The type and only known specimen, an adult ovigerous female (USNM 1752730), was collected from Roadside Cave, a small anchialine cavern in the Walsingham District of Bermuda (Fig. 1) located approximately 110 m from the nearest shore at Harrington Sound. Roadside Cave contains a tidal pool about 8 m deep and 1 m wide, which follows a fissure blocked at the bottom by breakdown rubble and is accessible through a hole at the base of a rock outcrop (Fig. 2). The pool has a tidal range approximately 57% that of the open sea, with a lag averaging 71 min. Salinities have been reported as 30.2‰ at the surface and 31.8‰ at 1 m (Bowman and Iliffe 1985; Fosshagen and Iliffe 1988).

Figure 1. 

Map showing the location of Roadside cave, the type locality of Tetragoniceps bermudensis sp. nov. A island of Bermuda B detail of Bermuda’s Harrington Sound area, with rectangle highlighting the Walsingham karst region. The approximate location of Roadside cave is indicated by the red dot.

Figure 2. 

Collection site at Roadside Cave, Walsingham District, Bermuda A entrance at the base of a rock outcrop B cave fissure, extending into C narrow passageway leading to D tidal pool.

Plankton collections were made with a 25 cm diameter, 50 µm mesh size net, on 5 April 2016 in Roadside Cave. The net was trawled near the bottom of the pool by a diver. Samples were kept in glass jars, alive, until identification under a dissecting microscope. Individuals were then fixed in DESS following Yoder et al. (2006). DNA was extracted from the whole individual using a 25 µL Chelex extraction, as described by Estoup et al. (1996); however, the sequences of the studied Tetragoniceps specimen were found to be too low in quality (mixed peaks) for further study. The remaining exoskeleton was then transferred to glycerine on a glass slide and stored in alcohol as a voucher for morphological description.

After Chelex DNA extraction, the specimen was stained overnight with a 1:1 solution of Congo Red and Acid Fuchsin using procedures adapted from Michels and Büntzow (2010). The whole specimen was temporarily mounted onto a slide with glycerine, and self-adhesive plastic reinforcement rings were used to support the coverslip, as detailed by Kihara and da Rocha (2009).

Imaging was performed using a Leica TCS SP5 laser scanning microscope (Suppl. material 1) equipped with a Leica DM5000 B upright microscope and three visible-light lasers (DPSS 10 mW 561 nm; HeNe 10 mW 633 nm; Ar 100 mW 458, 476, 488 and 514 nm), combined with the LAS AF software v. 2.2.1. (Leica Application Suite Advanced Fluorescence). Images were obtained using the objectives HCX PL APO CS 10.0 × 0.40 DRY UV and HCX APO U-V-I 40.0 × 0.75 DRY UV, a 561 nm excitation wavelength with an 80% acousto-optic tuneable filter (AOTF).

Series of stacks were obtained, collecting overlapping optical sections throughout the whole preparation with an optimal number of sections according to the software. The acquisition resolution was 2048 × 2048 pixels, final images were obtained by maximum projection, and CLSM illustrations were composed and adjusted for contrast and brightness using Adobe Photoshop CS5.

Images for the morphological drawings, made in Adobe Photoshop 2024, were taken using an Olympus IX-83 inverted microscope, using multiple objectives (20× to 100×) to capture the full habitus of the holotype and the details of the appendages, and checked for consistency with microscopy observations and confocal images at various magnifications. Individual photographs were stacked by layers to ensure that morphological characters were properly imaged.

To test the interrelationships of the new taxon and congeneric species, an exploratory maximum-parsimony (MP) phylogenetic analysis of Tetragoniceps was performed in TNT v. 1.5 (Goloboff et al. 2008). The analysis was based on a morphological character matrix including all known females of Tetragoniceps (Data S1) and based on the salient characters for the genus listed by Coull (1973). Protogoniceps hebraeus Por, 1964 was chosen as the outgroup. Protogoniceps is a monospecific genus within Tetragonicipitidae, sharing with Tetragoniceps key characters including the presence of an unguiform antennular projection, a first antennular segment at least 2.5 times as long as the second, and a non-foliaceous female P₅ (Fiers 1995). Moreover, Protogoniceps was regarded by Por (1964) as having a morphology close to that of the common ancestor of Tetragonicipitidae based on its mosaic of traits found in disparate genera within the family: a rostrum and swimming leg armatures similar to those of Pteropsyllus T. Scott, 1906, a spur located on the second antennular segment comparable to that of Phyllopodopsyllus T. Scott, 1906, and 9-segmented antennules also found in Tetragoniceps (Por 1964).

MP analyses were carried out using the Traditional Search option under equal weights (EW) and default settings (random seed = 1, 10 replicates, and using a tree bisection reconnection algorithm). The four most parsimonious trees were collapsed into a strict consensus topology. Resampling (100 replicates) was then conducted through standard bootstrapping (sampling with replacement) and jackknifing using a default removal probability of 36.

A₁, antennule; A₂, antenna; ae, aesthetasc; benp., baseoendopodite; enp., endopodite; exp., exopodite; P₁–P₆, pereiopods 1–6.

Order Harpacticoida Sars, 1903

Family Tetragonicipitidae Lang, 1944

 Tetragoniceps bermudensis sp. nov.

https://zoobank.org/8004F50D-6530-460E-AFAF-EB9B6F7EBFF6

Type locality.

Bermuda, Roadside Cave (32.3468, −64.7131).

Type material.

Holotype, ovigerous female bearing egg sac. National Museum of Natural History (USNM 1752730).

Etymology.

The specific name bermudensis refers to the place where the new species was found followed by the Latin suffix -ensis, i.e. living in, or coming from, Bermuda. It is an adjective in the nominative singular.

Diagnosis.

A Tetragoniceps with caudal rami approximately 10 times longer than wide, P₅ with baseoendopodite and exopodite not fused into a single plate, cephalothorax with smooth dorsal surface, last segment of exopodites of P₂ to P₄ with 1 inner seta, endopodites of P₂ to P₄ with setal formula 1.021, and P₅ exopodite with 6 setae.

Description.

Female (Figs 3, 4). Total body length from base of rostrum to end of caudal rami approximately 750 µm. Body slender, subcylindrical, with no demarcation between metasome and urosome (Fig. 3), transparent when alive. Body surface smooth, with sensilla pattern as figured (Figs 3A, B, 4). Body with 9 somites (prosome with cephalosome and P1-bearing somite fused, forming a cephalothorax, and with P₂ to P₄ bearing somites; urosome with P₅-bearing somite, genital somite, 3 free urosomites, and anal somite with caudal rami). Posterior margin of body somites 4–8 with serrate hyaline frills.

Figure 3. 

Tetragoniceps bermudensis sp. nov., female. Confocal laser scanning microscopy images A habitus, in dorsal view B habitus, in lateral view C habitus, in ventral view. Scale bars: 100 µm.

Figure 4. 

Tetragoniceps bermudensis sp. nov., female. Confocal laser scanning microscopy images A cephalothorax, in dorsal view B anterior part of cephalothorax and associated appendages, in ventral view. Scale bar: 50 µm.

Urosome (Figs 3A–C, 5A, B) 5-segmented, comprising P₅-bearing somite, genital double-somite, 2 free abdominal somites, and anal somite. Genital field located medially, on anterior half of genital double-somite, with small median copulatory pore and paired genital pores laterally (Fig. 5A). Anal operculum convex, with a row of fine spinules on distal margin (Fig. 5B).

Figure 5. 

Tetragoniceps bermudensis sp. nov., female A urosome, in ventral view, showing medial egg sac B last two abdominal somites with caudal rami, in dorsal view C caudal ramus, in lateral view. Scale bar: 50 µm.

Caudal rami (Figs 3A–C, 5B, C) approximately 10 times as long as wide at the widest portion (proximal end). Each ramus with 6 setae. Seta I completely reduced. Seta II laterally within the proximal half of each ramus. Seta III of similar length as seta II and located laterally within the terminal 20% of each ramus. Dorsal seta VII longer than seta V, multi-articulated. Setae IV–VI arising apically, with IV and VI reduced in size and <30% the length of seta V.

Rostrum (Fig. 6A) rounded, symmetrical, and distally concave; it bears 2 sensilla located near the outer margin.

Figure 6. 

Tetragoniceps bermudensis sp. nov., female A rostrum and antennule B antenna. Scale bar: 50 µm.

Antennule (Fig. 6A) 9-segmented. First segment elongate, with dentiform projection at outer distal corner. Aesthetascs on fourth and ninth segment. Armature formula 1-[1], 2-[8], 3-[8], 4-[2+ae], 5-[1], 6-[3], 7-[2], 8-[1], 9-[4+ae].

Antenna (Fig. 6B) with small, rectangular coxa and with long basis. Exopodite 1-segmented, with 2 long bipinnate setae apically and 1 shorter unipinnate seta laterally. Endopod 2-segmented. First endopodal segment unarmed. Second endopodal segment armed with 8 geniculate setae arising apically, the outer of which is basally fused with a slender seta, and armed with a small spinule on inner margin.

Mandible (Fig. 7A) with large sclerotised gnathobase. Pars incisiva with several acute teeth and several accessory spinules and dentations. Basis with 3 setae. Exopod short, 1-segmented, with 1 small outer seta and 2 terminal setae. Endopod 1-segmented, with 1 lateral and 5 terminal setae.

Figure 7. 

Tetragoniceps bermudensis sp. nov., female A mandible B maxillule C maxilla D maxilliped. Scale bars: 50 µm.

Maxillule (Fig. 7B) with praecoxa with arthrite armed with 6 distal spines, 2 bare terminal setae, and 1 subterminal bare seta. Coxal endite with 2 bare and 3 bipinnate terminal setae. Coxal epipodite represented by 1 bare seta. Basis with 4 bare, 1 unipinnate, and 2 geniculate terminal setae. Endopodite absent. Exopodite with 1 simple and 2 bipennate setae, and a row of long inner setules.

Maxilla (Fig. 7C), syncoxa with 3 endites, the first (proximal) bilobed, proximal lobe with two, distal lobe very reduced and armed with 1 seta. Second endite with 1 unipinnate and 1 bare seta. Third endite with 1 unipinnate and 2 bare setae, one shorter than the others. Allobasis with a claw-like spine, 1 long unipinnate seta, 1 long bare seta, and 2 short bare setae. Endopodite 1-segmented, with 6 terminal setae.

Maxilliped (Fig. 7D) subchelate. Syncoxa with 2 pinnate setae distally. Basis showing the following proximo-distally: a row of several spinules, a long spine, a long seta, and a distal spinule. Endopodite 2-segmented, second segment with 1 terminal claw, 1 terminal unipinnate seta, and 1 subterminal bare seta.

P₁ (Fig. 8A) coxa bare, basis with 1 bare outer seta. Endopodite 2-segmented, exopodite 3-segmented. First endopodal segment with outer setules, and 1 large inner bipinnate seta. Second endopodal segment with outer setules, and 2 geniculate unipinnate setae distally. First and second exopodal segments with 1 outer spine; first segment without, second segment with inner and outer setules. Third exopodal segment with 1 outer spine, 2 geniculate setae and 1 unipinnate seta distally, and both inner and outer margin setules. Setal formula listed in Table 1.

Table 1.

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Setal formula for pereiopods P₁–P₄ of Tetragoniceps bermudensis sp. nov.

Leg	Exopodite	Endopodite
P1	0.0.022	1.020
P2	0.1.133	1.021
P3	0.1.133	1.021
P4	0.1.321	1.021

Figure 8. 

Tetragoniceps bermudensis sp. nov., female pereiopods A P₁ B P₂ C P₃ D P₄ E P₅. Scale bar: 50 µm.

P₂ (Fig. 8B) coxa bare, basis with 1 bare outer seta and armed with 2 short spinules surrounding the endopodite. Endopodite 2-segmented, exopodite 3-segmented. First endopodal segment with 1 large inner seta. Second endopodal segment with inner setules, 1 outer spine and 2 bipinnate setae distally. First exopodal segment with a distal spinose outgrowth, 1 large outer spine, and an outer spinule. Second exopodal segment with a distal spinose outgrowth, 1 large outer spine, 1 pinnate inner seta, and a row of outer margin spinules. Third exopodal segment with 3 outer spines, 1 pinnate inner seta, 1 outer apical spine, 2 distal bipinnate setae, and rows of both inner and outer margin spinules. Setal formula listed in Table 1.

P₃ (Fig. 8C) coxa bare, basis with 1 bare outer seta. Endopodite 2-segmented, exopodite 3-segmented. First endopodal segment with 1 large inner seta. Second endopodal segment with 1 distal outer spine and 2 bipinnate setae distally. First exopodal segment with a distal spinose outgrowth and 1 large outer spine. Second exopodal segment with a distal spinose outgrowth and 1 large outer spine, 1 pinnate inner seta, and a row of outer spinules. Third exopodal segment with 3 large outer spines, 1 pinnate inner seta, 1 subapical bipinnate seta, 2 distal unipinnate setae, and a row of outer margin spinules. Setal formula listed in Table 1.

P₄ (Fig. 8D) coxa bare, basis with 1 bare outer seta and armed with short spinules surrounding the endopodite. Endopodite 2-segmented, exopodite 3-segmented. First endopodal segment with 1 inner unipinnate seta and short distal spinules. Second endopodal segment with 1 outer spine and 2 bipinnate setae distally. Exopodite approximately 3 times as long as the endopodite. First exopodal segment with a distal spinose outgrowth and 1 large outer spine. Second exopodal segment with a distal spinose outgrowth and 1 large outer spine, 1 pinnate inner seta, and rows of both inner and outer spinules. Third exopodal segment with 1 small outer spine, 1 unipinnate inner seta, 2 bare setae subapically, 3 unipinnate setae distally, and rows of both inner and outer spinules. Setal formula listed in Table 1.

P₅ (Fig. 8E), baseoendopodite very pronounced, armed with 1 outer basal seta; endopodal lobe with 1 long, bare seta apically and 1 small seta subapically, and with 3 setae on the inner margin. Exopodite pronounced, elongate, with 6 setae.

P₆ (Fig. 5A) each with rudimentary lobe carrying 2 bare setae, fused to basal plate.

Our MP strict consensus tree yielded a topology in which a monophyletic group, comprising T. galapagoensis plus a clade uniting T. unguis and T. brownei is the sister-taxon of all other species of Tetragoniceps (Fig. 9A). Tetragoniceps bermudensis sp. nov. was recovered in a polytomy with T. bergensis, T. pacificus, T. brevicauda, T. truncata, T. longicaudata, and the following clades: one consisting of T. prima, T. dubia, T. bookhouti, and T. malleolatus; and one comprising T. arenicolus and T. scotti (Fig. 9A).

Figure 9. 

Maximum parsimony-based analyses of the genus Tetragoniceps, showing the recovered placements of T. bermudensis sp. nov. (in bold) A strict consensus of four most parsimonious trees based on equal weights (EW) analysis B standard bootstrap analysis using the same settings as in (A) and 100 replicates C jackknifing analysis using the same settings as in (A), with 100 replicates and removal probability of 36. For each resampling analysis, numbers at nodes denote respective support values.

Resampling support values for both bootstrapping and jackknifing were found to be low for all recovered ingroup nodes (<50). All individual node support values under bootstrapping and jackknifing are presented in Fig. 9B and Fig. 9C, respectively. Under both bootstrapping (Fig. 9B) and jackknifing (Fig. 9C) analyses, which yielded identical topologies, T. bermudensis sp. nov. was recovered as the sister taxon of T. pacificus with low support values (<12). The clade comprising T. prima, T. dubia, T. bookhouti, and T. malleolatus, that comprising T. arenicolus and T. scotti, and that comprising T. galapagoensis, T. unguis, and T. brownei were found to be robust to both bootstrapping and jackknifing, albeit with low support and minor internal changes in topology (Fig. 9B, C). However, in both resampling analyses the T. galapagoensis + (T. unguis + T. brownei) clade collapsed into a polytomy with the other lineages within the genus, instead of being recovered as their sister-taxon (Fig. 9B, C).

Salient female characters of Tetragoniceps (Coull 1973) offer grounds for a broader morphological comparison of T. bermudensis sp. nov. and other members of the genus (Table 2). The P₂–P₄ endopods of T. bermudensis sp. nov. and a group of “Indo-Pacific” species (T. unguis, T. galapagoensis, and T. pacificus) share the same setal formula (1.021): another species from the Indian Ocean, T. brownei, has a similar P₂–P₄ endopodal formula of 1.020 (Table 2). In addition, T. unguis, T. galapagoensis, and T. pacificus share with T. bermudensis sp. nov. the presence of three setae on the exopod of A₂, and a P₅ with distinct baseoendopodite and exopodite; furthermore, T. galapagoensis has in common with T. bermudensis sp. nov. a baseoendopodite of P₅ armed with six setae (Table 2). Even closer similarities in setal armatures occur between T. bermudensis sp. nov. and T. pacificus, which also shares with the new taxon the presence of six setae on the exopodite of P₅. Since both T. pacificus and T. bermudensis sp. nov. have a 9-segmented A₁, the only salient characters established by Coull (1973) for which T. pacificus differs from the new Bermudian taxon are the length:width ratio of the caudal rami and the setal armatures of segments 1 and 3 in the exopods of P₂–P₄ (Table 2).

Among Tetragoniceps, caudal rami approximately 10 times longer than wide are only known in the newly described Bermudian species and in T. bergensis, reported from Norwegian waters (Por 1965). Three setae on the exopod of A₂ and a P₅ with distinct baseoendopodite and exopodite, as found in T. bermudensis sp. nov., also occur in a broader set of North Atlantic species (T. brevicauda, T. scotti, T. truncata, T. longicaudata, and T. bergensis) which share with T. bermudensis sp. nov. a 9-segmented A₁; the latter character is not found in “Indo-Pacific” Tetragoniceps, except for T. pacificus (Table 2).

These partly overlapping similarities of T. bermudensis sp. nov. with Pacific congeneric species on the one hand, and with North Atlantic ones the other, suggest that the new species may combine plesiomorphic characters retained by distinct, geographically widespread lineages of Tetragoniceps. This hypothesis is in accord with the recurrent archaisms expressed by Bermuda’s endemic anchialine cave faunas (Sket and Iliffe 1980; Hart et al. 1985; Iliffe and Kornicker 2009) and suggests that T. bermudensis sp. nov. may record an early-diverging taxon within the genus, or at least relative to its other Atlantic species.

Our MP consensus tree (Fig. 9A) is consistent with such a relatively basal placement. The only group of Tetragoniceps species recovered as more basal than the polytomy encompassing T. bermudensis sp. nov. is the clade formed by T. galapagoensis, T. brownei, and T. unguis, of which T. galapagoensis represents the earliest diverging member (Fig. 9A). This topology suggests that salient traits uniting T. bermudensis sp. nov. with T. galapagoensis and/or Protogoniceps, notably including a 9-segmented A₁, three setae on the exopodite of A₂, and distinct (non-confluent) baseoendopodite and exopodite of P₅, are plesiomorphic for Tetragoniceps. This scenario is consistent with these same character states being widely retained among species in the genus (Table 2, Suppl. material 2). By contrast, confluence of the baseoendopodite and exopodite of P₅ only occurs in the monophyletic group formed by T. dubia, T. bookhouti, and T. malleolatus and represents a probable synapomorphy of this clade (Fig. 9, Suppl. material 2). The other recovered clades shown in Fig. 9 are not united by any strong candidate synapomorphies, even though their members share multiple overlapping similarities in their setal armatures (Table 2, Suppl. material 2). However, these phylogenetic results must be considered preliminary given the few salient characters available for Tetragoniceps, the low resampling support values of our recovered topology (Fig. 9), and the lability of morphological traits within the genus (Table 2). Almost no genetic data is presently available for Tetragonicipitidae (Kim et al. 2023), and additional molecular evidence is necessary to build a robust phylogeny of the family.

Tetragoniceps bermudensis sp. nov. represents the first record of Tetragoniceps in Bermuda, the first known anchialine species in the genus, and the second record of an anchialine species of Tetragonicipitidae globally (Karanovic et al. 2001). The new taxon brings the total of anchialine copepod species in Bermuda to 22, distributed across 16 genera. The single known, geographically localised occurrence of T. bermudensis sp. nov. suggests a correspondingly limited area and a probable endemic status, consistent with the high degree of endemism typical of Bermuda’s cave-dwelling fauna (Iliffe and Calderón-Gutiérrez 2021). Although Roadside cave is in a relatively undisturbed area, persistent threats include urban development, vandalism, dumping, littering and pollution, and sediment disturbance due to unlawful access by humans and domesticated animals (Mammola et al. 2019; Iliffe and Calderón-Gutiérrez 2021). These risks emphasize the need for formal protection of Roadside cave, and for robust enforcement of existing measures (Glasspool 2014) for the safeguard of Bermuda’s anchialine fauna.

The authors have declared that no competing interests exist.

No ethical statement was reported.

GM acknowledges support from a NERC C-CLEAR DTP studentship [RG96579]. PMA was supported by the project “Massive mEIOfauna DiscoverY of new Species of our oceans and SEAs (MEIODYSSEA) funded by the Ocean Shot Research Grant Program of the Sasakawa Peace Foundation, supported by the Nippon Foundation.

Conceptualization: LBB, GM. Data curation: GM, LBB. Formal analysis: GM. Funding acquisition: GM, PMA. Investigation: GM. Methodology: TCK, GM, SK, LBB. Software: YJN. Supervision: LBB. Validation: TCK, LBB, GM, PMA, SK. Visualization: YJN, TCK, SK, GM. Writing – original draft: GM. Writing – review and editing: LBB, PMA, TCK, SK, GM.

Giovanni Mussini https://orcid.org/0009-0002-1093-5865

Sahar Khodami https://orcid.org/0000-0002-7944-4004

Terue C. Kihara https://orcid.org/0000-0002-7292-251X

Pedro Martinez Arbizu https://orcid.org/0000-0002-0891-1154

Leocadio Blanco-Bercial https://orcid.org/0000-0003-0658-7183

All of the data that support the findings of this study are available in the main text or Supplementary Information.

This is publication number 75 that uses data from the Senckenberg am Meer Confocal Laserscanning Microscope Facility (SGN-SaM-cLSM): additional digital data relating to this publication is available in the Zenodo repository with the following DOI: https://doi.org/10.5281/zenodo.14920270.