The partial mitochondrial genome of the enigmatic Bermuda fireworm Odontosyllis enopla Verrill, 1900 and its phylogenetic implications

The partial mitochondrial genome of the enigmatic Bermuda fireworm Odontosyllis enopla Verrill, 1900 and its phylogenetic implications | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The partial mitochondrial genome of the enigmatic Bermuda fireworm Odontosyllis enopla Verrill, 1900 and its phylogenetic implications Brendan A. Cruz, Lynette D. Wyant, Aydanni D. Gonzalez, Joshua M. Kovalcik, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7724154/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The Bermuda fireworm, Odontosyllis enopla Verrill, 1900 , is a marine polychaete that displays a unique bioluminescent mating ritual. Despite the first sighting of O. enopla over 534 years ago, the availability of molecular data has been limited. Several syllid mitogenomes are currently available; however, there are only three published genes for O. enopla ; two partial mitochondrial genes (16S [508bp] and cox1 [653bp]; 1,161bp total) and one partial nuclear gene (18S [1,339bp]). This study bioinformatically mined previously published transcriptomes of O. enopla for mitochondrial reads and subsequently assembled and annotated a partial mitochondrial genome (10,172bp). The partial mitogenome includes nine (of 13) protein-coding genes, two ribosomal RNAs, and seven (of 22) complete tRNAs. We place the Bermuda fireworm in phylogenetic context using all available syllid mitogenomes, analyze intraspecific variation among three female O. enopla partial mitogenomes, and propose two putative locations for the mitochondrial origin of replication. Evolutionary Biology Bioluminescent Eusyllinae gene overlap intraspecific variation mitogenome origin of replication polychaete Syllidae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Odontosyllis enopla Verrill, 1900, more commonly known as the Bermuda fireworm, is a remarkable annelid belonging to the family Syllidae (Figure 1). These tube-dwelling worms, found on sandy coral substrates in benthic habitats (Fischer & Fischer, 1995), display an incredible bioluminescent mating ritual that was first seen and recorded by Christopher Columbus in 1492 (Brugler et al., 2018) and first described by Addison Emery Verrill in 1900 (Verrill, A.E. 1900). The Bermuda fireworm is a unique annelid due to its distinct mating behaviors and physiology. In preparation for breeding, fireworms of both sexes undergo morphological changes, including enlargement and pigmentation of their four eyes to enhance visual sensitivity. This is particularly pronounced in males for detecting the females' bioluminescence. This sets the stage for a highly synchronized mating swarm, where selective pressures from predation favor precise timing to minimize vulnerability of isolated or early individuals. Females initiate the ritual by rising to the surface and swimming in slow circles while emitting a continuous bluish-green glow from secreted luminous mucus and releasing their gametes. Triggered by this display, males swim towards the glowing females, producing quick, consecutive bioluminescent flashes while releasing their own gametes into the surrounding water (Verdes et al., 2022; Fischer & Fischer, 1995). The bioluminescence peaks in the green portion of the visible spectrum, with wavelengths between 504-507 nanometers. Their visual system is most sensitive to the aforementioned wavelength, which was shown by electroretinogram (ERG) recordings in response to light at multiple different wavelengths (Wilkens & Wolken, 1981). The similarities between the light detected and the light emitted suggest that fireworms are visually tuned to detect mating signals from bioluminescence. The fireworm synchronizes its mating ritual with the lunar cycle with swarming episodes coinciding with the first day after full moons during the summer and early autumn months (Brugler et al., 2018). Utilizing specialized setae, this benthic organism will swim to the surface 57 ± 1 minutes after the astronomical sunset to begin its mating ritual (Fischer & Fischer, 1995). This mating swarm has been observed up to five nights following the full moon, after which both male and female fireworms return to their benthic habitats (Fischer & Fischer, 1995). The fertilized zygotes undergo cell division and after fourteen hours become trochophores that can freely swim through the water columns (Fischer & Fischer, 1995). Evidence suggests that the common ancestor of all the bioluminescent syllid species was not bioluminescent itself; in fact, bioluminescence within Odontosyllis evolved independently twice (Verdes et al., 2022). While the bioluminescence of this species is of great interest to researchers, of even greater importance is the reversible epitoky metamorphosis in both males and females. The fireworm undergoes different physiological changes to prepare for the mating event. Some of the most noticeable physiological changes the fireworm undergoes include the enlargement of the eyes in males, the growth of setae used for rapid swimming to reach the surface, and tissues protruding from the body of the females holding the oocytes (Fischer & Fischer, 1995; Brugler et al., 2018). After the mating swarm, the swimming setae are shed although there does not appear to be a specific timeline for this reversal. Some fireworms shed the setae in as little as five days after swarming while others had at least some remaining setae after 35 days. Additional research needs to be conducted regarding reversal of the size of the male eyes. Research thus far indicates that there was no size reduction in the male eye size two months after swarming and therefore, could simply be a general manifestation of sexual maturity (Fischer & Fischer, 1995). To date, there are only three published genes for the Bermuda fireworm, totaling 2,500bp. Of the three genes published, two are mitochondrial genes consisting of the large subunit ribosomal RNA (16S) and cytochrome c oxidase subunit I ( cox1 ). The third gene is the nuclear small subunit ribosomal RNA (18S). The majority of available mapped mitochondrial genomes for annelids are from the groups Errantia and Sedentaria. The family Syllidae, of which the fireworm is a member, is known to have a highly varied mitochondrial genome in terms of gene order (Aguado et al., 2016). This manuscript presents the partial mitochondrial genome for the Bermuda fireworm and places it in phylogenetic context amongst its relatives. To our knowledge, the only study that has included Odontosyllis enopla in a phylogenetic context was a three gene phylogeny by Verdes et al. (2022) where the authors listed O. enopla under the species ID ‘ Odontosyllis sp. 9’ and specimen code ‘OenoTR.’ We also analyze intraspecific variation among three female O. enopla partial mitogenomes and propose two putative locations for the mitochondrial origin of replication, which, to date, has not been definitively identified in the Bermuda fireworm or its closest relatives. Materials and Methods Background on Transcriptome Acquisition Per Brugler et al. 2018 , total RNA was isolated from the whole body of three female Odontosyllis enopla worms with a modified RNeasy Tissue Kit (Qiagen) protocol. Isolates were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA) with a 350bp insert size and run at the NY Genome Center on an Illumina HiSeq 2500 (2 x 125bp) allocating one-eighth of a lane for each isolate. The run generated 37,063,191 (Individual #1), 39,513,743 (Individual #2), and 34,329,885 (Individual #3) raw reads. After trimming adaptors and low-quality regions, assembly with Trinity yielded 176,598 (Individual #1), 207,006 (Individual #2) and 283,041 (Individual #3) contigs (including splice variants). These represented 44,426 (Individual #1), 49,458 (Individual #2) and 61,002 (Individual #3) open reading frames (> 100 amino acids) predicted by Transdecoder, and included > 99.0% of the 2,748 core KOGs. Bioinformatics Mitochondrial reads were bioinformatically extracted from the transcriptomes of three female Odontosyllis enopla worms using MitoFinder v1.4 (Allio et al., 2020 ). MitoFinder employed MEGAHIT v3.0 (Li et al., 2015 ) for mitogenome assembly and tRNAscan-SE (Chan & Lowe, 2019 ) for tRNA annotation. A read coverage plot is presented in Fig. 2 . The following command was used to run MitoFinder on an iMac: ./mitofinder --megahit --override --new-genes -j [file name] -1 [left_reads.fastq.gz] -2 [right_reads.fastq.gz] -r [genbank_reference.gb] -o [genetic_code] -p [threads] -m [memory] -t trnascan. Eusyllis blomstrandi (GenBank Accession Number KX752423; 14,712bp in length) was used as the reference, and Translation Table 5 (Invertebrate Mitochondrial Code) was used as the genetic code. Newly assembled mitogenomes were annotated using the MITOS Web Server (Bernt et al., 2013 ). Of the three Odontosyllis enopla worms, individual #3 (specimen ID: oe3) yielded the longest single mitochondrial contig at 10,172bp and thus this mitogenome is described herein. We utilized MEGA X (Kumar et al., 2018 ) to obtain intraspecific genetic distance estimates (p-distances) among the three partial O. enopla mitogenomes. Phylogenetic analysis The partial mitogenome of Odontosyllis enopla (GenBank Accession number PP998669) was combined with mitogenomes presented in Aguado et al. 2015 ( Ramisyllis multicaudata and Trypanobia cryptica ), Aguado et al., 2016 ( Eusyllis blomstrandi , Myrianida brachycephala , Streptosyllis sp., Typosyllis antoni , and Typosyllis sp.), Aguado et al., 2022 ( Ramisyllis kingghidorahi ), Cejp et al. 2023 ( Clavisyllis tenjini ), and Chae et al. 2023 ( Syllis sp.) for a total of 11 mitogenomes. Each of the 9 protein-coding genes ( cob , atp8 , cox1 - 3 , nad1 - 3 , nad6 ) and two ribosomal RNAs (12S and 16S) from all 11 mitogenomes were placed in individual AliView v1.23 (Larsson, 2014 ) files and individually aligned using MAFFT LINS-i v7 (Katoh et al., 2019 ). GBlocks v0.91b was applied to each individual gene region to remove poorly aligned positions and divergent regions. Each individual gene region was subsequently concatenated into a single file using Seqotron v1.0.1 (Fourment & Holmes, 2016 ), treating the mitogenome as a single locus. GBlocks reduced the length of the multiple sequence alignment to 7,097bp (alignment available upon request to corresponding author MRB). The Akaike Information Criterion (AIC) within jModelTest v2.1.10 (Darriba et al., 2012 ; Guindon & Gascuel, 2003 ) selected the GTR + I + G model of sequence evolution (p-inv: 0.2800; gamma: 1.0260). A maximum likelihood based phylogenetic tree was built using the command-line version of PhyML v3.1 (Guindon et al. 2010 ). PhyML parameters included a tree topology search consisting of the best of NNIs and SPRs, a BioNJ starting tree, and 1,000 bootstrap replicates. The resulting phylogenetic tree was visualized using FigTree v1.4.4 (by Andrew Rambaut; https://github.com/rambaut/figtree/releases ). The tree was rooted with Streptosyllis sp. (KX752422) based on a phylogenetic analysis conducted by Aguado et al. 2016 using full mitogenomes (but see DeSalle et al., 2023 ). Origin of Replication The DNA Skew Graphing tool (GraphDNA; Thomas et al. 2007 ), available online via the Viral Bioinformatics Research Centre ( https://4virology.net/ ), was used to search representative mitochondrial genomes for abrupt changes in base composition bias that are characteristic of the origin of replication. In particular, we used the ‘DNA Walker’ graphing option (per Lobry 1996 ; Fig. 3 ). After locating a putative origin of replication, we utilized the default parameters in the DNA Folding Form on the UNAFold web server (Zuker 2003 ) to locate a stable stem-loop configuration containing a characteristic T-rich loop (Fig. 4 ), which is a common feature within the origin of replication. Results The partial mitogenome of the Bermuda fireworm Odontosyllis enopla is 10,172bp in length and contains 9 of the 13 protein-coding genes ( cob , atp8 , cox1 - 3 , nad1 - 3 , nad6 ), two ribosomal RNAs (12S, 16S) and 8 transfer RNAs (Met, Val, Leu, Ala, Ile, Lys, Tyr, Gln) (Fig. 5 ). We were unable to bioinformatically recover nad4L , atp6 , and nad4 - 5 . The partial mitogenome can be accessed under GenBank Accession number PP998669. Gene order for the partial mitogenome of Odontosyllis enopla is as follows: tRNA[Met]-12S-tRNA[Val]-16S-tRNA[Leu]-tRNA[Ala]- nad1 -tRNA[Ile]-tRNA[Lys]- nad3 - nad2 - cox1 - cox2 - atp8 -tRNA[Tyr]- cox3 -tRNA[Gln]- nad6 - cob (Table 1 ). Of the genes that were recovered, gene order for Odontosyllis enopla matches that of Eusyllis blomstrandi (GenBank Accession number NC_031402; the E. blomstrandi mitogenome is 14,712bp in length). We are missing the following stretch of genes: atp6 - nad5 - nad4L - nad4 . MitoFinder found no evidence of circularization of the 10,172bp fragment. Similar to other invertebrates, the O. enopla mitogenome is AT rich (A: 3,880, T: 3,075, G: 1,886, C: 1,331). We obtained 6,120bp of comparable sequence data from the three O. enopla mitogenomes that yielded eight variable sites. Individual #1 (specimen ID: oe1) had four unique substitutions, individual #2 (specimen ID: oe2) had three unique substitutions, and individual #3 (specimen ID: oe3) had one unique substitution (Table 2 ). A maximum likelihood-based phylogenetic tree based on nine protein-coding genes ( cob , atp8 , cox1-3 , nad1-3 , nad6 ) and two ribosomal RNAs (12S, 16S) showed Odontosyllis enopla grouping sister to a clade comprised of Eusyllis blomstrandi (NC_031402) and Clavisyllis tenjini (NC_077651) with 98.8 node support (Fig. 6 ). Table 1 Gene order and length of Odontosyllis enopla mitochondrial protein coding genes, ribosomal RNAs, transfer RNAs, and intergenic regions (IGRs). Gene Length 12S 607 tRNA-Val 62 IGR 221 16S 730 IGR 31 tRNA-Leu 62 IGR 123 tRNA-Ala 62 IGR 442 NAD1 924 tRNA-Ile 61 IGR 3 tRNA-Lys 63 IGR 2 NAD3 353 IGR 60 NAD2 921 IGR 81 COX1 1533 IGR 45 COX2 684 IGR 64 ATP8 162 tRNA-Tyr 62 COX3 ** 780 tRNA-Gln** 61 IGR 2 NAD6 * 504 COB * 1131 IGR 28 * NAD6 overlaps with COB ** COX3 overlaps with tRNA-Gln Table 2 Genetic distance estimates (p-distances) among the three Odontosyllis enopla mitogenomes (based on 6,120bp of comparable sequence data). A total of eight variable sites were identified. Individual #1 (specimen ID: oe1); Individual #2 (specimen ID: oe2); Individual #3 (specimen ID: oe3). oe1 oe2 oe1 -- -- oe2 0.00114 (= 0.114%) -- oe3 0.000817 (= 0.0817%) 0.000654 (= 0.0654%) Discussion & Conclusion Prior to this publication, there were only two partial mitochondrial sequences for Odontosyllis enopla available on GenBank: the large subunit ribosomal RNA gene (16S; 508bp) and the cytochrome c oxidase subunit 1 gene ( cox1 ; 653bp). Combined, these sequences are 1,161bp in length. Our newly obtained sequence data (10,172bp) adds more than 8.76 times the amount of mitochondrial DNA than was previously available. Odontosyllis enopla was included in a three gene phylogeny by Verdes et al ( 2022 ) where the authors listed O. enopla under the species ID ‘ Odontosyllis sp. 9’ and specimen code ‘OenoTR.’ In that phylogeny, Odontosyllis was recovered as paraphyletic. O. enopla and congeners (‘Clade 2’ per Verdes et al 2022 ) grouped sister to Nudisyllis . The clade consisting of ‘Clade 2’ Odontosyllis + Nudisyllis grouped sister to ‘Clade 1’ Odontosyllis + Eusyllis + Pionosyllis . Mitochondrial genomes of Nudisyllis and Pionosyllis were not available at the time of this analysis; however, the complete mitogenome of Eusyllis blomstrandi (GenBank Accession number NC_031402) was indeed available and was included in our phylogenetic analysis. The phylogenetic reconstruction placed Odontosyllis enopla sister (ML bootstrap support: 98.8) to a clade containing Eusyllis blomstrandi (NC_031402) and Clavisyllis tenjini (NC_077651). These three species are all classified in the subfamily Eusyllinae. Additionally, all three taxa share the same mitochondrial gene order. We were unable to bioinformatically recover nad4L , atp6 , and nad4 - 5 . These four genes are found in tandem ( atp6 - nad5 - nad4L - nad4 ) in the mitogenomes of Clavisyllis tenjini (NC_077651), Eusyllis blomstrandi (NC_031402), Myrianida brachycephala (NC_031403; Autolytinae), and Streptosyllis sp. (KX752422; Anoplosyllinae). In C. tenjini , E. blomstrandi , and M. brachycephala, these four genes are located between cob and 12S. In Streptosyllis sp, these four genes have been translocated between cox2 and cox3 . In Ramisyllis multicaudata (NC_027699) and Trypanobia cryptica (KR534503), both members of the Syllinae, nad5 has been bisected out of the 4-gene segment and moved to a different location (between cox3 and 16S). In Typosyllis antoni (NC_031404) and Typosyllis sp. (KX752425), also members of the Syllinae, both nad5 and atp6 have been bisected out of the 4-gene segment and moved to different locations ( nad5 is between nad3 and nad1 , while atp6 is between 12S and cox1 ). Given how variable this 4-gene segment is in terms of structure and placement within these mitogenomes, there is a possibility that these four genes were lost during a rearrangement event. Having said that, MitoFinder found no evidence of circularization. A more plausible explanation is that this 4-gene segment was not transcriptionally active when the three female Odontosyllis enopla worms were collected and preserved. If the latter is correct, this may indicate where the heavy-strand origin of replication (OriH) is located (just upstream of 12S) as cells typically manufacture significant amounts of ribosomal DNA (12S & 16S) and can terminate transcription after these genes are successfully copied. These results also suggest that the 4-gene segment ( atp6 - nad5 - nad4L - nad4 ) may not play a significant role in the bioluminescence display, gamete formation, or sexual reproduction in general. As sequence data are not available to determine whether OriH is indeed upstream of 12S, we searched the nine (of 13) protein-coding genes, two ribosomal RNAs, and seven (of 22) complete tRNAs (totaling 10,172bp) for evidence of another putative origin of replication. A DNA Walk analysis identified a switchback in cardinal direction in a ~ 440bp non-coding region between tRNA[Ala] and nad1 (Fig. 3 ). After locating this putative origin of replication, we utilized the default parameters in the DNA Folding Form on the UNAFold web server to locate a lengthy stem-loop configuration containing a characteristic T-rich loop (Fig. 4 ), which is a common feature within the origin of replication. Selifanova et al. ( 2023 ) presented the complete mitogenome of the polychaete Polydora cf. ciliata (OQ078742; 17,645bp; Family Spionidae) and located the origin of replication (i.e., the Control Region or D-Loop) between nad3 and tRNA[Leu]. Chae et al. ( 2023 ) described the mitochondrial genome of Syllis sp. (ON312495; 17,092bp; Family Syllidae) and identified a 1,291bp putative control region between tRNA-W and tRNA-G (surrounding gene order: ...cox1- nad4L-tRNA[Gln]-tRNA[Trp]-Control Region-tRNA[Gly]-tRNA[Leu]-atp6-cox3...). Aguado et al. ( 2016 ) simply noted that "In the five mt genomes ( Streptosyllis sp., Eusyllis blomstrandi , Myrianida brachycephala , Typosyllis antoni and Typosyllis sp.), the longest noncoding regions are AT rich and are suggested to be the putative control regions." As the available literature does not provide a consensus on where the origin of replication is located, additional sequence data are needed (four protein-coding genes and 15 tRNAs) to determine whether the origin of replication is indeed located upstream of 12S or is in a ~ 440bp non-coding region between tRNA[Ala] and nad1 in the Odontosyllis enopla mitogenome. Declarations Acknowledgements MRB is a Research Associate at the American Museum of Natural History and the Smithsonian Institution’s National Museum of Natural History and gratefully acknowledges these affiliations. Conflict of interest The authors have declared that no competing interests exist. Funding Financial support was provided to MRB by the Port Royal Sound Foundation, to BAC, HC, and ADG through USCB’s Summer Research Experience Scholarship Program, and to ADG through the University of South Carolina SMART Program. Author contributions Conceptualization: MRB. Data curation: BAC, LDW, ADG, MC, JJA, MRB. Formal analysis and interpretation: all authors. Investigation: BAC, MRB. Visualization: LDW, ADG, MC, LCH, HC, JCR, AC. Writing – original draft: BAC, LDW, ADG, JMK, MC, LCH, HC, JCR, AC, JJA, MRB. Writing – review & editing: all authors. Author ORCIDs BAC: 0009-0008-4422-6489 LDW: 0009-0001-7700-6940 ADG: 0009-0007-7049-1019 JMK: 0009-0004-4425-5096 MAC: 0009-0007-6980-4292 LCH: 0009-0002-7307-6308 HC: 0009-0008-5821-9335 JCR: 0009-0002-9297-8008 AC: 0009-0007-8700-5726 JJA: 0009-0002-5983-5523 AP: 0009-0009-2354-1399 DTP: 0000-0002-2060-3226 MRB: 0000-0003-3676-1226 Data availability Mitogenomic data are available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov) under accession number PP998669. The phylogenetic tree can be found on FigShare at the following URL: https://doi.org/10.6084/m9.figshare.30138370. The transcriptomic sequence data that support the findings of this study are openly available in GenBank of NCBI under BioProject accession number PRJNA448700, SRA accession numbers SRX4382063-SRX4382065, and BioSample accession numbers SAMN08863502, SAMN08863511, and SAMN08863514. References Aguado MT, Glasby CJ, Schroeder PC, Weigert A, Bleidorn C, 2015. 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Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7724154","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":521269005,"identity":"f3c7e4fe-6a97-482f-9a1b-08b7ed8cede1","order_by":0,"name":"Brendan A. Cruz","email":"","orcid":"https://orcid.org/0009-0008-4422-6489","institution":"University of South Carolina Beaufort","correspondingAuthor":false,"prefix":"","firstName":"Brendan","middleName":"A.","lastName":"Cruz","suffix":""},{"id":521269178,"identity":"fedf7087-0016-4d64-98df-ec362360178d","order_by":1,"name":"Lynette D. 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08:32:21","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102042,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7724154/v1/baa6deab323a26b0a7a7bcff.html"},{"id":92391092,"identity":"09869b0f-b3f0-4689-a809-83d83016258a","added_by":"auto","created_at":"2025-09-29 08:40:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":265323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOdontosyllis enopla \u003c/em\u003e(photo credit: coauthor James B. Wood)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7724154/v1/48c3daea0f76eeb686a1314a.png"},{"id":92390804,"identity":"3c2fb7a3-0c96-4fd4-91e2-436102ea1eb2","added_by":"auto","created_at":"2025-09-29 08:32:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":85775,"visible":true,"origin":"","legend":"\u003cp\u003eA read coverage plot showing the number of individual reads (y-axis) that mapped to the different parts of the assembled mitogenome (x-axis; 10,172 bp in length).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7724154/v1/2b73b9c126357fef2950bf6b.png"},{"id":92390809,"identity":"46288828-0599-4215-ab06-a589192dcdef","added_by":"auto","created_at":"2025-09-29 08:32:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":171894,"visible":true,"origin":"","legend":"\u003cp\u003eA DNA Walk of the partial mitochondrial genome of \u003cem\u003eOdontosyllis enopla\u003c/em\u003e. Abrupt changes in base composition bias (switchbacks) are characteristic of the origin of replication. A) Full mitogenome walk (window size: 75), B) Zoomed-in image of switchback (window size: 20), C) Zoomed-in image of switchback (window size: 10), D) Zoomed-in image of switchback with nucleotide positions and cardinal direction changes indicated (window size: 15).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7724154/v1/565b5cebd490f90af84fb19a.png"},{"id":92390808,"identity":"6931f5a3-f954-40c3-8a42-85d62f93cf03","added_by":"auto","created_at":"2025-09-29 08:32:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39146,"visible":true,"origin":"","legend":"\u003cp\u003eThe most thermodynamically stable stem-loop structure (dG = -22.02) as output by the UNAFold web server. Note the long AT rich stem containing a characteristic T-rich loop, which is a common feature within the origin of replication.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7724154/v1/2761685bdfedf792be809470.png"},{"id":92390812,"identity":"5b50d4e6-f5ed-4b16-bd39-91bdc6f2516f","added_by":"auto","created_at":"2025-09-29 08:32:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":369738,"visible":true,"origin":"","legend":"\u003cp\u003ePartial mitochondrial genome map of \u003cem\u003eOdontosyllis enopla\u003c/em\u003e. We were unable to bioinformatically recover \u003cem\u003enad4L\u003c/em\u003e, \u003cem\u003eatp6\u003c/em\u003e, and \u003cem\u003enad4\u003c/em\u003e-\u003cem\u003e5\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7724154/v1/ca2f445954828a492777b61f.png"},{"id":92390811,"identity":"18a79318-8f46-4c92-bac3-baf3d458d0a6","added_by":"auto","created_at":"2025-09-29 08:32:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":102734,"visible":true,"origin":"","legend":"\u003cp\u003eA maximum likelihood-based phylogenetic tree based on nine protein-coding genes (\u003cem\u003ecob\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003ecox1\u003c/em\u003e-\u003cem\u003e3\u003c/em\u003e, \u003cem\u003enad1\u003c/em\u003e-\u003cem\u003e3\u003c/em\u003e, \u003cem\u003enad6\u003c/em\u003e) and two ribosomal RNAs (12S, 16S). Node support is based on 1,000 bootstrap replicates. The tree was rooted with \u003cem\u003eStreptosyllis \u003c/em\u003esp. based on a phylogenetic analysis conducted by Aguado et al. (2016) using full mitogenomes. The following sequences were used: \u003cem\u003eOdontosyllis enopla\u003c/em\u003e PP998669 (this study), \u003cem\u003eRamisyllis multicaudata\u003c/em\u003e NC_027699 and \u003cem\u003eTrypanobia cryptica\u003c/em\u003e KR534503 (Aguado et al. 2015), \u003cem\u003eEusyllis blomstrandi \u003c/em\u003eNC_031402, \u003cem\u003eMyrianida brachycephala \u003c/em\u003eNC_031403, \u003cem\u003eStreptosyllis \u003c/em\u003esp. KX752422, \u003cem\u003eTyposyllis antoni \u003c/em\u003eNC_031404, and \u003cem\u003eTyposyllis \u003c/em\u003esp. KX752425 (Aguado et al., 2016), \u003cem\u003eRamisyllis kingghidorahi\u003c/em\u003e NC_065765 (Aguado et al., 2022), \u003cem\u003eClavisyllis tenjini\u003c/em\u003e NC_077651 (Cejp et al. 2023), and \u003cem\u003eSyllis \u003c/em\u003esp. ON312495 (Chae et al. 2023).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7724154/v1/82c3756d2a14e68c3bb60aa4.png"},{"id":92391994,"identity":"e343c30f-b05d-4a34-808e-f76f89b099ea","added_by":"auto","created_at":"2025-09-29 08:48:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1611846,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7724154/v1/0176d684-a543-4fcf-b3cb-47e84eeef3bd.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eThe partial mitochondrial genome of the enigmatic Bermuda fireworm \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOdontosyllis enopla\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Verrill, 1900 and its phylogenetic implications\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eOdontosyllis enopla\u0026nbsp;\u003c/em\u003eVerrill, 1900, more commonly known as the Bermuda fireworm, is a remarkable annelid belonging to the family Syllidae (Figure 1). These tube-dwelling worms, found on sandy coral substrates in benthic habitats (Fischer \u0026amp; Fischer, 1995), display an incredible bioluminescent mating ritual that was first seen and recorded by Christopher Columbus in 1492 (Brugler et al., 2018) and first described by Addison Emery Verrill in 1900 (Verrill, A.E. 1900).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Bermuda fireworm is a unique annelid due to its distinct mating behaviors and physiology. In preparation for breeding, fireworms of both sexes undergo morphological changes, including enlargement and pigmentation of their four eyes to enhance visual sensitivity. This is particularly pronounced in males for detecting the females' bioluminescence. This sets the stage for a highly synchronized mating swarm, where selective pressures from predation favor precise timing to minimize vulnerability of isolated or early individuals. Females initiate the ritual by rising to the surface and swimming in slow circles while emitting a continuous bluish-green glow from secreted luminous mucus and releasing their gametes. Triggered by this display, males swim towards the glowing females, producing quick, consecutive bioluminescent flashes while releasing their own gametes into the surrounding water (Verdes et al., 2022; Fischer \u0026amp; Fischer, 1995). The bioluminescence peaks in the green portion of the visible spectrum, with wavelengths between 504-507 nanometers. Their visual system is most sensitive to the aforementioned wavelength, which was shown by electroretinogram (ERG) recordings in response to light at multiple different wavelengths (Wilkens \u0026amp; Wolken, 1981). The similarities between the light detected and the light emitted suggest that fireworms are visually tuned to detect mating signals from bioluminescence. The fireworm synchronizes its mating ritual with the lunar cycle with swarming episodes coinciding with the first day after full moons during the summer and early autumn months (Brugler et al., 2018). Utilizing specialized setae, this benthic organism will swim to the surface 57 ± 1 minutes after the astronomical sunset to begin its mating ritual (Fischer \u0026amp; Fischer, 1995). This mating swarm has been observed up to five nights following the full moon, after which both male and female fireworms return to their benthic habitats (Fischer \u0026amp; Fischer, 1995). The fertilized zygotes undergo cell division and after fourteen hours become trochophores that can freely swim through the water columns (Fischer \u0026amp; Fischer, 1995).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEvidence suggests that the common ancestor of all the bioluminescent syllid species was not bioluminescent itself; in fact, bioluminescence within \u003cem\u003eOdontosyllis\u0026nbsp;\u003c/em\u003eevolved independently twice (Verdes et al., 2022). While the bioluminescence of this species is of great interest to researchers, of even greater importance is the \u003cem\u003ereversible\u0026nbsp;\u003c/em\u003eepitoky metamorphosis in both males and females. The fireworm undergoes different physiological changes to prepare for the mating event. Some of the most noticeable physiological changes the fireworm undergoes include the enlargement of the eyes in males, the growth of setae used for rapid swimming to reach the surface, and tissues protruding from the body of the females holding the oocytes (Fischer \u0026amp; Fischer, 1995; Brugler et al., 2018). After the mating swarm, the swimming setae are shed although there does not appear to be a specific timeline for this reversal. Some fireworms shed the setae in as little as five days after swarming while others had at least some remaining setae after 35 days. Additional research needs to be conducted regarding reversal of the size of the male eyes. Research thus far indicates that there was no size reduction in the male eye size two months after swarming and therefore, could simply be a general manifestation of sexual maturity (Fischer \u0026amp; Fischer, 1995).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo date, there are only three published genes for the Bermuda fireworm, totaling 2,500bp. Of the three genes published, two are mitochondrial genes consisting of the large subunit ribosomal RNA (16S) and cytochrome c oxidase subunit I (\u003cem\u003ecox1\u003c/em\u003e). The third gene is the nuclear small subunit ribosomal RNA (18S). The majority of available mapped mitochondrial genomes for annelids are from the groups Errantia and Sedentaria. The family Syllidae, of which the fireworm is a member, is known to have a highly varied mitochondrial genome in terms of gene order (Aguado et al., 2016). This manuscript presents the partial mitochondrial genome for the Bermuda fireworm and places it in phylogenetic context amongst its relatives. To our knowledge, the only study that has included \u003cem\u003eOdontosyllis enopla\u003c/em\u003e in a phylogenetic context was a three gene phylogeny by Verdes et al. (2022) where the authors listed \u003cem\u003eO. enopla\u003c/em\u003e under the species ID ‘\u003cem\u003eOdontosyllis\u0026nbsp;\u003c/em\u003esp. 9’ and specimen code ‘OenoTR.’ We also analyze intraspecific variation among three female \u003cem\u003eO. enopla\u003c/em\u003e partial mitogenomes and propose two putative locations for the mitochondrial origin of replication, which, to date, has not been definitively identified in the Bermuda fireworm or its closest relatives.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\n\u003ch3\u003eBackground on Transcriptome Acquisition\u003c/h3\u003e\n\u003cp\u003ePer Brugler et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, total RNA was isolated from the whole body of three female \u003cem\u003eOdontosyllis enopla\u003c/em\u003e worms with a modified RNeasy Tissue Kit (Qiagen) protocol. Isolates were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA) with a 350bp insert size and run at the NY Genome Center on an Illumina HiSeq 2500 (2 x 125bp) allocating one-eighth of a lane for each isolate. The run generated 37,063,191 (Individual #1), 39,513,743 (Individual #2), and 34,329,885 (Individual #3) raw reads. After trimming adaptors and low-quality regions, assembly with Trinity yielded 176,598 (Individual #1), 207,006 (Individual #2) and 283,041 (Individual #3) contigs (including splice variants). These represented 44,426 (Individual #1), 49,458 (Individual #2) and 61,002 (Individual #3) open reading frames (\u0026gt;\u0026thinsp;100 amino acids) predicted by Transdecoder, and included\u0026thinsp;\u0026gt;\u0026thinsp;99.0% of the 2,748 core KOGs.\u003c/p\u003e\n\u003ch3\u003eBioinformatics\u003c/h3\u003e\n\u003cp\u003eMitochondrial reads were bioinformatically extracted from the transcriptomes of three female \u003cem\u003eOdontosyllis enopla\u003c/em\u003e worms using MitoFinder v1.4 (Allio et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). MitoFinder employed MEGAHIT v3.0 (Li et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) for mitogenome assembly and tRNAscan-SE (Chan \u0026amp; Lowe, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) for tRNA annotation. A read coverage plot is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The following command was used to run MitoFinder on an iMac: ./mitofinder --megahit --override --new-genes -j [file name] -1 [left_reads.fastq.gz] -2 [right_reads.fastq.gz] -r [genbank_reference.gb] -o [genetic_code] -p [threads] -m [memory] -t trnascan. \u003cem\u003eEusyllis blomstrandi\u003c/em\u003e (GenBank Accession Number KX752423; 14,712bp in length) was used as the reference, and Translation Table\u0026nbsp;5 (Invertebrate Mitochondrial Code) was used as the genetic code. Newly assembled mitogenomes were annotated using the MITOS Web Server (Bernt et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Of the three \u003cem\u003eOdontosyllis enopla\u003c/em\u003e worms, individual #3 (specimen ID: oe3) yielded the longest single mitochondrial contig at 10,172bp and thus this mitogenome is described herein. We utilized MEGA X (Kumar et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) to obtain intraspecific genetic distance estimates (p-distances) among the three partial \u003cem\u003eO. enopla\u003c/em\u003e mitogenomes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e\u003cp\u003eThe partial mitogenome of \u003cem\u003eOdontosyllis enopla\u003c/em\u003e (GenBank Accession number PP998669) was combined with mitogenomes presented in Aguado et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e (\u003cem\u003eRamisyllis multicaudata\u003c/em\u003e and \u003cem\u003eTrypanobia cryptica\u003c/em\u003e), Aguado et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e (\u003cem\u003eEusyllis blomstrandi\u003c/em\u003e, \u003cem\u003eMyrianida brachycephala\u003c/em\u003e, \u003cem\u003eStreptosyllis\u003c/em\u003e sp., \u003cem\u003eTyposyllis antoni\u003c/em\u003e, and \u003cem\u003eTyposyllis\u003c/em\u003e sp.), Aguado et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e (\u003cem\u003eRamisyllis kingghidorahi\u003c/em\u003e), Cejp et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e (\u003cem\u003eClavisyllis tenjini\u003c/em\u003e), and Chae et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e (\u003cem\u003eSyllis\u003c/em\u003e sp.) for a total of 11 mitogenomes. Each of the 9 protein-coding genes (\u003cem\u003ecob\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003ecox1\u003c/em\u003e-\u003cem\u003e3\u003c/em\u003e, \u003cem\u003enad1\u003c/em\u003e-\u003cem\u003e3\u003c/em\u003e, \u003cem\u003enad6\u003c/em\u003e) and two ribosomal RNAs (12S and 16S) from all 11 mitogenomes were placed in individual AliView v1.23 (Larsson, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) files and individually aligned using MAFFT LINS-i v7 (Katoh et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). GBlocks v0.91b was applied to each individual gene region to remove poorly aligned positions and divergent regions. Each individual gene region was subsequently concatenated into a single file using Seqotron v1.0.1 (Fourment \u0026amp; Holmes, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), treating the mitogenome as a single locus. GBlocks reduced the length of the multiple sequence alignment to 7,097bp (alignment available upon request to corresponding author MRB).\u003c/p\u003e\u003cp\u003eThe Akaike Information Criterion (AIC) within jModelTest v2.1.10 (Darriba et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Guindon \u0026amp; Gascuel, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) selected the GTR\u0026thinsp;+\u0026thinsp;I\u0026thinsp;+\u0026thinsp;G model of sequence evolution (p-inv: 0.2800; gamma: 1.0260). A maximum likelihood based phylogenetic tree was built using the command-line version of PhyML v3.1 (Guindon et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). PhyML parameters included a tree topology search consisting of the best of NNIs and SPRs, a BioNJ starting tree, and 1,000 bootstrap replicates. The resulting phylogenetic tree was visualized using FigTree v1.4.4 (by Andrew Rambaut; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/rambaut/figtree/releases\u003c/span\u003e\u003cspan address=\"https://github.com/rambaut/figtree/releases\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The tree was rooted with \u003cem\u003eStreptosyllis\u003c/em\u003e sp. (KX752422) based on a phylogenetic analysis conducted by Aguado et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e using full mitogenomes (but see DeSalle et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eOrigin of Replication\u003c/h3\u003e\n\u003cp\u003eThe DNA Skew Graphing tool (GraphDNA; Thomas et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), available online via the Viral Bioinformatics Research Centre (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://4virology.net/\u003c/span\u003e\u003cspan address=\"https://4virology.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), was used to search representative mitochondrial genomes for abrupt changes in base composition bias that are characteristic of the origin of replication. In particular, we used the \u0026lsquo;DNA Walker\u0026rsquo; graphing option (per Lobry \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After locating a putative origin of replication, we utilized the default parameters in the DNA Folding Form on the UNAFold web server (Zuker \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) to locate a stable stem-loop configuration containing a characteristic T-rich loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which is a common feature within the origin of replication.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe partial mitogenome of the Bermuda fireworm \u003cem\u003eOdontosyllis enopla\u003c/em\u003e is 10,172bp in length and contains 9 of the 13 protein-coding genes (\u003cem\u003ecob\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003ecox1\u003c/em\u003e-\u003cem\u003e3\u003c/em\u003e, \u003cem\u003enad1\u003c/em\u003e-\u003cem\u003e3\u003c/em\u003e, \u003cem\u003enad6\u003c/em\u003e), two ribosomal RNAs (12S, 16S) and 8 transfer RNAs (Met, Val, Leu, Ala, Ile, Lys, Tyr, Gln) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We were unable to bioinformatically recover \u003cem\u003enad4L\u003c/em\u003e, \u003cem\u003eatp6\u003c/em\u003e, and \u003cem\u003enad4\u003c/em\u003e-\u003cem\u003e5\u003c/em\u003e. The partial mitogenome can be accessed under GenBank Accession number PP998669. Gene order for the partial mitogenome of \u003cem\u003eOdontosyllis enopla\u003c/em\u003e is as follows: tRNA[Met]-12S-tRNA[Val]-16S-tRNA[Leu]-tRNA[Ala]-\u003cem\u003enad1\u003c/em\u003e-tRNA[Ile]-tRNA[Lys]-\u003cem\u003enad3\u003c/em\u003e-\u003cem\u003enad2\u003c/em\u003e-\u003cem\u003ecox1\u003c/em\u003e-\u003cem\u003ecox2\u003c/em\u003e-\u003cem\u003eatp8\u003c/em\u003e-tRNA[Tyr]-\u003cem\u003ecox3\u003c/em\u003e-tRNA[Gln]-\u003cem\u003enad6\u003c/em\u003e-\u003cem\u003ecob\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of the genes that were recovered, gene order for \u003cem\u003eOdontosyllis enopla\u003c/em\u003e matches that of \u003cem\u003eEusyllis blomstrandi\u003c/em\u003e (GenBank Accession number NC_031402; the \u003cem\u003eE. blomstrandi\u003c/em\u003e mitogenome is 14,712bp in length). We are missing the following stretch of genes: \u003cem\u003eatp6\u003c/em\u003e-\u003cem\u003enad5\u003c/em\u003e-\u003cem\u003enad4L\u003c/em\u003e-\u003cem\u003enad4\u003c/em\u003e. MitoFinder found no evidence of circularization of the 10,172bp fragment. Similar to other invertebrates, the \u003cem\u003eO. enopla\u003c/em\u003e mitogenome is AT rich (A: 3,880, T: 3,075, G: 1,886, C: 1,331). We obtained 6,120bp of comparable sequence data from the three \u003cem\u003eO. enopla\u003c/em\u003e mitogenomes that yielded eight variable sites. Individual #1 (specimen ID: oe1) had four unique substitutions, individual #2 (specimen ID: oe2) had three unique substitutions, and individual #3 (specimen ID: oe3) had one unique substitution (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A maximum likelihood-based phylogenetic tree based on nine protein-coding genes (\u003cem\u003ecob\u003c/em\u003e, \u003cem\u003eatp8\u003c/em\u003e, \u003cem\u003ecox1-3\u003c/em\u003e, \u003cem\u003enad1-3\u003c/em\u003e, \u003cem\u003enad6\u003c/em\u003e) and two ribosomal RNAs (12S, 16S) showed \u003cem\u003eOdontosyllis enopla\u003c/em\u003e grouping sister to a clade comprised of \u003cem\u003eEusyllis blomstrandi\u003c/em\u003e (NC_031402) and \u003cem\u003eClavisyllis tenjini\u003c/em\u003e (NC_077651) with 98.8 node support (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGene order and length of \u003cem\u003eOdontosyllis enopla\u003c/em\u003e mitochondrial protein coding genes, ribosomal RNAs, transfer RNAs, and intergenic regions (IGRs).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLength\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e607\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etRNA-Val\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e221\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16S\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e730\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etRNA-Leu\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e123\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etRNA-Ala\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e442\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eNAD1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e924\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etRNA-Ile\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etRNA-Lys\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eNAD3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e353\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eNAD2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e921\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e81\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCOX1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1533\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCOX2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e684\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e64\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eATP8\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e162\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etRNA-Tyr\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCOX3\u003c/em\u003e**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e780\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etRNA-Gln**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e61\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eNAD6\u003c/em\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e504\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCOB\u003c/em\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1131\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIGR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003e*\u003cem\u003eNAD6\u003c/em\u003e overlaps with COB\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003e**\u003cem\u003eCOX3\u003c/em\u003e overlaps with tRNA-Gln\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGenetic distance estimates (p-distances) among the three \u003cem\u003eOdontosyllis enopla\u003c/em\u003e mitogenomes (based on 6,120bp of comparable sequence data). A total of eight variable sites were identified. Individual #1 (specimen ID: oe1); Individual #2 (specimen ID: oe2); Individual #3 (specimen ID: oe3).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eoe1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eoe2\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eoe1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eoe2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00114 (=\u0026thinsp;0.114%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e--\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eoe3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.000817 (=\u0026thinsp;0.0817%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.000654 (=\u0026thinsp;0.0654%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion \u0026 Conclusion","content":"\u003cp\u003ePrior to this publication, there were only two partial mitochondrial sequences for \u003cem\u003eOdontosyllis enopla\u003c/em\u003e available on GenBank: the large subunit ribosomal RNA gene (16S; 508bp) and the cytochrome c oxidase subunit 1 gene (\u003cem\u003ecox1\u003c/em\u003e; 653bp). Combined, these sequences are 1,161bp in length. Our newly obtained sequence data (10,172bp) adds more than 8.76 times the amount of mitochondrial DNA than was previously available.\u003c/p\u003e\u003cp\u003e\u003cem\u003eOdontosyllis enopla\u003c/em\u003e was included in a three gene phylogeny by Verdes et al (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) where the authors listed \u003cem\u003eO. enopla\u003c/em\u003e under the species ID \u0026lsquo;\u003cem\u003eOdontosyllis\u003c/em\u003e sp. 9\u0026rsquo; and specimen code \u0026lsquo;OenoTR.\u0026rsquo; In that phylogeny, \u003cem\u003eOdontosyllis\u003c/em\u003e was recovered as paraphyletic. \u003cem\u003eO. enopla\u003c/em\u003e and congeners (\u0026lsquo;Clade 2\u0026rsquo; per Verdes et al \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) grouped sister to \u003cem\u003eNudisyllis\u003c/em\u003e. The clade consisting of \u0026lsquo;Clade 2\u0026rsquo; \u003cem\u003eOdontosyllis\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eNudisyllis\u003c/em\u003e grouped sister to \u0026lsquo;Clade 1\u0026rsquo; \u003cem\u003eOdontosyllis\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eEusyllis\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003ePionosyllis\u003c/em\u003e. Mitochondrial genomes of \u003cem\u003eNudisyllis\u003c/em\u003e and \u003cem\u003ePionosyllis\u003c/em\u003e were not available at the time of this analysis; however, the complete mitogenome of \u003cem\u003eEusyllis blomstrandi\u003c/em\u003e (GenBank Accession number NC_031402) was indeed available and was included in our phylogenetic analysis.\u003c/p\u003e\u003cp\u003eThe phylogenetic reconstruction placed \u003cem\u003eOdontosyllis enopla\u003c/em\u003e sister (ML bootstrap support: 98.8) to a clade containing \u003cem\u003eEusyllis blomstrandi\u003c/em\u003e (NC_031402) and \u003cem\u003eClavisyllis tenjini\u003c/em\u003e (NC_077651). These three species are all classified in the subfamily Eusyllinae. Additionally, all three taxa share the same mitochondrial gene order.\u003c/p\u003e\u003cp\u003eWe were unable to bioinformatically recover \u003cem\u003enad4L\u003c/em\u003e, \u003cem\u003eatp6\u003c/em\u003e, and \u003cem\u003enad4\u003c/em\u003e-\u003cem\u003e5\u003c/em\u003e. These four genes are found in tandem (\u003cem\u003eatp6\u003c/em\u003e-\u003cem\u003enad5\u003c/em\u003e-\u003cem\u003enad4L\u003c/em\u003e-\u003cem\u003enad4\u003c/em\u003e) in the mitogenomes of \u003cem\u003eClavisyllis tenjini\u003c/em\u003e (NC_077651), \u003cem\u003eEusyllis blomstrandi\u003c/em\u003e (NC_031402), \u003cem\u003eMyrianida brachycephala\u003c/em\u003e (NC_031403; Autolytinae), and \u003cem\u003eStreptosyllis\u003c/em\u003e sp. (KX752422; Anoplosyllinae). In \u003cem\u003eC. tenjini\u003c/em\u003e, \u003cem\u003eE. blomstrandi\u003c/em\u003e, and \u003cem\u003eM.\u003c/em\u003e brachycephala, these four genes are located between \u003cem\u003ecob\u003c/em\u003e and 12S. In \u003cem\u003eStreptosyllis\u003c/em\u003e sp, these four genes have been translocated between \u003cem\u003ecox2\u003c/em\u003e and \u003cem\u003ecox3\u003c/em\u003e. In \u003cem\u003eRamisyllis multicaudata\u003c/em\u003e (NC_027699) and \u003cem\u003eTrypanobia cryptica\u003c/em\u003e (KR534503), both members of the Syllinae, \u003cem\u003enad5\u003c/em\u003e has been bisected out of the 4-gene segment and moved to a different location (between \u003cem\u003ecox3\u003c/em\u003e and 16S). In \u003cem\u003eTyposyllis antoni\u003c/em\u003e (NC_031404) and \u003cem\u003eTyposyllis\u003c/em\u003e sp. (KX752425), also members of the Syllinae, both \u003cem\u003enad5\u003c/em\u003e and \u003cem\u003eatp6\u003c/em\u003e have been bisected out of the 4-gene segment and moved to different locations (\u003cem\u003enad5\u003c/em\u003e is between \u003cem\u003enad3\u003c/em\u003e and \u003cem\u003enad1\u003c/em\u003e, while \u003cem\u003eatp6\u003c/em\u003e is between 12S and \u003cem\u003ecox1\u003c/em\u003e). Given how variable this 4-gene segment is in terms of structure and placement within these mitogenomes, there is a possibility that these four genes were lost during a rearrangement event. Having said that, MitoFinder found no evidence of circularization. A more plausible explanation is that this 4-gene segment was not transcriptionally active when the three female \u003cem\u003eOdontosyllis enopla\u003c/em\u003e worms were collected and preserved. If the latter is correct, this may indicate where the heavy-strand origin of replication (OriH) is located (just upstream of 12S) as cells typically manufacture significant amounts of ribosomal DNA (12S \u0026amp; 16S) and can terminate transcription after these genes are successfully copied. These results also suggest that the 4-gene segment (\u003cem\u003eatp6\u003c/em\u003e-\u003cem\u003enad5\u003c/em\u003e-\u003cem\u003enad4L\u003c/em\u003e-\u003cem\u003enad4\u003c/em\u003e) may not play a significant role in the bioluminescence display, gamete formation, or sexual reproduction in general.\u003c/p\u003e\u003cp\u003eAs sequence data are not available to determine whether OriH is indeed upstream of 12S, we searched the nine (of 13) protein-coding genes, two ribosomal RNAs, and seven (of 22) complete tRNAs (totaling 10,172bp) for evidence of another putative origin of replication. A DNA Walk analysis identified a switchback in cardinal direction in a\u0026thinsp;~\u0026thinsp;440bp non-coding region between tRNA[Ala] and \u003cem\u003enad1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After locating this putative origin of replication, we utilized the default parameters in the DNA Folding Form on the UNAFold web server to locate a lengthy stem-loop configuration containing a characteristic T-rich loop (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which is a common feature within the origin of replication. Selifanova et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) presented the complete mitogenome of the polychaete \u003cem\u003ePolydora\u003c/em\u003e cf. \u003cem\u003eciliata\u003c/em\u003e (OQ078742; 17,645bp; Family Spionidae) and located the origin of replication (i.e., the Control Region or D-Loop) between \u003cem\u003enad3\u003c/em\u003e and tRNA[Leu]. Chae et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) described the mitochondrial genome of \u003cem\u003eSyllis\u003c/em\u003e sp. (ON312495; 17,092bp; Family Syllidae) and identified a 1,291bp putative control region between tRNA-W and tRNA-G (surrounding gene order: ...cox1- nad4L-tRNA[Gln]-tRNA[Trp]-Control Region-tRNA[Gly]-tRNA[Leu]-atp6-cox3...). Aguado et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) simply noted that \"In the five mt genomes (\u003cem\u003eStreptosyllis\u003c/em\u003e sp., \u003cem\u003eEusyllis blomstrandi\u003c/em\u003e, \u003cem\u003eMyrianida brachycephala\u003c/em\u003e, \u003cem\u003eTyposyllis antoni\u003c/em\u003e and \u003cem\u003eTyposyllis\u003c/em\u003e sp.), the longest noncoding regions are AT rich and are suggested to be the putative control regions.\" As the available literature does not provide a consensus on where the origin of replication is located, additional sequence data are needed (four protein-coding genes and 15 tRNAs) to determine whether the origin of replication is indeed located upstream of 12S or is in a\u0026thinsp;~\u0026thinsp;440bp non-coding region between tRNA[Ala] and \u003cem\u003enad1\u003c/em\u003e in the \u003cem\u003eOdontosyllis enopla\u003c/em\u003e mitogenome.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMRB is a Research Associate at the American Museum of Natural History and the Smithsonian Institution\u0026rsquo;s National Museum of Natural History and gratefully acknowledges these affiliations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinancial support was provided to MRB by the Port Royal Sound Foundation, to BAC, HC, and ADG through USCB\u0026rsquo;s Summer Research Experience Scholarship Program, and to ADG through the University of South Carolina SMART Program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: MRB. Data curation: BAC, LDW, ADG, MC, JJA, MRB. Formal analysis and interpretation: all authors. Investigation: BAC, MRB. Visualization: LDW, ADG, MC, LCH, HC, JCR, AC. Writing \u0026ndash; original draft: BAC, LDW, ADG, JMK, MC, LCH, HC, JCR, AC, JJA, MRB. Writing \u0026ndash; review \u0026amp; editing: all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor ORCIDs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBAC: 0009-0008-4422-6489\u003c/p\u003e\n\u003cp\u003eLDW:\u0026nbsp;0009-0001-7700-6940\u003c/p\u003e\n\u003cp\u003eADG: 0009-0007-7049-1019\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJMK: 0009-0004-4425-5096\u003c/p\u003e\n\u003cp\u003eMAC: 0009-0007-6980-4292\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLCH: \u0026nbsp; 0009-0002-7307-6308\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHC: 0009-0008-5821-9335\u003c/p\u003e\n\u003cp\u003eJCR: 0009-0002-9297-8008\u003c/p\u003e\n\u003cp\u003eAC: 0009-0007-8700-5726\u003c/p\u003e\n\u003cp\u003eJJA: 0009-0002-5983-5523\u003c/p\u003e\n\u003cp\u003eAP: 0009-0009-2354-1399\u003c/p\u003e\n\u003cp\u003eDTP: 0000-0002-2060-3226\u003c/p\u003e\n\u003cp\u003eMRB: 0000-0003-3676-1226\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitogenomic data are available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov) under accession number PP998669. The phylogenetic tree can be found on FigShare at the following URL: https://doi.org/10.6084/m9.figshare.30138370. The transcriptomic sequence data that support the findings of this study are openly available in GenBank of NCBI under BioProject accession number PRJNA448700, SRA accession numbers SRX4382063-SRX4382065, and BioSample accession numbers SAMN08863502, SAMN08863511, and SAMN08863514.\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\n\u003cli\u003eAguado MT, Glasby CJ, Schroeder PC, Weigert A, Bleidorn C, 2015. The making of a branching annelid: an analysis of complete mitochondrial genome and ribosomal data of Ramisyllis multicaudata. Scientific Reports, 5(1), p.12072. https://www.nature.com/articles/srep12072\u003c/li\u003e\n\u003cli\u003eAguado MT, Richter S, Sontowski R, Golombek A, Struck TH, Bleidorn C (2016). Syllidae mitochondrial gene order is unusually variable for Annelida. 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Molecular Phylogenetics and Evolution 69(2): 313-319. https://doi.org/10.1016/j.ympev.2012.08.023\u003c/li\u003e\n\u003cli\u003eBrugler MR, Aguado MT, Tessler M, Siddall ME (2018) The transcriptome of the Bermuda fireworm Odontosyllis enopla (Annelida: Syllidae): A unique luciferase gene family and putative epitoky-related genes. \u003cem\u003ePLoS ONE\u003c/em\u003e 13(8): e0200944. https://doi.org/10.1371/journal.pone.0200944\u003c/li\u003e\n\u003cli\u003eCejp B, Jimi N, Aguado MT, 2023. Another piece for the syllid puzzle: A new species from Japan and its mitochondrial genome reveal the enigmatic Clavisyllis (Phyllodocida: Syllidae) as a member of Eusyllinae. \u003cem\u003eZootaxa\u003c/em\u003e, \u003cem\u003e5244\u003c/em\u003e(4), pp.341-360. https://www.mapress.com/zt/article/view/zootaxa.5244.4.2\u003c/li\u003e\n\u003cli\u003eChae JY, Kim J, Kang TW, Lee JI, Lee HH, Kim MS, 2023. 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Multiple outgroups can cause random rooting in phylogenomics. \u003cem\u003eMolecular Phylogenetics and Evolution\u003c/em\u003e, \u003cem\u003e184\u003c/em\u003e, p.107806. https://doi.org/10.1016/j.ympev.2023.10780\u003c/li\u003e\n\u003cli\u003eFischer A, Fischer U (1995) On the Life-Style and Life-Cycle of the Luminescent Polychaete Odontosyllis enopla (Annelida: Polychaeta). \u003cem\u003eInvertebrate Biology \u003c/em\u003e114(3): 236\u0026ndash;247. https://doi.org/10.2307/3226878 \u003c/li\u003e\n\u003cli\u003eFourment M, Holmes EC (2016) Seqotron: a user-friendly sequence editor for Mac OS X. BMC Research Notes 9: 1-4. https://doi.org/10.1186/s13104-016-1927-4 \u003c/li\u003e\n\u003cli\u003eGuindon, S., Dufayard, J. F., Lefort, V., Anisimova, M., Hordijk, W., \u0026amp; Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. 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Genome Biology and Evolution, 15(12), p.evad219. https://doi.org/10.1093/gbe/evad219\u003c/li\u003e\n\u003cli\u003eStecher G, Tamura K, Kumar S (2020) Molecular evolutionary genetics analysis (MEGA) for macOS. Molecular Biology and Evolution 37(4): 1237-1239. https://doi.org/10.1093/molbev/msz312 \u003c/li\u003e\n\u003cli\u003eThomas JM, Horspool D, Brown G, Tcherepanov V, Upton C, 2007. GraphDNA: a Java program for graphical display of DNA composition analyses. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e, 8(1), p.21. https://doi.org/10.1186/1471-2105-8-21\u003c/li\u003e\n\u003cli\u003eVerdes A, \u0026Aacute;lvarez-Campos P, Nygren M, Guillermo D, Dimitri D (2022) Molecular Phylogeny and evolution of bioluminescence in Odontosyllis (Annelida, Syllidae). \u003cem\u003eBioOne Complete: Invertebrate Systematics\u003c/em\u003e 36(7), 622\u0026ndash;630. https://doi.org/10.1071/IS22007 \u003c/li\u003e\n\u003cli\u003eVerrill AE (1900) Additions to the Turbellaria, Nemertina, and Annelida of the Bermudas, with revisions of some New England genera and species. \u003cem\u003eTransactions of the Connecticut Academy of Arts and Sciences \u003c/em\u003e10(2): 595-671. https://www.biodiversitylibrary.org/page/27731368 \u003c/li\u003e\n\u003cli\u003eWilkens LA, Wolken JJ (1981). Electroretinograms from Odontosyllis enopla (polychaeta; syllidae): Initial observations on the visual system of the bioluminescent fireworm of Bermuda. \u003cem\u003eMarine Behaviour and Physiology\u003c/em\u003e, 8(1), 55\u0026ndash;66. https://doi.org/10.1080/10236248109387003\u003c/li\u003e\n\u003cli\u003eZuker M, 2003. Mfold web server for nucleic acid folding and hybridization prediction. \u003cem\u003eNucleic Acids Research\u003c/em\u003e, 31(13), pp.3406-3415. https://doi.org/10.1093/nar/gkg595\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of South Carolina Beaufort","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bioluminescent, Eusyllinae, gene overlap, intraspecific variation, mitogenome, origin of replication, polychaete, Syllidae","lastPublishedDoi":"10.21203/rs.3.rs-7724154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7724154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Bermuda fireworm, \u003cem\u003eOdontosyllis enopla\u003c/em\u003e Verrill, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1900\u003c/span\u003e, is a marine polychaete that displays a unique bioluminescent mating ritual. Despite the first sighting of \u003cem\u003eO. enopla\u003c/em\u003e over 534 years ago, the availability of molecular data has been limited. Several syllid mitogenomes are currently available; however, there are only three published genes for \u003cem\u003eO. enopla\u003c/em\u003e; two partial mitochondrial genes (16S [508bp] and \u003cem\u003ecox1\u003c/em\u003e [653bp]; 1,161bp total) and one partial nuclear gene (18S [1,339bp]). This study bioinformatically mined previously published transcriptomes of \u003cem\u003eO. enopla\u003c/em\u003e for mitochondrial reads and subsequently assembled and annotated a partial mitochondrial genome (10,172bp). The partial mitogenome includes nine (of 13) protein-coding genes, two ribosomal RNAs, and seven (of 22) complete tRNAs. We place the Bermuda fireworm in phylogenetic context using all available syllid mitogenomes, analyze intraspecific variation among three female \u003cem\u003eO. enopla\u003c/em\u003e partial mitogenomes, and propose two putative locations for the mitochondrial origin of replication.\u003c/p\u003e","manuscriptTitle":"The partial mitochondrial genome of the enigmatic Bermuda fireworm Odontosyllis enopla Verrill, 1900 and its phylogenetic implications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 08:32:16","doi":"10.21203/rs.3.rs-7724154/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"24eb9454-5295-41b9-9eee-db48aaf409e5","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55414793,"name":"Evolutionary Biology"}],"tags":[],"updatedAt":"2025-09-29T08:32:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 08:32:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7724154","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7724154","identity":"rs-7724154","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}