updated assessment of the phylogeny of the clade of deep-sea polynoids that are mainly found at vents, seeps, and organic falls. Our results (Fig. 1, Suppl. material 1: Figs S1, S2) are largely similar to the three gene phylogeny in Zhou et al. (2018: fig. 7), though our rooting is different. We show that B. cupreus, which has branchiae, is the sister group to all of the other ingroup taxa. Pettibone (1985b) had recognized that the branchiae of B. cupreus were quite different from those of the only known branchiate polynoid at that time, Branchipolynoe, and placed it in its own subfamily Branchiplicatinae. We found Branchinotogluma to be paraphyletic, as has been reported by others recently (Zhang et al. 2018a, b;Zhou et al. 2018;Wu et al. 2019). Branchinotogluma occurs in seven places across our phylogeny (Fig. 1). The type species of Branchinotogluma is B. hessleri, which occupies an isolated position in Fig. 1. If this position is maintained with further phylogenetic investigation, then membership of Branchinotogluma may become quite restricted. Support for some keys nodes is low though (Fig.  1), so no taxonomic changes are recommended at this time for Branchinotogluma.
A clade of mainly non-branchiate polynoids Levensteiniella spp., Lepidonotopodium spp., B. guaymasensis and T. branchiata (Fig. 1) was sister group to B. segonzaci and nested within a clade of various branchiate polynoids, suggesting they may have lost this feature. Thermopolynoe branchiata does have branchiae, but in his description Miura (1994) pointed out that this species was unique among branchiate polynoids in having well-developed bracts encircling the notopodia and in the position of branchiae. Thermopolynoe branchiata has arborescent branchiae as in Branchinotogluma, Branchipolynoe and Peinaleopolynoe, but segments with two groups of branchiae are split into anterior and posterior groups as opposed to upper and lower groups (Miura 1994). It is thus clear that branchiae have evolved and been lost several times in Polynoidae. The placement of L. fimbriatum as sister taxon to T. branchiata and the other three included Lepidonotopodium terminals as sister group to Levensteiniella suggest Lepidonotopodium will require revision. Further taxon sampling of the other species in Lepidonotopodium is warranted and support for some key nodes is too low at present.
In a recent phylogenetic study of deep-sea Polynoidae, Bonifácio and Menot (2018) made the polynoid subfamilies that comprise the majority of deep-sea polynoids found at vents, seeps and whalefalls, namely Branchinotogluminae, Branchiplicatinae, Branchipolynoinae, and Lepidonotopodinae, junior synonyms of Macellicephalinae. The sampling of these taxa for a morphology/molecular sequence data analysis (Bonifácio and Menot 2018: fig. 2) that they had for these subfamilies (L. fimbriatum, B. sandersi, B. symmytilida and Peinaleopolynoe sp.) resulted in a clade referred to as 'clade b1' that formed the sister group to the rest of the Macellicephalinae. Clade b1 also included B. guaymasensis that was placed in Macellicephalinae when first described by Pettibone (1989). Our results shown here clearly suggest that the members of Branchinotogluminae, Branchiplicatinae, Branchipolynoinae, and Lepidonotopodinae, as well as B. guaymasensis and Levensteiniella (initially described as Macellicephalinae), form a well-supported clade. Here, we refer to this clade, clade b1 of Bonifácio and Menot (2018), with the oldest available subfamily name and reinstate Lepidonotopodinae.