More Knot Worms: Four New Polygordius (Annelida) Species from the Paciﬁc and Caribbean

: Polygordius is a clade of marine annelids mainly seen in coarse-grained habitats. They are notable for their smooth bodies, lacking in chaetae or obvious segments, and they resemble Nematoda or Nemertea. Though Polygordius taxa are found in all oceans of the world, identifying species based only on morphological characters can be challenging due to their relatively uniform external appearances. Diversity within the clade has likely been markedly underestimated. Where morphological characters are inconspicuous or even unreliable, molecular methods can provide clarity in delimiting species. In this study, morphological methods (examination under light and scanning electron microscopy) were integrated with molecular analyses (sequencing of Cytochrome c oxidase subunit I, 16S rRNA and Histone H3 gene fragments) to establish the systematic placement of Polygordius specimens collected from Australia, Belize, French Polynesia, Indonesia, Japan, and the U.S. west coast. These analyses revealed three new species of Polygordius from the Paciﬁc Ocean ( P. erikae n. sp., P. kurthcarolae n. sp., and P. kurthsusanae n. sp.) as well as one new species from the Caribbean Sea ( P. jenniferae n. sp.). These new species are formally described, and a previously known Japanese species, P. paciﬁcus Uchida, 1936, is redescribed. This study establishes the ﬁrst molecular data set for Polygordius species from the Paciﬁc region, as well as the ﬁrst formal description of a Caribbean species of Polygordius . Phylogenetic relationships within Polygordius are summarized and discussed. ◦ followed cycles of at ◦ C (40 annealing ◦ (40 ◦ ﬁnal ◦ (5 initial 35 cycles of denaturation ◦ C (40 s), ◦ C (40 s), at ◦ C (50 s), ﬁnal at ◦ C (5 min). COI ampliﬁcation, proﬁles (1) initial denaturation at 95 ◦ C (3 min), followed by 40 cycles of denaturation at 95 ◦ C (40 s), annealing at 42 ◦ C (45 s), elongation at 72 ◦ C (50 s), and ﬁnal extension at 72 ◦ C (5 min); (2) initial denaturation at 94 ◦ C (3 min), followed by 5 cycles of denaturation at 94 ◦ C (30 s), annealing at 47 ◦ C (45 s), elongation at ◦ (60 30 cycles of denaturation at 94 ◦ C (30


Introduction
Polygordius Schneider, 1868 [1], the only genus in Polygordiidae Czerniavsky, 1881 [2], is a clade whose members are all smooth-bodied marine annelids found living in the interstitial spaces of relatively coarse sediments ranging from the intertidal zone to continental slope environments worldwide [3,4]. Their tendency to tie themselves in knots when collected, and the etymological link to the legendary Gordian Knot, led us to coin the common name "knot worms" for this paper. Although 17 species and two subspecies have been described from the Atlantic, Pacific, Indian, and Southern Oceans, only 14 species are considered valid [3] (Table 1), and most of these have been described from the north Atlantic Ocean and Mediterranean Sea [5].

Materials and Methods
Distinguishing features of Polygordius include a rounded or conical prostomium that tapers anteriorly into paired palps of varying length and proximity to one another, which are innervated and function as sensory receptors [7,8]. The prostomium also bears paired dorsolaterally-positioned nuchal organs used for sensory perception [6]. Completing the head is the peristomium, and its division from the prostomium is marked by a deep or shallow "head fold" situated in front of the ventral mouth [7,9]. The trunk shows no signs of external segmentation and is reminiscent of nematodes or ribbon worms (Nemertea), though the septa of the segments can be seen internally with the appropriate lighting. The trunk, which can have 200 or more segments, terminates in a pygidium that may be either inflated or cylindrical, and in many species of Polygordius it is encircled by pygidial glands that serve an adhesive function in the sediments [10]. These glands vary in size, shape, and number between different species [7]. Pygidial appendages/cirri may also be present either terminally or subterminally, with the tip of the pygidium forming distinctive anal lobes [4].
Identifying Polygordius species based on morphological characters alone can be challenging. Their long, cylindrical bodies appear relatively similar to one another under visual inspection, and the distinguishing features useful for morphology-based discrimination of species are small, requiring examination under scanning electron microscopy [3]. Even annelid species with larger and more conspicuous external features may be difficult to describe based on morphological methods alone [11], and species richness and diversity within Annelida in general and Polygordius specifically are likely much greater than is presently documented due to such challenges [10,12]. Molecular data can reveal both synonymous and cryptic species [5] and, combined with morphology-based techniques, provides a powerful tool for delineating species with greater accuracy.
After the first adult Polygordius was described, P. lacteus Schneider, 1868, the genus spent much of its history misinterpreted as a primitive group owing to a lack of annelid features such as chaetae and parapodia [4]. Polygordius was the first clade to be placed in the taxon Archiannelida by Hatschek [13]. He established the group for what he presumed to be the earliest annelid lineages, but Archiannelida was subsequently revealed as an invalid, polyphyletic taxon [3,4,14]. Polygordius is now viewed as a derived group that has adapted well to life in the interstitial environment [11,15]. Recent molecular systematic studies relate them most closely to Protodrilida [15], or Phyllodocida [11] as part of Errantia.
Ramey-Balci et al. [5] published the first phylogenetic hypotheses within Polygordius, focusing on European and Atlantic species. Their work confirmed six valid species, while synonymizing two, thus clarifying the diversity present in the region of study and providing a foundation for the use of molecular methods to understand evolutionary relationships within Polygordius. However, molecular data for Polygordius from other regions are lacking, and they have not been established for species Diversity 2020, 12, 146 3 of 25 outside of the Atlantic Ocean. In this study, we have generated molecular and morphological data for three new species of Polygordius from the Pacific Ocean as well as the first known species from the Caribbean Sea. We provide data for several other specimens that we do not describe here, owing to a lack of complete specimens. We also redescribe Polygordius pacificus Uchida, 1935 [16] from Japan. We summarize the evolutionary relationships of these new species in relation to each other and the established species from the Atlantic Ocean.

Collection of Specimens
Specimens for all new species described in this study were collected from coarse sediments in locations across the Pacific Ocean and Caribbean Sea including French Polynesia, the east coast of Australia, Belize, and Japan. Specimens were collected over a depth range of 1-18 m and across a time span from 1996 to 2015 ( Figure 1).
Diversity 2020, 12, x FOR PEER REVIEW 3 of 29 species outside of the Atlantic Ocean. In this study, we have generated molecular and morphological data for three new species of Polygordius from the Pacific Ocean as well as the first known species from the Caribbean Sea. We provide data for several other specimens that we do not describe here, owing to a lack of complete specimens. We also redescribe Polygordius pacificus Uchida, 1935 [16] from Japan. We summarize the evolutionary relationships of these new species in relation to each other and the established species from the Atlantic Ocean.

Collection of Specimens
Specimens for all new species described in this study were collected from coarse sediments in locations across the Pacific Ocean and Caribbean Sea including French Polynesia, the east coast of Australia, Belize, and Japan. Specimens were collected over a depth range of 1-18 m and across a time span from 1996 to 2015 ( Figure 1).

Light and Scanning Electron Microscopy
Specimens of all new species described in this study were examined using light microscopy (Leica S8 Apo stereomicroscope and Canon EOS Rebel T6s camera, using Adobe Photoshop software, version 21.1.0) to measure more prominent morphological features (e.g., body length and width) and scanning electron microscopy (SEM) to examine minute features such as the number, size, and shape of the pygidial glands and anal lobes which are necessary for morphology-based discernment of species. Measurements were taken as follows: total length, from the base of the palps to the tip of the pygidium where it terminates in the anal lobes; prostomium length, which is the distance from the base of the palps to the top of the head fold (ventral transverse groove separating the prostomium and peristomium); body and pygidial width, measured at the widest section of the trunk and pygidium (at pygidial inflation) respectively; "glandular belt width", is the width of the ring of glands encircling the Diversity 2020, 12, 146 5 of 25 pygidium; gland width, measured in the middle region of several well-fixed glands; and the length of "elongate/enlarged anal lobes", which is the distance from the base of the anal lobe to the tip of the elongate/enlarged extension (= lobe + pygidial cirrus). In order to determine whether the prostomium is "conical" or "rounded" the fixed specimen must be lying relatively flat, ventral-side-up or laterally. If the prostomium length is > its width, it is considered "conical", whereas, if the prostomium width ≥ its length, it is "rounded". Specimens were prepared for SEM examination using a procedure adapted from the air-drying technique outlined by Murtey and Ramasamy [21]. Specimens were first moved from their respective preservatives (either ethanol or formalin) to water, then postfixed in osmium tetroxide for 80 min. Specimens were washed in water 4-5 times at 60-minute intervals followed by overnight immersion. After washing, specimens were dehydrated through a graded series of ethanol and hexamethyldisilazane (HMDS), beginning with immersion in 70% ethanol for 20 min, followed by two changes of 95% ethanol at 20-minute intervals, and two changes of 100% ethanol at 20-minute intervals. After immersion in pure ethanol, 50% HDMS was added to each specimen for a 20-minute interval, then 75% HDMS for a 20-minute interval, and finally three changes of 100% HDMS at 20-minute intervals. Specimens were allowed to dry overnight before being mounted on aluminum stubs, sputter-coated with gold-palladium (Au-Pd), and examined under a Zeiss EVO 10 scanning electron microscope.

DNA Extraction, Amplification, and Sequencing
DNA was extracted from samples of each specimen's trunk tissue using the Zymo Research DNA-Tissue Miniprep Kit. All successful extractions were completed using the protocol outlined by the manufacturer. Two mitochondrial genes were then amplified and sequenced for the majority of specimens in this study: the 16S rRNA gene (16S) and cytochrome c oxidase subunit I (COI). The nuclear gene Histone H3 (H3) was also amplified and sequenced in order to compare specimens with more fragmented DNA ( Table 2). The polymerase chain reaction (PCR) was used to amplify a section of each target gene in a 25 µL solution of H 2 O (8.5 µL), taq DNA polymerase (12.5 µL), primer sets (1 ul each), and template DNA (2 ul). (In some cases, volumes were adjusted based on the concentration of DNA in the extraction.) The following primer sets were used:  The following cycling profiles were used for 16S amplification: (1)  . Successful PCR products were purified using the ExoSAP-IT protocol (USB, Affymetrix), sequencing was conducted by Eurofins Genomics (Louisville, KY), and sequences were then assembled and edited using Geneious software (version 11.1.5).

Molecular Methods and Analysis
Sequences from previously published Polygordius species were obtained from GenBank (Table 2). Where sequences were available from multiple regions, then ones from nearest the type locality were chosen. Sequences were aligned using MAFFT [28] and then concatenated using SequenceMatrix v.1.8 [29]. A maximum likelihood (ML) analysis was performed on this data set using RAxML v.8.1.22 [30], partitioned by gene (and codon for COI), under the model GTR+G. Node support was assessed via the thorough bootstrapping (with 1000 pseudoreplicates). The protodrilid Protodrilus pythonius Di Domenico, Martínez, Lana and Worsaae, 2013 [26] was used as an outgroup, based on recent phylogenetic results [11,15]. Haplotype networks were generated for the COI data obtained for three of the new species using the TCS algorithm [31] in PopArt [32]. Uncorrected pairwise distances for COI were calculated using PAUP* [33]. Table 3 shows uncorrected pairwise distances for COI sequences for available data on GenBank for five known Polygordius species (from type localities) and the new samples for this study. Most Diversity 2020, 12, 146 7 of 25 distances among species are minimally 20% or more. The minimum distance found for any previously known species was between P. lacteus and P. neapolitanus Fraipont, 1887 [19] (15%). With respect to the new data, the undescribed Polygordius sp. from southern California was found to be 13% divergent from P. jenniferae n. sp. from Belize. Polygordius sp. from Indonesia was 17% divergent from P. jouinae (NE United States). Polygordius sp. from Washington was closest in distance and phylogeny ( Figure 2) to a clade of three species from Germany and Italy, with P. lacteus being closest in distance (19%). Polygordius eschaturus from Brazil was 19% divergent from P. pacificus (Japan). The Pacific clade of three new Polygordius species from Australia and French Polynesia (P. erikae n. sp. P. kurthcarolae n. sp. and P. kurthsusanae n. sp.) were all at least 20% divergent from each other. These distances support the recognition of eight new species level taxa in Polygordius. One of these is recognized here as the known species P. pacificus and redescribed below. Sequences for Polygordius sp. (California), Polygordius sp. (Indonesia), and Polygordius sp. (Washington) were each obtained from incomplete individuals that were lacking pygidia, so these are not formally described here. The other species did have at least one complete specimen and so these are formally described below.

Species Delimitation and Phylogeny
For the new species with multiple individuals, P. erikae n. sp., P. kurthcarolae n. sp., and P. kurthsusanae n. sp., haplotype networks for COI were made and these are shown in Figure 2. The two sequences for P. erikae n. sp. were both from the same locality (Lord Howe Island, NSW, Australia) but were 2% divergent from each other. We do not consider this to be enough to separate them, since this distance, while marked for specimens from the same locality, is within what is "normal" annelid intraspecific variation in annelids [34]. The maximum distance among the four COI sequences for P. kurthsusanae n. sp., all in one sand sample from Moorea, French Polynesia was also relatively high, for intraspecific distance, at 2.5%. Polygordius kurthcarolae n. sp. has a type locality on the Great Barrier Reef (Australia) and the two COI sequences from the type locality were identical. A Polygordius from Tetiaroa Atoll, French Polynesia, was 2% divergent from these Australian sequences, so to be consistent, we regard this specimen to be P. kurthcarolae n. sp., even over 6000 km away.
The maximum likelihood phylogeny based on the three gene fragments (mostly missing H3) is shown in Figure 2. The outgroup Protodrilus pythonius is not shown to save space, but the root position is based on its inclusion. A well-supported clade comprising P. triestinus (Croatia), P. jouinae (NE United States), and Polygordius sp. (Indonesia) was recovered as the sister group to the remaining Polygordius, but support was low. Within this major clade, P. eschaturus (Brazil) was the well supported sister group to P. pacificus (Japan) and this clade was then the sister group to the remaining Polygordius, which had two main subclades. One comprised the remaining European species from Germany and Italy, P. appendiculatus, P. lacteus, and P. neapolitanus, which formed a clade that was sister group to Polygordius sp. (Washington). The remaining clade of Polygordius was well-supported and comprised mainly Pacific terminals, including the three new Pacific species (P. erikae n. sp., P. kurthcarolae n. sp., and P. kurthsusanae n. sp.). These formed a poorly supported clade, though there was strong support for P. kurthcarolae n. sp. and P. kurthsusanae n. sp. as sister taxa. The new species from Belize (P. jenniferae n. sp.) was the well-supported sister group to Polygordius sp. (California). Overall, the tree had only a few poorly supported nodes, so the lack of any clear biogeographic patterns is somewhat surprising. The Atlantic/European/Mediterranean terminals were placed in three regions of the tree and none were close to the Caribbean P. jenniferae n. sp., which showed fairly low COI divergence from Polygordius sp. (California). The Australian/French Polynesian species did form a discrete clade. The phylogeny lacks sequence data for many of the known Polygordius species and further sequence data may make for more comprehensible biogeographical patterns. This paper includes the first data for Pacific and Caribbean Polygordius after the initial molecular study that focused on European/Atlantic region [5]. Obtaining sequences for Polygordius known from regions such as Antarctica and the Indian Ocean will be valuable for continuing phylogenetic studies. Table 3. Uncorrected interspecific distances for Polygordius taxa used in this study based on COI data. Sequences for holotypes are used for the new species and specimens from type localities are used for previously published data (see Table 2). Distances in bold are discussed in the text.

Etymology
Polygordius jenniferae n. sp. is named after the lead author's sister.

Remarks
The new species from Australia and French Polynesia (P. erikae n. sp., P. kurthcarolae n. sp. and P. kurthsusanae n. sp.) formed a distinct clade (Figure 2), within which each species was distinguished by at least 20% divergence from the others (Table 3). Polygordius erikae n. sp. is not markedly similar to either P. kurthcarolae n. sp. or P. kurthsusanae n. sp. but its pygidial glands and subterminal pygidial cirri resembles P. appendiculatus, P. ijimai (see remarks of P. jenniferae n. sp., discussing interpretation of morphological characters for this species), and P. kiarama (Tables 3 and 4). It can be clearly distinguished from these species by the following characteristics including~40-52 oval glands, 2 ventro-lateral cirri, 5 anal lobes, and a rounded prostomium (Table 4). In comparison, P. appendiculatus is easily distinguished from the new species in that it has~30 round glands, a palp: prostomium length ratio of 2:1, and pigment/"eyespots" (Table 4). Polygordius ijimai has three pygidial cirri including two inserted ventro-laterally and one dorsal-median, whereas, P. erikae n. sp. has two ventral-lateral cirri (Table 4) Polygordius erikae n. sp. is most similar to P. kiarama, for which there is unfortunately no DNA data available. Although both species have oval pygidial glands, there are fewer in number for P. kiarama (20 vs. 40-52), which also has 7-8 anal lobes compared to 5 for P. erikae n. sp. The prostomium of P. kiarama is conical and blunt (not pointed), whereas it is rounded in P. erikae. (Table 4).

Etymology
Polygordius kurthcarolae n. sp. is named to commemorate Carol Marie Kurth in honor of her wedding. We celebrate her tying the knot with a new knot worm.

Remarks
Polygordius kurthcarolae n. sp. is the well supported sister taxon to P. kurthsusanae n. sp. ( Figure  2) though they are morphologically very distinct. Polygordius kurthcarolae n. sp. (Great Barrier Reef, Australia) is distinguished from most other Polygordius species by having pygidial glands and subterminal pygidial appendages (Table 1). Morphologically similar species include P. appendiculatus, P. kiarama, and P. ijimai (see remarks of P. jenniferae n. sp., discussing interpretation of morphological characters for this latter species), which also have pygidial glands and subterminal pygidial appendages (Tables 3,4). Polygordius kurthcarolae n. sp. can be distinguished from these three

Etymology
Polygordius kurthsusanae n. sp. is named in honor of Susan Anne Kurth, who along with her sister Carol, "tied the knot" in 2019.

Remarks
Polygordius kurthsusanae n. sp. (French Polynesia) is the well-supported sister taxon to P. kurthcarolae n. sp. (Figure 2), but can be distinguished from it and most other Polygordius species in that it lacks pygidial glands and appendages (Table 1). Morphologically, the most similar species also lacking pygidial glands and appendages include P. arafura from Australia, P. jouinae from New Jersey, USA, and P. triestinus from the Adriatic Sea (Table 1). Polygordius kurthsusanae n. sp. can, however, be clearly distinguished from these three species (Table 5). Polygordius arafura, for which there is unfortunately no DNA available, has shorter palps (0.04-0.06 mm) with a palp to prostomium length ratio of~0.5:1 compared to 0.25 mm and~1.5:1 respectively, for P. kurthsusanae n. sp. ( Table 5). The median fold in P. arafura is subtriangular with a deep-cleft and is subtriangular without a deep-cleft in the new species.

Remarks
The type locality for Polygordius pacificus is near the Misaki Marine Biological Station, Honshu Japan. This is the same type locality as P. ijimai [35]. In the original description it was also noted that P. pacificus also sampled at the coast of the Japanese Sea [16]. The type material has apparently been lost (Eijiroh Nishi pers. comm.). The samples of Polygordius pacificus used in this study were also from Honshu, though 450 km south of the type locality. Given the distance from the type locality we do not nominate any of our specimens as a neotype.
Based on findings in the present study, P. pacificus is distinguished from most other Polygordius species by having pygidial glands and terminal pygidial appendages (Table 1). Morphologically similar species/subspecies include P. pacificus floreanensis, Polygordius eschaturus, P. eschaturus brevipapillosus, and P. madrasensis Aiyar and Alikunhi, 1944 [36], which also have pygidial glands and terminal pygidial appendages (Table 1). Of these, P. pacificus is most similar to P. pacificus floreanensis, P. eschaturus, and P. eschaturus brevipapillosus since they all have elongate pygidial glands (Table 6), whereas, P. madrasensis has oval glands (Aiyar and Alikunhi 1944 [36]). Polygordius pacificus can be easily distinguished from P. pacificus floreanensis by its rounded prostomium and~2:1 palp to prostomium length ratio, whereas the prostomium of P. pacificus floreanensis is conical with a palp to prostomium length ratio of~1:1 (Table 6). Unfortunately, examination of the single type specimen available for P. pacificus floreanensis could not provide details regarding the number of pygidial glands and anal lobes useful for species distinction, since SEM is needed to observe these and was not possible. Polygordius pacificus has 2-3 elongate/enlarged anal lobes similar to both Polygordius eschaturus and P. eschaturus brevipapillosus which have 2 and 2-3 elongate lobes respectively. The elongate lobes of these two species, however, are much longer (length 50-70 and 80 µm, respectively) than those of P. pacificus (length 40 µm), (Table 6). It is important to note that terminal pygidial cirri were absent in the original species description of P. pacificus (Table 6). This is not surprising, given that we only observed these with SEM. Furthermore, their morphology varied depending on fixation/condition of the specimen(s). In a relatively well-fixed individual, three of the six lobes appear enlarged ( Figure 12B,C, 3 enlarged) rather than elongate ( Figure 12G,H, 2 elongate). In the case of enlarged lobes, the tip of the lobe/pygidial cirrus is situated such that it is directed/turned "inward and downward" covering the anal opening ( Figure 12B,C). The genetic distance matrix indicated that the divergence value between P. pacificus and P. eschaturus is 19%, making P. eschaturus a sister group to P. pacificus within their clade, but clearly denoting separate species. Table 6. Characters of P. pacificus Uchida, 1935 [16] and specimens designated in the present study as compared to morphologically most similar species/subspecies including P. pacificus floreanensis, P. eschaturus, and P. eschaturus brevipapillosus. Morphological information was taken from the original species descriptions unless otherwise indicated.