An Elusive New Genus and Species of Subterranean Amphipod (Hadzioidea: Eriopisidae) from Barrow Island, Western Australia ^†

Abstract

Barrow Island, off the coast of north-west Western Australia, supports a rich subterranean amphipod fauna amid major resource development. Previous biological surveys for the purpose of species documentation and environmental impact assessment have helped to uncover a once overlooked genus of amphipod from the family Eriopisidae. Here, we describe this new genus and one new species, Aenigmata megabranchia Stringer and King gen. et sp. nov., using a combination of molecular and morphological data, and present a key to Western Australian subterranean eriopisid genera. The new genus represents a distinct, genetically divergent lineage that can be distinguished from all other eriopisid genera by the following characters: the shape and setation of the mouthparts, the enlarged coxal gills, the straight posterior margin of the first coxa, and an almost entirely cleft telson. This research enhances our knowledge of the Australian Eriopisidae, emphasises the importance of Barrow Island as a key location for subterranean amphipod fauna, and will assist in the future recognition of the species for conservation.

1. Introduction

Barrow Island is a small, karstic continental island off the north-west coast of Western Australia (WA) that supports a rich endemic biodiversity. In recognition of its uniqueness and importance as a refuge for native wildlife, Barrow Island was listed as a Class A Nature Reserve in 1910 [1]. Barrow Island has further supported an operating oilfield since the 1960s, together with the more recent establishment of natural gas processing facilities in 2009 [2,3]. These resource developments have prompted expanded biological sampling to inform environmental impact assessments and ecological monitoring programs [3,4], which have substantially increased flora and fauna records and have facilitated new species descriptions [1,5,6,7].

Subterranean fauna, in particular, are exceedingly prevalent on Barrow Island, with the area known internationally for its extraordinary biodiversity [4,8,9]. The subterranean aquatic fauna (stygofauna) inhabits water-filled limestone voids and primarily consists of crustaceans, including amphipods. Species from three amphipod families have been described in the region; Bogidiellidae Hertzog, 1936 [10] (one species [11]), Eriopisidae Lowry & Myers, 2013 [12] (three species [6]), and Hadziidae S. Karaman, 1943 [13] (one species [14]), with one further undescribed species of Paramelitidae Bousfield, 1977 [15] also documented [4]. The Australian Eriopisidae, specifically, is prominent across WA subterranean habitats and comprises Nedsia Barnard & Williams, 1995 [16] from Barrow Island, the Pilbara (WA), and North West Cape peninsula (WA), Norcapensis Bradbury & Williams, 1997 [17] from North West Cape, and Pilbarana Stringer & King, 2022 [18] from the Pilbara. The family also includes marine and estuarine eastern Australian species of Eriopisella Chevreux, 1920 [19], Netamelita J. L. Barnard, 1962 [20], and Victoriopisa Karaman & Barnard, 1979 [21]. Recently, an evaluation of biological survey reports (associated with resource development), collected specimens, and molecular analyses revealed a new eriopisid genus on Barrow Island with a limited distribution. This genus had remained overlooked, and was typically and incorrectly attributed to Nedsia in collections.

Given the need to formally recognise the exceptional diversity of Australia’s subterranean fauna [22], the aim of this study was to document and describe this new genus and one new species based on an integrative approach using a combination of morphological and molecular data. This study provides essential taxonomic information necessary for the documentation of species for environmental impact assessment and conservation, contributes to a broader collaborative project aiming to improve the detection and identification of subterranean fauna [23,24,25], and emphasises the significance of Barrow Island as a location for Eriopisidae in Australia.

2. Materials and Methods

2.1. Study Site and Specimen Collection

Barrow Island is a semi-arid continental island located 56 km off the Pilbara coast of WA, which occupies an area of 202 km², making it the largest island in the north-west shelf and the second largest in WA [3]. The island mainly consists of karstic limestone outcrops and deposits that are covered by sands and gravels, and the groundwater is anchialine, with a freshwater lens overlying seawater [8]. Stygofauna are collected from the groundwater via pre-existing PVC bore holes, some drilled for the purpose of subterranean fauna sampling for biological surveys and impact assessment, using haul nets with an attached collection tube. An examination of the reports compiled from these environmental surveys revealed a potential new undescribed genus and species (largely through molecular lineage reporting) of subterranean amphipod, primarily from a single bore (X62M), which is located to the north of the island (Figure 1). This bore was targeted on a December 2020 collection trip to Barrow Island, and specimens of the new genus were sampled, permitting molecular sequencing and morphological analysis for description. The amphipods were collected using haul nets with a diameter of 45 or 95 mm and alternating mesh sizes of 50 and 150 μm, and then stored in 100% ethanol and frozen at −20 °C for optimal tissue preservation. Additional specimens (collected from the same site: X62M) were also obtained from the Western Australian Museum’s crustacean collection.

2.2. DNA Extraction, Sequencing, and Phylogenetics

To evaluate the phylogenetic placement of the collected specimens, DNA was extracted from two or three pereopods and pleopods (removed from the right side of the animal where possible) per amphipod specimen using the Gentra Puregene DNA Purification Kit (Gentra Systems, Minneapolis, MN, USA) for tissue according to the manufacturer’s protocol. Partial sequences (approximately 670–700 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) gene were amplified using polymerase chain reaction (PCR) with the universal primers LCOI490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) [27]. PCR amplifications were carried out in 25 μL reactions consisting of 10x AmpliTaq Gold Buffer (Applied Biosystems, Foster City, CA, USA), 2.5 mM MgCl₂, 2.5 mM of each dNTP, 0.5 μM of each primer, 0.1 U of AmpliTaq Gold Polymerase, and approximately 1–5 ng of DNA. The thermal cycling conditions involved an initial hold at 94 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 47 °C for 30 s, and extension at 72 °C for 90 s, with a final extension at 72 °C for 6 min. PCR clean up and bidirectional sequencing was carried out by The Australian Genome Research Facility (AGRF).

Forward and reverse sequences were trimmed, edited, and assembled into consensus sequences using Geneious v.9.0.5 (https://www.geneious.com) and aligned with MUSCLE [28]. Five sequences obtained from specimens of the new amphipod (either generated in this study or acquired from external sources) were included in the final COI alignment, along with sequences of additional WA eriopisid species from the genera Nedsia, Norcapensis, and Pilbarana downloaded from GenBank (see Table S1 for accession numbers). COI sequences from two specimens of Hadzia branchialis (Bradbury & Williams, 1996) [14] (Hadziidae), which were extracted and sequenced here according to the above methods, were further incorporated as an outgroup to evaluate the relationship between the WA North West Cape and Barrow Island Hadzia species and the new genus. Bayesian phylogenetic analyses were performed in BEAST2 v.2.6.7 [29] (with bModelTest [30] used for nucleotide substitution model selection), and conducted with a strict clock, birth–death tree prior to account for intraspecific sampling [31], and run for 20 million generations. Two partitions were applied to the alignment: one for nucleotides at the first and second codon positions, and one for the third codon position. Convergence of the stationary distribution was assessed with Tracer v1.7.1 [32] for ESS > 200, and a maximum clade credibility tree was produced using TreeAnnotator [29]. For comparison, maximum likelihood analyses were carried out with IQ-TREE v.1.6.12 [33] using MFP + MERGE for substitution model selection and 1000 ultrafast bootstrap replicates [34,35]. The Hadzia sequences were not included in the maximum likelihood analyses due to their substantial genetic distance from the ingroup. All tree outputs were visualised and edited in FigTree v.1.4.2 (available at: http://beast.bio.ed.ac.uk/).

Additionally, MEGA v.11.0.11 [36] was used to estimate COI divergences with the Kimura 2-parameter (K2P) model [37] (see the ‘Results’ and ‘Remarks’ sections for pertinent results). All COI sequences included in this study are available on GenBank (Table S1).

2.3. Morphology

The morphological examination involved appendage micro-dissections, light microscopy, and illustrations using a Nikon Eclipse 80i (Tokyo, Japan) compound microscope with a camera lucida attachment. The type material was dissected along the left side and mounted on temporary slides in glycerol for taxonomic examination. Total body length was measured through the lateral midline from the head to the telson, and family and higher-level classification followed [12]. The type material is lodged at the Western Australian Museum. The morphological examination, along with the molecular sequence data produced above, and the General Lineage Species Concept [38,39] were used to define the new genus and species.

3. Results

A comprehensive examination of the material based on morphological and molecular data confirm the occurrence of an undescribed eriopisid genus, Aenigmata gen. nov., with one new species on Barrow Island. The phylogenetic analyses (Figure 2 and Figure S1) recovered Aenigmata gen. nov. as a distinct, well-resolved group firmly placed within the WA Eriopisidae, and most closely related to the Pilbara genus Pilbarana. The divergence estimates reveal that Aenigmata gen. nov. is approximately 18.5–20% divergent from species of Nedsia, Norcapensis, and Pilbarana, which is comparable to COI divergence values employed in past studies to delimit amphipod genera [40,41]. Morphological analyses similarly indicate that Aenigmata gen. nov. falls within the Eriopisidae principally due to the following characters: the lack of sternal gills, the absence of sexually dimorphic second gnathopods, the presence of a single basofacial seta on the peduncle of the first uropod, and a biramous and elongated third uropod [12].

Taxonomy

• Superfamily Hadzioidea S. Karaman, 1943 (Bousfield 1983)
• Family Eriopisidae Lowry & Myers, 2013
• Remarks

The Eriopisidae is a cosmopolitan distributed family of marine, epigean, and subterranean amphipods attributed to the Hadzioidea by [12]. The main characters used to differentiate Eriopisidae from other amphipod families include the following: antennae 1–2 without calceoli; antenna 1 longer than antenna 2; antenna 2 peduncular article 1 not enlarged; gnathopod 2 similar in males and females (not sexually dimorphic); uropod 1 with or without robust basofacial setae; uropod 3 biramous, with endopod shorter than exopod; and telson deeply cleft [12]. Few characters separate the eriopisids from the related families Maeridae Krapp-Schickel, 2008 [42] and Melitidae Bousfield, 1973 [43]; nevertheless, maerids and melitids, unlike eriopisids, possess sexually dimorphic second gnathopods. The WA subterranean eriopisid genera, Nedsia and Norcapensis, were originally described within the Melitidae, but were later transferred to the Eriopisidae upon the establishment of the family [12]. There are now four subterranean eriopisid genera from the arid region of WA (Nedsia, Norcapensis, Pilbarana, and Aenigmata gen. nov.), with recent studies incorporating morphological and molecular sequence data helping to increase our understanding of the distinct differences among the genera [6,18]. Below, we present a new key specifically to the WA genera of Eriopisidae.

Key to Western Australian subterranean genera of Eriopisidae

1.

Gnathopod 2 propodus enlarged in both males and females, at least 3 times length of gnathopod 1 propodus...........................................................................................................2

–

Gnathopod 2 propodus no more than 2 times length of gnathopod 1 propodus..........3

2.

Distinct antennal sinus; pereonite 1 with concave posterodistal corner, pereonites 2–7 laterally square-shaped, as long as broad, vermiform body shape; uropod 1 peduncle 2 times length of rami; uropod 3 outer ramus second article apically concave................................................................................................................................Pilbarana

–

No distinct antennal sinus; pereonite 1 lacking concave posterodistal corner, pereonites 2–7 around 2 times as long as broad, robust body shape; uropod 1 peduncle approximately equal in length to rami; uropod 3 outer ramus second article not apically concave .....................................................................................................................Norcapensis

3.

Coxal gills elongated, around 2 or more times length of pereonites (Figure 3A); pereopods 5–7 bases with distinct lobe at posterodistal corner (Figure 4E–G)......................................................................................................................................Aenigmata gen. nov.

–

Coxal gills shorter than 2 times length of pereonites; pereopods 5–7 bases without lobe at posterodistal corner ...................................................................................................Nedsia

Figure 3. Aenigmata megabranchia sp. nov., holotype male WAM C84784, 5.5 mm. (A) Whole animal with scale; (B) antenna 1; (C) antenna 2; (D) upper lip; (E) lower lip; (F) mandible; (G) maxilla 1; (H) maxilla 2; (I) maxilliped.

Figure 3. Aenigmata megabranchia sp. nov., holotype male WAM C84784, 5.5 mm. (A) Whole animal with scale; (B) antenna 1; (C) antenna 2; (D) upper lip; (E) lower lip; (F) mandible; (G) maxilla 1; (H) maxilla 2; (I) maxilliped.

Figure 4. Aenigmata megabranchia sp. nov., holotype male WAM C84784, 5.5 mm. (A) Gnathopod 1; (B) gnathopod 2; (C) pereopod 3; (D) pereopod 4; (E) pereopod 5; (F) pereopod 6; (G) pereopod 7.

Figure 4. Aenigmata megabranchia sp. nov., holotype male WAM C84784, 5.5 mm. (A) Gnathopod 1; (B) gnathopod 2; (C) pereopod 3; (D) pereopod 4; (E) pereopod 5; (F) pereopod 6; (G) pereopod 7.

• Aenigmata Stringer & King gen. nov.
• urn:lsid:zoobank.org:act:0F7D34AB-0999-43CD-A2BC-F59BCB99F8CB
• Type species: Aenigmata megabranchia sp. nov.
• Diagnosis

Head with indistinct antennal sinus. Maxilla 1 inner plate triangular in shape, with row of simple setae along entire inner margin. Maxilla 2 inner plate apically constricted, with marginal row of simple setae and second oblique row of simple setae on inner margin and extending onto face; outer plate heavily setose apically, with scattered plumose setae. Maxilliped inner plate broader than palp articles; outer plate ovate, broader than palp articles, with smooth edge. Coxa 1 with posterior margin straight. Coxal gills present on coxae 2–6, large and oblong-shaped, around 2 or more times length of pereonites. Pereopods 5–7 bases expanded posteriorly, distinct lobe at posterodistal corner. Uropod 1–2 sparsely setose, lacking setae along length of rami. Uropod 3 strongly extended, not sexually dimorphic, parviramous (inner ramus reduced); outer ramus flattened and leaf-shaped; inner ramus short and scale-like. Telson almost entirely cleft into two lobes.

• Description

Head with indistinct antennal sinus; eyes absent. Antenna 1 just longer than half body length. Antenna 2 flagellum with first article around 2 times length of remaining flagellum articles. Mandibular palp elongate and of three articles. Maxilla 1 inner plate triangular in shape, with row of simple setae along entire inner margin. Maxilla 2 inner plate apically constricted, with marginal row of simple setae and second oblique row of simple setae on inner margin and extending onto face; outer plate heavily setose apically, with scattered plumose setae. Maxilliped inner plate broader than palp articles; outer plate ovate, broader than palp articles, with smooth edge. Pereonites 1–7 laterally squareshaped, as broad as long, giving a vermiform appearance. Coxae 1–7 short, broader than long, reduced compared to pereonites (lengths or depths distinctly shorter than pereonite lengths); coxae 1–2 largely ovoid in shape, coxa 1 with posterior margin straight; coxae 3–4 subrectangular; coxae 5–6 with small anterior lobe and associated setae; coxa 7 simple and rounded. Oostegites present on coxae 2–5 in females. Coxal gills present on coxae 2–6, large and oblong-shaped, around 2 or more times length of pereonites; sternal gills absent. Gnathopods 1–2 subchelate, not sexually dimorphic. Gnathopod 1 carpus with multiple rows of setae; propodus with palm transverse, with rows of robust bifurcated setae and short simple setae at palm corner and along palm margin. Gnathopod 2 propodus with row of setae on lateral face, palm oblique, not enlarged. Pereopods 5–7 bases expanded posteriorly, distinct lobe at posterodistal corner. Uropod 1–2 sparsely setose, lacking setae along length of rami. Uropod 1 peduncle around 1.5 times length of rami, with one robust basofacial seta. Uropod 2 peduncle similar length to inner ramus. Uropod 3 much larger than uropods 1–2 and strongly extended, not sexually dimorphic, parviramous; outer ramus of two articles, flattened and leaf-shaped; inner ramus short and scale-like. Telson almost entirely cleft into two lobes.

• Etymology

Derived from ‘enigma’, in reference to the genus being ‘hidden’ amongst other amphipods and with a very limited distribution. Gender: feminine.

• Remarks

Morphologically, Aenigmata gen. nov. is distinct from all other Australian eriopisid genera and can be identified based on the shape and setation of the maxillae and maxilliped, the elongated coxal gills, the straight posterior margin of the first coxa, the lack of setae on the uropods, and the almost entirely cleft telson. Aenigmata gen. nov. is most similar morphologically to the subterranean eriopisid genera Nedsia, Norcapensis, and Pilbarana, rather than the east coast marine genera, with Nedsia (which also occurs on Barrow Island) exhibiting the most comparable characters: smaller body size, second gnathopods only marginally larger than the first, and leaf-shaped third uropods. Nevertheless, Aenigmata gen. nov. can be easily distinguished, as Nedsia has a robust rather than vermiform body shape, shorter and rounded coxal gills, an ovate rather than triangular-shaped first maxilla inner plate, a maxilliped with a serrated outer plate (not broader than palp articles), and pereopods 5–7 bases expanded, without lobes at the posterodistal corners. Aenigmata gen. nov. can also be differentiated from Norcapensis and Pilbarana, as species from these two genera have a larger body size and possess enlarged second gnathopods and cylindrical third uropods.

Significant similarities in the mouthpart morphology, which is clearly distinct between Aenigmata gen. nov. and the other Australian Eriopisidae, were evident between Aenigmata gen. nov. and two species of Hadzia (originally described in the genus Liagoceradocus), H. subthalassicus (Bradbury & Williams, 1996) from Barrow Island and H. branchialis (Bradbury & Williams, 1996) from North West Cape, WA. To verify that Aenigmata gen. nov. was indeed a new genus, type specimens of these two species were examined, and specimens of H. branchialis (due to the lack of material of H. subthalassicus) were sequenced for inclusion in the eriopisid phylogenetic analysis (Figure 2). Clear morphological differences were apparent between the Hadzia species and Aenigmata gen. nov., with Hadzia, in particular, possessing a magniramous (inner and outer rami approximately equally extended) rather than a parviramous third uropod. The molecular analyses, moreover, reflected no close relationship between H. branchialis and Aenigmata gen. nov., with estimates of approximately 41% COI divergence between the sequences, indicating the presence of a new genus.

• Aenigmata megabranchia Stringer & King sp. nov.
• Figure 3, Figure 4 and Figure 5
• urn:lsid:zoobank.org:act:70A76716-3FE2-44DE-AC13-71162E13BC9E
• Material examined
• Holotype: male, WAM C84784 (LN00302.1), bore X62M, Barrow Island, WA, 20°43′56″ S 115°25′34″ E, coll. N.B. Stevens, M. van der Heyde, and M. Jones, 1 December 2020.

Paratypes: female, WAM C84785 (LN00302.2; GenBank COI: PQ497467), collection data as for holotype; 2 males, WAM C84786 (LN00302), collection data as for holotype; male, WAM C84787 (LN00299.1; GenBank COI: PQ497466), collection data as for holotype; male, WAM C84788 (LN00299.2), collection data as for holotype; female, WAM C84789 (LN00299.3), collection data as for holotype; female, WAM C84790 (LN00299), collection data as for holotype; male, WAM C84791 (LN00446), collection data as for holotype; female, WAM C84792 (LN00305), collection data as for holotype; 1 male, 1 female, 2 juveniles, WAM C84793 (LN00307), collection data as for holotype; male, WAM C84794 (LN00312), collection data as for holotype; male, WAM C76707 (LN33580) bore X62M, Barrow Island, WA, 20°43′56″ S 115°25′34″ E, coll. N.B. Stevens and M. Jones, 16 July 2019; 1 male, 1 female, WAM C76708 (LN37571), bore X62M, Barrow Island, WA, 20°43′56″ S 115°25′34″ E, coll. N.B. Stevens and M. Jones, 16 July 2019; 13 males, 7 females, 1 juvenile, WAM C84795, bore X62M, ca. 6 km NW of Terminal Tanks, Barrow Island, WA, 20°43′57.3″ S 115°25′33.0″ E, coll. J. Cairnes and J. Alexander, 23 June 2016; male, WAM C84796, bore X62M, ca. 6 km NW of Terminal Tanks, Barrow Island, WA, 20°43′57.3″ S 115°25′33.0″ E, coll. J Cairnes and J. Alexander, 23 June 2016.

• Description: Holotype, male, 5.5 mm

Head. Rostrum weak to obsolete; lateral cephalic lobes indistinct, no antennal sinus present; eyes absent. Antenna 1 elongate, slightly longer than half body length, longer than antenna 2; peduncular article 3 approximately one third length of article 1 and 2; flagellum around 2 times length of peduncle, 18 articles, with one ventral aesthetasc on proximal margin of most articles; accessory flagellum of two articles, second article tiny. Antenna 2 around two thirds length of antenna 1; peduncle longer than flagellum, articles 4–5 subequal in length; flagellum of five articles, with first article approximately 2 times length of remaining flagellum articles; calceoli absent. Upper lip evenly rounded, setose apically. Lower lip inner lobes present. Mandibular palp of three articles, article 1 approximately one third length of articles 2 and 3; terminal article linear and tapered, with long apical and medial setae; molar small and triturative. Maxilla 1 inner plate triangular, with row of simple setae along entire inner margin; outer plate with eight denticulate robust setae; palp of two articles, distal article longest, with apical setae. Maxilla 2 inner plate apically constricted, with marginal row of simple setae, a second oblique row of simple setae extending onto face, and four rows of alternating apical simple and plumose setae; outer plate heavily setose apically, with scattered plumose setae. Maxilliped inner plate broader than palp articles, width of plate increasing towards apex, moderately setose, with simple and robust tooth-like setae; outer plate ovate, with smooth edge, broader than palp articles, and moderately setose, with two long apical setae.

Pereon. Pereonites 1–7 with slightly lobed posterodistal corners, 5–7 with associated seta. Coxae 1–2 largely ovoid-shaped, coxa 1 with posterior margin straight rather than rounded as in coxa 2; coxae 3–4 subrectangular; coxae 5–6 with small anterior lobe and associated setae; coxa 7 simple and rounded; coxae 2–6 with large, simple oblong-shaped gills, around 2 or more times length of pereonites, coxa 6 gill shortest. Thoracic segments lacking sternal gills. Gnathopod 1 smaller than gnathopod 2; carpus around 3 times as long as broad with multiple rows or groups of long setae, longer than propodus; propodus with palm transverse, with row of robust bifurcated setae and row of short simple setae at palm corner and along palm margin. Gnathopod 2 carpus shorter than propodus with multiple rows of long setae; propodus just broader than carpus, distinct row of setae on lateral face, palm oblique with robust setae along palm margin, one long robust seta at palm corner. Pereopods 3–4 similar, bases moderately elongate, approximately 3.5 times as long as broad, not expanded posteriorly. Pereopods 5–7 lacking larger robust setae, each pereopod increasingly longer; bases expanded posteriorly, remaining longer than broad, with distinct lobe at posterodistal corner.

Pleon. Epimera 1–3 without scattered setae or spines along ventral margins and lateral face; epimera 1–2 with rounded posterodistal margins and associated seta; epimeron 3 posterodistal margin slightly squared and with associated seta. Uropod 1 sparsely setose; peduncle around 1.5 times length of rami, with one robust basofacial seta, lacking row of setae on dorsal margin; rami lacking setae along length. Uropod 2 smaller than uropod 1, sparsely setose; peduncle similar in length to inner ramus, lacking row of setae on dorsal margin; rami lacking setae along length, outer ramus shorter than inner ramus. Uropod 3 much larger than uropod 1 and strongly extended, parviramous; outer ramus flattened and leaf-shaped with two articles, first article longer than second; inner ramus short and scale-like. Telson longer than broad, almost entirely cleft into two lobes, with lateral setae and long apical penicillate setae.

• Etymology

Named for the very large (‘mega’) coxal gills (‘branchia’), which are distinct for the species.

• Remarks

Aengimata megabranchia sp. nov. appears to have a markedly restricted distribution, with specimens collected from only a single bore on Barrow Island, despite intensive sampling efforts over the last 20 years across the island. Nonetheless, one COI sequence (PQ497470) obtained from an external consulting company and included in the phylogenetic analysis was generated from an egg collected from an additional bore, Ledge01 (see Figure 1, Table S1), which suggests a wider distribution. However, to our knowledge, no additional specimens of A. megabranchia sp. nov. have been collected at this site. Molecular data revealed limited divergence between the Ledge01 COI sequence and the X62M A. megabranchia sp. nov. sequences (PQ497466–PQ497469), with only 0.15–0.3% divergence estimated, which is comparable to the level of divergence estimated among the X62M-only sequences.

4. Discussion

Here, we have described a new genus and one new species, Aengimata megabranchia gen. et sp. nov., of eriopisid amphipod from Barrow Island based on morphological and molecular information. This work increases the number of Australian eriopisid genera to seven (with four genera occurring exclusively within WA subterranean habitats), which comprise a total of 24 described species. Aenigmata gen. nov. has a narrow distribution [44,45], and only a single endemic species is recognised. This contrasts significantly with the more widespread distribution of species from the eriopisid genus Nedsia, which is prevalent across Barrow Island, North West Cape, and the Pilbara. Additionally, Nedsia is found in the same bore as A. megabranchia sp. nov., which has led to misidentification in past collections [25], while previous publications and reports relating to environmental impact assessment have referred to A. megabranchia sp. nov. as a species belonging to the family Melitidae (e.g., ‘sp. 4’ in [4]). This study, therefore, has important implications for the identification and documentation of the species collected in biological surveys for environmental impact assessment and monitoring. Moreover, previously described subterranean amphipod species on Barrow Island are listed as vulnerable and are specially protected under the WA Biodiversity Conservation Act 2016. Therefore, formal description allows for comparable status and conservation management of A. megabranchia sp. nov.