# Genetic variability of New Caledonian Boeckella De Guerne & Richard, 1889 (Copepoda: Calanoida), with description of a new species

Coline Royaux, Nicolas Charpin, Nicolas Rabet

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Coline Royaux, Nicolas Charpin, Nicolas Rabet. Genetic variability of New Caledonian Boeckella De Guerne & Richard, 1889 (Copepoda: Calanoida), with description of a new species. Journal of Crustacean Biology, 2024, 44 (1), pp.ruae001. 10.1093/jcbiol/ruae001. hal-04681440

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Distributed under a Creative Commons Attribution 4.0 International License
Running head: ROYAUX *ET AL.*: GENETIC VARIABILITY OF NEW CALEDONIAN
*BOECKELLA*

# Genetic variability of New Caledonian *Boeckella* De Guerne & Richard, 1889 (Copepoda: Calanoida), with description of a new species

Coline Royaux¹,², Nicolas Charpin³ and Nicolas Rabet¹

¹ Sorbonne Universités, Muséum national d'Histoire naturelle, UCN, UA, CNRS, IRD, Biologie des organismes et écosystèmes aquatiques, BOREA, UMR 8067, CP26 75231, 75005 Paris, France

²Muséum national d'Histoire naturelle, UMR BOREA, Station de Biologie Marine de Concarneau, 29900 Concarneau, France

³Association Vies d'Ô douce, 98809 Mont Dore, New Caledonia

Version of Record, first published online 23 January 2024, with fixed content and layout in compliance with Art. 8.1.3.2 ICZN. LSID: urn:lsid:zoobank.org:pub: ADD4CB9F-061D-4A52-9B56-59C3FD851791.

Correspondence: C. Royaux: e-mail: coline.royaux@mnhn.fr – royaux.c@gmail.com

## ABSTRACT

Thirteen new freshwater populations of the copepod genus *Boeckella* De Guerne & Richard, 1889 were found during three expeditions to New Caledonia ('La Planète Revisitée,' 2016-2018). The 12 populations from the Plaine des Lacs, which show remarkable genetic diversity among themselves, were identified as *B. spinogibba* Defaye, 1998, the only species of its genus known from New Caledonia until now. The sole exception, the population from Mont-Dore 22 km further east, appeared genetically and morphologically distinct from the others and is described herein as a new species. The two species are distinguished from each other by the shapes of the male and female P5, female Th5, and body colour. A previously published key is amended to separate the species. Our concatenated COI+28S phylogeny places the two New Caledonia species as a new branch within *Boeckella*, distinct from the branches consisting of South American, Antarctic and Australasian species. All 13 *Boeckella* populations inhabit the extreme south of New Caledonia, an area known for endemism and high heavy metal concentrations in the soil. Extensive mining activity in this metal-rich area, begun in 1873, is now leading to conflict with conservation goals. By using aerial
photographs to trace the fate of the pond that is the type locality of *B. spinogibba*, we confirmed that it disappeared between 2007 and 2014 as a result of the expansion of the Goro nickel mine.

**KEY WORDS:** *Boeckella spinogibba*, conservation, Crustacea, micro-endemism, freshwater zooplankton, mining perturbation, morphology, phylogeny, population genetics

INTRODUCTION

The main island of New Caledonia (Grande Terre) separated from Australia around 80 mya. It later became submerged until approximately 37 mya, when uplift caused the re-emersion of Grande Terre along with rocks from the upper mantle. Laterization of these mantellic rocks formed ultramafic massifs abundant in heavy metals across the island (Dubois *et al.*, 1974; Pelletier, 2007; Grandcolas *et al.*, 2008). Ultramafic massifs are mainly located in the southeast of New Caledonia (Fig. 1), a site of high endemism (Nattier *et al.*, 2013; Isnard *et al.*, 2016). Intensive mining of, e.g., nickel and cobalt, constitutes a major threat to the area's biodiversity (L'Huillier & Jaffré, 2010; Pitoiset, 2016).

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Localities sampled in New Caledonia during the three ‘La Planète Revisitée’ expeditions (2016–2018). Red represents localities where *Boeckella* was found.</figcaption></figure>
New Caledonia is globally acknowledged to be a major biodiversity hotspot despite its relatively small surface area (18,576 km²). The fauna of its lentic freshwater bodies is, however, relatively little-known. Freshwater ponds are diverse environments; the turbidity of the water, size of the pool, flora outside and inside, and average water temperature are some of the characteristics that vary over time and space and condition the fauna inhabiting them. The fauna of New Caledonia freshwater ponds includes such invertebrates as the coleopteran *Phalilus oberthuri* Guignot, 1935, the ostracod *Cyprinotus cingalensis* Brady, 1886, and the branchiopod *Lynceus insularis* Olesen, Pöllabauer, Sigvardt & Rogers, 2016 [Olesen *et al.* 2016] (Inventaire National du Patrimoine Naturel, 2023). When such bodies are temporary, their cyclic drying-out prevents most members of the aquatic fauna from colonizing them permanently so that most fishes and some macro-crustaceans are excluded. As planktonic crustaceans in temporary freshwater can produce diapausing eggs, they often represent a prominent fraction of biodiversity in these environments (Brock *et al.*, 2003).

The diversity of freshwater zooplankton remains inadequately studied globally, even more so in New Caledonia. To fill in knowledge gaps there, many temporary ponds and sinkholes all across Grande Terre were sampled during three ‘La Planète Revisitée’ expeditions between 2016 and 2018 (Fig. 1). Preliminary examinations of these samples identified many previously unknown populations of freshwater crustaceans. More specifically, the copepod genus *Boeckella* De Guerne & Richard, 1889 was exclusively found on ultramafic massifs known for hosting high endemicity. Only one species of *Boeckella* had previously been reported in New Caledonian temporary freshwater ponds: *B. spinogibba* Defaye, 1998. It was described and reported from a single locality on the ultramafic massifs of the south of Grande Terre (Defaye, 1998) and has not been further studied or sought for since.

We aimed to describe part of the taxonomic and genetic diversity of *Boeckella* in New Caledonia. This genus is currently composed of 42 valid species, including 23 from Australasia and 19 from South America, various sub-Antarctic islands, and Antarctica (Supplementary material Fig. S1; Maturana *et al.*, 2021; Walter & Boxshall, 2021). Extensive sampling conducted between 2016 and 2018 resulted in the identification of new populations of *Boeckella* in temporary ponds, one of which is geographically isolated in Mont-Dore, approximately 22 km away from the others, which inhabit ponds in the Plaine des Lacs. The morphological and genetic features of the *Boeckella* populations from both locations were investigated, including a population genetics analysis based on the cytochrome C oxidase I (COI) gene in these populations. The left antennule (A1) of *B. spinogibba* is redescribed.
Some of these populations are in ponds located in the RAMSAR area defined by the Convention on Wetlands (Fig. 1) and very close to the Goro mine, the largest nickel mine in New Caledonia. This facility is an open-pit complex where nickel and cobalt minerals are extracted. It has notably expanded since the description of *B. spinogibba* in 1998, and the effect of such expansion on the fate of the type locality of *B. spinogibba* is here tracked through time.

# MATERIALS AND METHODS

## Study sites and sampling

The survey of 469 ponds on Grande Terre was conducted between 2016 and 2018. Ponds filled with water were sampled using a plankton net (NHBS, Bonn, Germany) with a frame diameter of 25 cm and a 55 cm-long bag of 200 µm nylon mesh with a filter at the tip. The net was either tossed and pulled from the edge of the pond or towed while swimming in the pond. Samples were preserved in 80-90% ethanol at 4 °C as soon as possible to avoid alteration of genetic material. Individuals of *Boeckella* were found in 13 temporary ponds. Twelve of these were in the Plaine des Lacs (Figs. 1, 2A–E) in a small area of 2.5 km² at around 290 m elevation situated between 22°16.2’S and 22°15’S latitude and between 166°56.4’E and 166°57.6E longitude (sites HYNC918, HYNC920, HYNC925, HYNC927, HYNC931, HYNC4102, HYNC4103, HYNC4104, HYNC4105, HYNC4109, HYNC4110 and HYNC4111) (Table 1). The remaining pond was in Mont-Dore (site HYNC928, 22°12.9’S, 166°43.6’E, 117 m elevation; Figs 1, 2F–G).
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Sampled ponds in the Plaine des Lacs where species of *Boeckella* were found. Pond HYNC918 dry in November 2016 (A); filled in June 2018 (B). Pond HYNC920 in November 2016 (C); and in June 2018 (D). Pond HYNC931 in June 2018 (E). Sampled pond (HYNC928) in Mont-Dore where *Boeckella sibleti* sp. nov. was found (type locality). Dry in November 2016 (F); filled in June 2018 (G).</figcaption></figure>

<table><caption>**Table 1.** *Boeckella* sampling sites, coordinates and aliases.</caption><thead><tr><th>Locality (pond)<br>single identifier</th><th>Area in New<br>Caledonia</th><th>Latitude</th><th>Longitude</th><th>Other names<br>given to the<br>locality</th></tr></thead><tbody><tr><td>HYNC918</td><td>Plaine des Lacs</td><td>22°16.1982S</td><td>166°57.5412E</td><td>HYNC4101</td></tr><tr><td>HYNC920</td><td>Plaine des Lacs</td><td>22°15.7692S</td><td>166°57.126E</td><td>HYNC4005</td></tr><tr><td>HYNC925</td><td>Plaine des Lacs</td><td>22°15.6642S</td><td>166°57.1554E</td><td>HYNC4107;<br>Lac en long</td></tr><tr><td>HYNC927</td><td>Plaine des Lacs</td><td>22°15.726S</td><td>166°57.1938E</td><td>HYNC4108</td></tr><tr><td>HYNC931</td><td>Plaine des Lacs</td><td>22°15.5502S</td><td>166°56.901E</td><td>HYNC4106;<br>DT53</td></tr><tr><td>HYNC4102</td><td>Plaine des Lacs</td><td>22°15.5556S</td><td>166°56.4846E</td><td></td></tr><tr><td>HYNC4103</td><td>Plaine des Lacs</td><td>22°16.125S</td><td>166°56.4996E</td><td></td></tr><tr><td>HYNC4104</td><td>Plaine des Lacs</td><td>22°16.101S</td><td>166°56.8638E</td><td></td></tr><tr><td>HYNC4105</td><td>Plaine des Lacs</td><td>22°15.7542S</td><td>166°56.865E</td><td></td></tr><tr><td>HYNC4109</td><td>Plaine des Lacs</td><td>22°15.7182S</td><td>166°57.201E</td><td></td></tr><tr><td>HYNC4110</td><td>Plaine des Lacs</td><td>22°15.7686S</td><td>166°57.207E</td><td></td></tr><tr><td>HYNC4111</td><td>Plaine des Lacs</td><td>22°15.7176S</td><td>166°57.2382E</td><td>Doline croissante</td></tr><tr><td>HYNC928</td><td>Mont-Dore</td><td>22°12.93S</td><td>166°43.662E</td><td>HYNC4004</td></tr></tbody></table>
Morphological analysis

Specimens were dissected under a binocular dissecting microscope (SZX7, Olympus, Tokyo, Japan) and microscopically observed in glycerol or 96% ethanol. Photographs serving as the basis for drawings and measurements were taken with a digital camera (MC170 HD, Leica, Wetzlar, Germany) attached to an optical compound microscope (BX51, Olympus); the drawings and measurements were made using Inkscape 1.2.2 (The Inkscape Project; https://inkscape.org/) and Gimp 2.10 (GNOME Project; https://www.gimp.org/).

Some specimens from the Plaine des Lacs and Mont-Dore were prepared for SEM examination and underwent sequential dehydration in 96% and 100% ethanol followed by critical point-drying in liquid CO₂ (Emitech K850, Quorum Technologies, Lewes, UK). After mounting on flat aluminium stubs (12.7 mm diameter) with double-sided conductive carbon adhesive tape, specimens were sputter-coated with platinum (EM ACE600, Leica) and examined at 15 kV accelerating voltage and at 90–5500× magnification in a scanning electron microscope (SU3500, Hitachi, Tokyo, Japan).

Abbreviations used in the text: Th (1–5), thoracic segment; A1, antennules; Ur (1–5), urosomites; P (1–5), legs; B1, coxa; B2, basis; Re (1–3), exopod; Ri (1–3), endopod.

All the sampled material except for specimens used destructively for SEM or sequencing is preserved in ethanol and deposited in the Muséum national d'Histoire naturelle, Paris (see Supplementary material Fig. S2 for registration numbers of samples).

DNA extraction and PCR

Apart from site HYNC4105, where only a few individuals of *Boeckella* were found, DNA was extracted from 5–10 individuals from each pond where this genus was detected (90 individuals in total). Lysis was performed on whole bodies, and DNA was extracted from tissues by the NucleoSpin® 96 robot using the NucleoSpin® 96 Tissue Core Kit (Macherey-Nagel, Düren, Germany).

In order to amplify the mitochondrial gene COI (cytochrome C oxidase I), Folmer primers LCO1490 and HCO2198 (Folmer *et al.*, 1994) were used with the following PCR (polymerase chain reaction) cycle: 5 min at 94 °C; 40 cycles of 30 sec at 94 °C, 30 sec at 50 °C, 1 min at 72 °C, and 8 min at 72 °C. To ensure proper amplification of the sequences, PCR products were subjected to gel electrophoresis. The expected size of amplified DNA strands was 658 bp without primers. Detailed protocols are available on protocols.io (DNA
extraction: https://dx.doi.org/10.17504/protocols.io.j8nlk4x7wg5r/v2;
https://dx.doi.org/10.17504/protocols.io.261ge4y4jv47/v3).

PCR:

## Sequencing and phylogenetic analysis

Properly amplified DNA sequences were sent to Eurofins Scientific (Luxembourg City, Luxembourg) for Sanger sequencing (Sanger et al., 1977). The obtained sequences were cleaned, complementary strands were aligned, and consensus sequences were determined using Galaxy (https://ecology.usegalaxy.eu; The Galaxy Community, 2022).

To obtain more genomic information on individuals from New Caledonia, library preps from DNA amplified from one individual from the Plaine des Lacs (Site HYNC931; DNA sample identifier M156) and 50 individuals from Mont-Dore (DNA sample identifier M223) were performed with a Nextera XT kit (Illumina; https://www.illumina.com/) using fragmentation and Illumina index adapter ligation. Equimolar pools of each library were established. Qualification and quantification of the final library was established before sequencing on Illumina Miseq at Plateforme iGenSeq, Institut du Cerveau (ICM) (Hôpital Pitié Salpêtrière, Paris) with 2×25 million reads cartridge of 300 bases each (30–45 libraries per run).

All raw sequences were assembled and sequencing errors corrected using Geneious (Geneious® 11.1.4, Dotmatics; https://www.geneious.com/). The consensus sequences are available in GenBank (see Supplementary material Fig. S3 for NCBI accession numbers). In addition, gene concatenations were performed by adding the assembled genes from the present study to the concatenation (ribosomal RNA large subunit 28S+COI) published by Maturana *et al.* (2021) and retrieved in Genbank. In total, the phylogeny was computed on 40 individual sequence concatenations (Supplementary material Fig. S4). Apart from the two species from New Caledonia, 16 other species of *Boeckella* were included in the phylogeny. *Calamoecia ampulla* (Searle, 1911) and *Parabroteas sarsi* (Daday, 1901) were used as an external group, as Adamowicz *et al.* (2010) demonstrated *Calamoecia* Brady, 1906 and *Parabroteas* Mrázek, 1901 formed a monophyletic group closest to *Boeckella* in the family Centropagidae. Genes were independently aligned with Geneious alignment in Geneious 11.1.4 (Dotmatics). The 28S was delimited by hand. The COI sequences were aligned and verified for the absence of a stop codon in the reading phase. The COI alignment was then subjected to a Gblock performed with the default options on the LIRMM web platform (Dereeper *et al.*, 2008). The concatenation was realized on PhyloSuite v1.2.3 (Zhang *et al.*, 2020; Xiang *et al.*, 2023). Evolution models were estimated with distinct codon positions for
COI using ‘model finder’ in PhyloSuite 1.2.3 (Kalyaanamoorthy *et al.*, 2017; Zhang *et al.*, 2020; Xiang *et al.*, 2023).

The concatenated 28S+COI phylogeny was constructed from a Bayesian analysis performed with MrBayes 3.2.7 (Ronquist *et al.*, 2012) using the models TIM+I+G (28S), TIM+G, TRN+I, and F81+I for each codon position (COI). Maximum likelihood analysis was performed with IQTree 2.2.0 (Minh *et al.*, 2020) using the models TIM+I+G (28S), TIM+G, TRN+I, and F81+I for each codon positions (COI), always with 1,000 bootstrap resamplings. The multilocus phylogenetic tree was formatted using FigTree 1.4.4 (https://tree.bio.ed.ac.uk/software/figtree) and Inkscape 1.2.2 (The Inkscape Project).

Two species-delimitation models were performed on the bPTP server (Bayesian Poisson Tree Processes; https://species.h-its.org/), one on the concatenated 28S+COI phylogenetic tree and one on a COI phylogenetic tree (Supplementary material Fig. S5). Both analyses were calibrated with 100,000 MCMC generations and exclusion of the outgroup.

## Population structure of *Boeckella spinogibba*

To assess the genetic structure of the populations of *Boeckella spinogibba* found in the Plaine des Lacs, an exact test of population differentiation and an analysis of molecular variance (AMOVA) were performed using R 4.1.2 (CRAN) packages mmod 1.3.3 (Winter, 2012) and ade4 1.7-18 (Dray & Dufour, 2007), respectively. Significance of the AMOVA was computed through randomised matrix permutations. An alignment was also produced using Galaxy (mafft method, tool align seqs 1.9.1.0; Katoh *et al.*, 2002) in order to compute a median-joining haplotype network (Bandelt *et al.*, 1999) using R package pegas 1.2 (Paradis, 2010). Median-joining haplotype networks represent genetic links between haplotypes. The median-joining algorithm occasionally computes hypothetical haplotypes (referred to as ‘median vectors’), that could potentially be unsampled sequences, to resolve the network. The network was computed using several tolerance (epsilon) parametrisations in order to obtain several possible representations of the links between haplotypes. The tolerance parameter set in a median-joining network affects the search for median vectors. The augmentation of the tolerance parameter beyond 2 had little impact on the organisation of the network.

All sequence cleaning and analyses conducted in Galaxy have been implemented as reusable workflows at: https://usegalaxy.fr/u/coline_royaux/w/sanger1--from-ab1-to-aligned-fasta-imported-from-uploaded-file; https://usegalaxy.eu/u/coline_royaux/w/sanger-5--from-hand-corrected-haplotypes-table-to-population-haplotypes-map-and-euclidean-distance-matrix.
## History of type locality of *Boeckella spinogibba*

The coordinates of the type locality of *B. spinogibba* provided by Defaye (1998) are precise within about 2 km². Aerial photographs of the region in question were downloaded from the Observatoire de l'environnement Nouvelle-Calédonie (www.oeil.nc), and satellite images were obtained from Google Earth Pro 7.3.2. The description of the surrounding environment by Defaye (1998) and these aerial and satellite images allowed us to find the pond and monitor its condition from 1976 to 2014.

## TAXONOMY

**Order Calanoida** G.O. Sars, 1903

**Family Centropagidae** Giesbrecht, 1892

*Boeckella* De Guerne & Richard, 1889

*Boeckella spinogibba* Defaye, 1998

(Fig. 3)

*Material examined:* 2 males examined in SEM from *B. spinogibba* in pond HYNC931 (MNHN-UI-2017-2310, MNHN-UI-2017-2311); 5 males examined in photonic microscope from *B. spinogibba* in pond HYNC931 (4 individuals; MNHN-UI-2017-2310 and MNHN-UI-2017-2311) and in pond HYNC920 (1 individual; MNHN-UI-2017-2303).

*Morphological remarks:* On the right A1 of males, Defaye (1998) described five types of sensory structure: aesthetascs (Fig. 3A–C, G); thick setae (Fig. 3B, C); thin setae (Fig. 3C, G); setae with hairs (Fig. 3F); and pectinate setae (Fig. 3D, E). Detailed examination with SEM of specimens of *B. spinogibba* in pond HYNC931 of Plaine des Lacs (Table 2) showed that the distribution of these sensory structures on the right antennule of males is different from the original description by Defaye (1998). Namely, the original description made no mention of the thin seta on segments 3, 12, 17, and 19; the supplementary pectinate seta and the seta with hairs on segment 19; and the two thin setae on segment 20. No hairs were observed on the thin setae of segments 3, 5, 9, and 14, contrary to the original description, which mentioned such structures (Defaye, 1998).
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Types of sensory structures on male right antennule of *Boeckella spibogibba*. Lanceolate aesthetasc on segment 7 (A). Thick seta next to aesthetasc on segment 11 (B); long and short thin setae with two aesthetascs and two thick seta on segments 9–11 (C); pectinate seta on segment 19 (D); pectinate edge of pectinate seta on segment 18 (E); long seta on segment 19 with thin setules on distal third (F); short seta and tiny thin seta next to aesthetasc on segment 12 (G).</figcaption></figure>

<table><caption>**Table 2.** Sensory armature of each segment (seg.) of the right antennules of male *Boeckella spinogibba* and *Boeckella sibleti* sp. nov.</caption><thead><tr><th>Seg.</th><th>Structures</th><th>Seg.</th><th>Structures</th><th>Seg.</th><th>Structures</th><th>Seg.</th><th>Structures</th></tr></thead><tbody><tr><td>1</td><td>1 aesthetasc<br>1 thin seta</td><td>7</td><td>2 thin setae<br>1 aesthetasc</td><td>13</td><td>1 thin seta<br>1 aesthetasc</td><td>19</td><td>2 pectinate setae<br>1 aesthetasc<br>1 thin seta<br>1 long, setulose seta</td></tr><tr><td>2</td><td>3 thin setae<br>1 aesthetasc</td><td>8</td><td>1 thin seta<br>1 thick seta</td><td>14</td><td>2 thin setae<br>1 aesthetasc</td><td>20</td><td>2 thin setae<br>2 long, setulose setae</td></tr><tr><td>3</td><td>2 thin setae<br>1 aesthetasc</td><td>9</td><td>1 aesthetasc<br>2 thin setae</td><td>15</td><td>2 thin setae<br>1 aesthetasc</td><td>21</td><td>2 long, setulose setae</td></tr><tr><td>4</td><td>2 thin setae</td><td>10</td><td>1 thick seta</td><td>16</td><td>2 thin setae<br>1 aesthetasc</td><td>22</td><td>3 long, setulose setae<br>2 thin setae<br>1 aesthetasc</td></tr><tr><td>5</td><td>2 thin setae<br>1 aesthetasc</td><td>11</td><td>1 thick seta<br>1 aesthetasc</td><td>17</td><td>1 pectinate seta<br>1 thin seta</td><td></td><td></td></tr><tr><td>6</td><td>1 thin seta</td><td>12</td><td>2 thin setae<br>1 aesthetasc</td><td>18</td><td>1 pectinate seta<br>1 thin seta</td><td></td><td></td></tr></tbody></table>
*Boeckella sibleti* sp. nov.

(Figs. 4–6)

*Material examined:* Holotype, MNHN-UI-2017-2299, male. Allotype, MNHN-UI-2017-2300, female. Paratypes, MNHN-UI-2017-2298, 25 males; MNHN-UI-2017-2297, 20 females. Topotypes, MNHN-UI-2017-2301, MNHN-UI-2017-2302, >1,000 males (length, without furcal setae, 1.00–1.14 mm) and females (length, without furcal setae, 1.07–1.28 mm).

*Type locality:* HYNC928, a pond near the Pirogues River in Mont-Dore, Southern Province, New Caledonia, 22°12.9′S, 166°43.68′E, elevation 117 m, collected 5 June 2018. The pond occupies a pseudo-karstic formation called a ‘doline’ or ‘sinkhole’ (Fig. 2F-G). Its water is rather heavily tannic and the surrounding extensive area of shrubby vegetation includes a large number of niaouli trees (bark tea tree, *Melaleuca quinquenervia*, Blake).

*Etymology:* Named in honour of Jean-Philippe Siblet, a fervent defender of nature conservation.

*Colouration:* Body red in both sexes.

*Male:* Holotype, MNHN-UI-2017-2299, length without furcal setae 1.13 mm. Body elongated as a tapered droplet. Cephalothorax 6-segmented with cephalosome longest, 5 free thoracic segments (Th1, closest to cephalosome, to Th5, closest to urosome) successively shorter towards urosome (Fig. 4A).

Antennules (A1) asymmetrical as in *B. spinogibba* (Defaye, 1998: figs. 1F, 4C); left one 25-segmented, reaching tips of caudal rami; right one 22-segmented, slightly longer than left, with geniculations between 18, 19th and 19, 20th segments (Fig. 4B). Mouthparts as in *B. spinogibba* (Defaye, 1998: figs. 2, 3A, 5A).

Th5 (Fig. 5A) symmetrical, with each ‘wing’ ending in pointed lobe nearly reaching posterior end of first urosomite. Wing ending with small terminal spine and bearing second small spine towards posterior margin.

Natatory legs P1 to P4 identical to those of *B. spinogibba* (Defaye, 1998: fig. 5B–E).

Urosome with 5 urosomites successively decreasing in length towards furca from Ur1 to Ur4 but with Ur5 longer than Ur4 (Fig. 4A, 5A).

P5 (Fig. 5B–D) asymmetrical, with left limb’s coxa (B1) twice as long as that of right limb. Left P5: Basis (B2) with medially swollen, finely denticular protrusion on inner margin, overlapping proximal margin of exopod (Re1), latter with distally denticulate terminal lateral spine. Endopod (Ri) atrophied but bearing fine hairs, inserted beneath B2’s protrusion before Re1. Re2 with proximolateral distally denticulate spine. Re3 elongated, spine-like with slight medial curve, denticulate, pointed at apex. Right P5: B2 without medial protrusion, Ri
elongated, digitiform, swollen at midlength, medially curved, rounded terminally. Re1 approximately half as long as Re2, bearing distal spine armed with approximately uniformly-sized denticles. Re2 with small medial bulge on inner margin and large, distally denticulate distolateral spine of almost equal length to segment. Re3 elongated, medially curved at midlength with slight medial bulge, ending in pointed apex.

*Female:* Allotype, MNHN-UI-2017-2300. Larger than male, length without furcal setae 1.23 mm.

Body shape slender, elongated. Cephalothoracic tagmosis as in male (Fig. 6A).
Mouthparts as in *B. spinogibba* (Defaye, 1998: figs. 2, 3A).
<figure><img src="image_5.png" /><figcaption>**Figure 4.** *Boeckella sibleti* sp. nov., male. Whole body without antennules, posterior view (A); right antennule, with thick setae and pectinate setae labelled, aesthetascs stippled, and long, setulose setae drawn with hairs, all others are thin setae; geniculation points indicated by black arrows (B). Scale bars = 100 µm.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 5.** *Boeckella sibleti sp. nov.*, male. Th4, Th5, and urosome, viewed from right side (A); P5, anterior view (B); left B2, Ri, and Re1, posterior view (C); distal part of left Re2, posterior view (D).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 6.** *Boeckella sibleti sp. nov.*, female. Whole body without antennules, posterior view (A); genital operculum with part of spermatophore, anterior view (B); Th3, Th4, Th5, and urosome with spermatophore and genital operculum, viewed from right side (C); P5, anterior view (D). Scale bars = 100 µm.</figcaption></figure>
Antennules 25-segmented, extending beyond caudal rami, identical to those of *B. spinogibba* (Defaye, 1998: fig. 1F).

Left, right lateral 'wings' of Th5 ending in 2 lobes (Fig. 6A, C), and each lobe bearing small spine on outer margin.

Natatory legs P1-P4 identical to those of *B. spinogibba* (Defaye, 1998: fig. 5B-E).

Urosome 3-segmented. Urosomite 1 (Ur1) longest, widest on medial transversal axis, narrowing towards anterior and posterior margins from posterior view (Fig. 6A), asymmetrical anterior genital operculum, latter with wide anterior part, thinner posterior part in anterior view (Fig. 6B). Ur1 also bearing spermatophore (Fig. 6C).

P5 (Fig. 6D) symmetrical, distolateral spines of Re1-2 equal in length. Distomedial spiniform process of Re2 extending beyond Re3, Re3 armed with small distolateral spine and 2 longer terminal spines, medial one of which longest and medially curved. Ri 3-segmented; Ri3 bearing 4 spines with fine hairs.

*Remarks:* The body of *B. sibleti sp. nov.* is red, whereas that of *B. spinogibba* is blue. The body shape of males is similar in both species, and the mouthparts and swimming legs appear identical between the species. The main differences are found in P5 of both sexes (Fig. 7), and, for the female, Th5 (see Table 3).

<figure><img src="image_8.png" /><figcaption>**Figure 7.** SEM images of male copulatory organ (P5) of *Boeckella sibleti sp. nov.* (A) and of *B. spinogibba (B)*. Major differences listed in Table 3 are indicated with a white arrow and a numeric identifier.</figcaption></figure>
<table><caption>**Table 3.** Morphology-based differential diagnosis of *Boeckella spinogibba* and *B. sibleti* sp. nov. See Scheihing *et al.* (2010: 47, fig. 2) for abbreviations of parts of legs. Numbers (no.) given to differences shown in Figure 8 of text are provided for male P5.</caption><thead><tr><th></th><th></th><th>Boeckella spinogibba</th><th>Boeckella sibleti</th><th>Numbers in Figure 8</th></tr></thead><tbody><tr><td rowspan="2">Female</td><td>Th5</td><td>Right</td><td>Ending in two lobes of same length</td><td>Ending in two lobes with ventral lobe slightly longer</td></tr><tr><td></td><td>Left</td><td>Ending in one lobe with a slight swelling bearing a small spine on the dorsal side</td><td>Ending in two lobes</td></tr><tr><td>P5</td><td>Endopodite 3</td><td>3 spines</td><td>4 spines</td><td></td></tr><tr><td rowspan="5">Male</td><td rowspan="5">P5</td><td>Right</td><td>Short spines on Re1 and Re2<br>Ri thin and long<br>Re3 slightly curved<br>No slit</td><td>Longer spines on Re1 and Re2<br>Ri thick and long<br>Re3 highly curved like a hook<br>Inner margin of Re2 with a small slit</td><td>no. 1, 2<br>no. 3<br>no. 4<br>no. 5</td></tr><tr><td>Left</td><td>Round process on B2 rather flat and bonded to B2</td><td>Round process on B2 rather plump and detached from B2</td><td>no. 6</td></tr></tbody></table>

Both *B. spinogibba* and *B. sibleti* sp. nov. bear three spines on the Re3 of the female P5 (according to Defaye, 1998: 697), this is ‘the usual setation pattern of [this exopodite in] *Boeckella*.’ By contrast, most of the Australian species of *Boeckella* (*B. pilkililli* Quinlan & Bayly, 2017, *B. montana* Bayly, 1964, *B. opaqua* Fairbridge, 1945, and *B. triarticulata* Thomson, 1883) bear five to seven spines on this segment (Bayly, 1964; Quinlan & Bayly, 2017).

The female P5 in *B. spinogibba* has three spines on the Ri3, whereas the females of *B. sibleti* sp. nov. have four spines. By contrast, Australasian species usually bear four to six spines on the Ri3 in females (Bayly, 1964; cf. *B. pilkililli* Quinlan & Bayly, 2017 and *B. shieli* Bayly, 1985; Quinlan & Bayly, 2017: fig. 4D; Bayly, 1985: fig. 1E).

## Phylogenetic analysis

Among the 90 DNA samples submitted to PCR, 84 were successfully amplified, sequenced, and cleaned. The haplotypes and NCBI accession numbers are provided in Supplementary material Fig. S3 (OQ117983–OQ118066). Pairwise genetic distances between each of the B_spi_COI01–B_spi_COI13 haplotypes and the single Mont-Dore haplotype (B_sib_COI01) ranged 8.51%–9.73%. Pairwise genetic distances among the B_spi_COI01–B_spi_COI13 haplotypes of 74 individuals were 2.9% or less. A unique haplotype was found at Mont-Dore in 10 individuals (Fig. 8, Supplementary material Fig. S2).
<table><caption>**Figure 8.** Matrix of cytochrome C oxydase I (COI) haplotype uncorrected genetic distance (%). All haplotypes of *Boeckella spinogibba* (Plaine des Lacs) and *B. sibleti* sp. nov. (Mont-Dore) are represented.</caption><thead><tr><th colspan="2"></th><th colspan="10">B. spinogibba haplotypes from the Plaine des Lacs</th></tr><tr><th></th><th></th><th>B_spi_COI01</th><th>B_spi_COI02</th><th>B_spi_COI03</th><th>B_spi_COI04</th><th>B_spi_COI05</th><th>B_spi_COI06</th><th>B_spi_COI07</th><th>B_spi_COI08</th><th>B_spi_COI09</th><th>B_spi_COI10</th><th>B_spi_COI11</th><th>B_spi_COI12</th><th>B_spi_COI13</th></tr></thead><tbody><tr><td rowspan="10">B. spinogibba haplotypes from the Plaine des Lacs</td><td>B_spi_COI02</td><td>2.13</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>B_spi_COI03</td><td>2.89</td><td>1.67</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>B_spi_COI04</td><td>2.43</td><td>1.22</td><td>1.98</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>B_spi_COI05</td><td>2.28</td><td>0.76</td><td>1.82</td><td>1.06</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>B_spi_COI06</td><td>2.74</td><td>2.13</td><td>1.37</td><td>2.43</td><td>2.28</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>B_spi_COI07</td><td>2.43</td><td>0.91</td><td>1.98</td><td>1.22</td><td>0.15</td><td>2.43</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>B_spi_COI08</td><td>2.58</td><td>1.37</td><td>2.13</td><td>0.15</td><td>1.22</td><td>2.58</td><td>1.37</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>B_spi_COI09</td><td>1.98</td><td>0.15</td><td>1.82</td><td>1.37</td><td>0.91</td><td>2.28</td><td>1.06</td><td>1.52</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>B_spi_COI10</td><td>0.61</td><td>2.13</td><td>2.89</td><td>2.43</td><td>2.28</td><td>2.43</td><td>2.43</td><td>2.58</td><td>1.98</td><td></td><td></td><td></td><td></td></tr><tr><td>B_spi_COI11</td><td>0.46</td><td>1.98</td><td>2.74</td><td>2.28</td><td>2.13</td><td>2.58</td><td>2.28</td><td>2.43</td><td>1.82</td><td>0.15</td><td></td><td></td><td></td></tr><tr><td>B_spi_COI12</td><td>2.28</td><td>0.15</td><td>1.82</td><td>1.37</td><td>0.76</td><td>2.28</td><td>0.91</td><td>1.52</td><td>0.30</td><td>2.28</td><td>2.13</td><td></td><td></td></tr><tr><td>B_spi_COI13</td><td>2.28</td><td>0.15</td><td>1.82</td><td>1.37</td><td>0.91</td><td>2.28</td><td>1.06</td><td>1.52</td><td>0.30</td><td>2.28</td><td>2.13</td><td>0.30</td><td></td></tr><tr><td colspan="2">B. sibleti sp. nov. haplotype from Mont-Dore</td><td>8.51</td><td>8.97</td><td>8.66</td><td>9.57</td><td>9.12</td><td>8.66</td><td>8.97</td><td>9.73</td><td>9.12</td><td>8.51</td><td>8.66</td><td>9.12</td><td>8.81</td></tr></tbody></table>

The consensus sequences for 28S were established with 2,087 reads for *B. sibleti* sp. nov. (M223; NCBI accession: OR612784) and 545 reads for *B. spinogibba* (M156; NCBI accession: OR612785).

The concatenated tree (28S+COI) based on the concatenation used in Maturana *et al.* (2021) has been reproduced with the additional concatenated sequences from New Caledonia (Fig. 9, Supplementary material Fig. S4). One concatenated sequence, identified as 'Hap_19' in Maturana *et al.* (2021), was excluded from the original data because, after verification, this sequence appeared likely to be a chimeric concatenation of two distinct species. Originally identified as *B. brevicaudata* (Brady, 1875), this sequence has been corrected in NCBI (accession MZ757027.3) since the publication of Maturana *et al.* (2021) and is now identified as *B. vallentini* (Scott, 1914). The final alignment obtained includes 1045 bp (673 for 28S, 372 for COI). The results are very similar to those obtained by Maturana *et al.* (2021) except that, in addition to the Australia/New Zealand group and the group of *Boeckella* from South America, Antarctica, and various sub-Antarctic Islands, the two New Caledonian
species together form a third robust monophyletic group within this genus.

<figure><img src="image_9.png" /><figcaption>**Figure 9.** Concatenated phylogeny (28S+COI) of *Boeckella* in New Caledonia, other *Boeckella* species from South America, Antarctica, Australia, and New Zealand, and the outgroup *Calamoecia ampulla* - *Parabroteas sarsi* (nodes: probability support values according to Bayesian/bootstrap value for Maximum Likelihood).</figcaption></figure>

The raw results from the two bPTP analyses are available in Supplementary material Figure S6), and the COI phylogenetic tree used for one of the analyses is also available there (Supplementary material Fig. S5). *Boeckella spinogibba* and *B. sibleti* sp. nov. are distinct in the two models, with good support.
Population structure

The AMOVA test performed on populations of *B. spinogibba* from the Plaine des Lacs showed these populations had a genetic variance rate of 17.24% within ponds and 82.76% among ponds (P = 0.001). Exact tests of population differentiation showed that the *Boeckella* populations were generally significantly genetically differentiated among themselves.

The haplotype network of *B. spinogibba* (Fig. 8, 10) shows the mutations, distribution, and abundance of haplotypes in the sampled localities. The network presented in Figure 10 has a tolerance parameter of 2; this network was chosen for its closeness to the distance matrix (Fig. 8). For the sake of clarity, the network is presented without alternative links, and six median vectors have been removed as they do not directly interact with several of the sampled haplotypes (see Supplementary material Fig. S7 for the network with all alternative links and median vectors). The network on Supplementary material Fig. S7 has a tolerance parameter of 1. Its main difference with Figure 10 concerns the link between haplotypes B Spi COI03 and B Spi COI06. The median-joining network was unable to resolve the links between these two haplotypes and the rest of the network as regards the real number of mutations between the sequences (e.g., 18 mutations between B Spi COI01 and B Spi COI06 versus 22 mutations displayed on Fig. 10 and 30 displayed in Supplementary material Fig. S7).
<figure><img src="image_10.png" /><figcaption>**Figure 10.** Haplotype network of 74 individuals of *Boeckella spinogibba* from 11 ponds in the Plaine des Lacs; haplotype names, originally of the form *B_spi_COIXX*, are abbreviated to hXX for the purpose of this representation (A). Satellite images and locations of sampled localities where species of *Boeckella* were found (B). Closer view of satellite images of locations where *B. spinogibba* was found in the Plaine des Lacs (outlined by a white rectangle in Fig. 10B). Localities are represented with colours and patterns corresponding to those in the haplotype network (Fig. 10A), except locality HYNC4105, which is represented in grey as no sequences were obtained from copepods from this pond (C).</figcaption></figure>

The pond at site HYNC4102 displayed the highest haplotype richness (four haplotypes), whereas six other ponds contained only one haplotype each (sites HYNC918, HYNC925, HYNC927, HYNC928, HYNC931, and HYNC4109; Supplementary material Fig.
S2). The median-joining model inferred four main haplotypes, missed in the sampling (white diamonds in Fig. 10), being needed to articulate the structure of the network: one between haplotypes B_spin_COI1 and B_spin_COI11; one between B_spin_COI2, B_spin_COI4, and B_spin_COI5; one between B_spin_COI3 and B_spin_COI6; and one at the centre of the network between the B_spin_COI3-B_spin_COI6 and the B_spin_COI1-B_spin_COI11-B_spin_COI10 haplotype complexes. The other missing haplotypes inferred by the model support alternative links (black diamonds in Fig. 10, Supplementary material Fig. S7).

## History of the type locality of *Boeckella spinogibba*

Defaye (1998: 687) described the type locality of *B. spinogibba* at the time of collection in 1996 as 'a small pond in the Plaine des Lacs [...] on the route between Grand Lac and the fork to Parc Boisé, the pond nearest to the fork.' As the area has been damaged by the Goro mine, roads on recent satellite images (Fig. 11C) are very different from those in 1996. Aerial photographs nonetheless allowed the pond to be located at 22°17.652'S, 166°58.044'E, while also showing that it was filled in by the mine between 2007 and 2014 (filled with water: Fig. 11A, B; filled with tailings from the mine: Fig. 11C).

<figure><img src="image_11.png" /><figcaption>**Figure 11.** Aerial photographs and satellite images of the type locality of *Boeckella spinogibba* in the Goro mine area, New Caledonia in 1992 (A), 2007 (B), and 2014 (C).</figcaption></figure>
DISCUSSION

*Boeckella* in New Caledonia

The genus *Boeckella* is generally considered a Gondwanan taxon (Bayly & Morton, 1978; Bayly, 1992; Maturana *et al.*, 2021) with three centres of distribution. It occurs in South America from the Andes Mountains in Colombia to the plains of southern Chile and Argentina, on sub-Antarctic islands and in coastal Antarctica; and in Australasia, especially in South Australia and New Zealand (Maturana *et al.*, 2019). *Boeckella triarticulata* Thomson, 1883 in Mongolia (Bayly, 1992) and Italy (Alfonso & Belmonte, 2008) is the only species present outside the Gondwanan area. Our concatenated COI+28S phylogeny places the two New Caledonian species as a monophyletic branch in the phylogeny of the genus, distinct from other such groups composed of South American and Australian species (Fig. 9). This suggests that the evolutionary history of the genus is more complex than previously suspected and merits further investigation.

No further information has been published on *Boeckella* in New Caledonia since the original observation of *B. spinogibba* from a single pond by Defaye (1998). We, however, found several populations of the species in the Plaine des Lacs which permitted the redescription of the left male antennule (A1) (Fig. 3) and the acquisition of genomic information on the species. We also found a population of *Boeckella* at Mont-Dore, 22.5 km from the nearest originally sampled population of *B. spinogibba*, that showed 8-9.6% genetic divergence from the latter in the COI gene and clear morphological differentiation, particularly in the male fifth leg, the copulatory organ (Fig. 7, Table 3) and its different body colour. According to the principles of integrative taxonomy (Padial *et al.*, 2010), several criteria, including evidence of genetic, morphological, and geographical differentiation, must be fulfilled to confirm that the species are distinct. Geographically, although the present two species are separated from each other, they are yet close enough to possibly indicate micro-endemism, something that is common in New Caledonia among, for example, plants and insects (Caesar *et al.*, 2017) and freshwater invertebrates such as gastropods (Haase & Bouchet, 1998). Such phenomenon seems to be particularly prominent on ultramafic soils (Pillon *et al.*, 2021), which is consistent with the unusual environmental conditions in New Caledonia, notably a high pH and high concentrations of heavy metals in the surface waters (Marques *et al.*, 2008). In New Caledonia, *Boeckella* has been found only in temporary freshwater ponds on ultramafic soils despite a fairly large amount of sampling in other types of ponds. This is a peculiar habitat, with water that is toxic for most plant and animal species and particularly poor in nutrients (Kierczak *et al.*, 2021). We still lack enough data on the
distribution of *Boeckella* in New Caledonia to confirm the extent of its micro-endemism.

The body colouration of copepods is mainly due to carotenoids, and freshwater copepods tend to have higher carotenoid concentrations than marine copepods. As highlighted in the review by Vilgrain *et al.* (2023), body colouration can vary widely in individuals depending on biotic and abiotic factors, making its study challenging. *Boeckella spinogibba* is blue, which is typical for copepods occurring at the water surface where this colour may be useful for camouflage, especially so for *B. spinogibba*, which lives in ponds with bright blue water (Fig. 2D). Red colouration like that of *B. sibleti sp. nov.* occurs in copepods from all ecosystems (marine and freshwater, shallow and deep) and can provide many physiological and ecological advantages, including, protection from UV light, antioxidant activity, and higher reproduction rates (Powers *et al.*, 2019; Vilgrain *et al.*, 2023).

Genetic divergence can occur over time when connectivity and the genetic flows that depend on it are no longer maintained, (Rundle & Nosil, 2005). Indeed, poor connectivity between different populations can lead to speciation (Schluter, 2001). The small size and weight of freshwater zooplankton permits their transportation, either as eggs, larvae, or adult individuals, by other animals, mostly wading birds, which may get eggs stuck in their feathers or ingest them accidentally (Incagnone *et al.*, 2015; Fontaneto, 2019; Parry *et al.*, 2023). In New Caledonia, *Boeckella* has only been found in ultramafic ponds, which are mostly oligotrophic if not dystrophic, with low prey density. Wading birds occur where there is a high density of prey (Bryant, 1979; Herteux *et al.*, 2019), which greatly limits the potential for dispersion of zooplanktonic species from these ultramafic ponds by birds. Moreover, *B. spinogibba* showed high genetic variability within a very small, 2.5 km² area, with up to 2.9% COI differentiation between individuals from different ponds and with highly structured populations in terms of haplotype distribution (Fig. 8, 10). AMOVA revealed that 83% of the observed genetic variance is attributed to variance among ponds, with only 17% being within ponds. The exact test also identified significant genetic divergence among ponds. As it has also been observed in *B. poppei* populations from Signy Islands, South Orkney Islands (Maturana *et al.*, 2020), these results tend to confirm weak connectivity for this genus in New Caledonia, which can support the hypothesis of an allopatric speciation between *B. spinogibba* and *B. sibleti sp. nov*.

Updated key by Quinlan & Bayly (2017)

Quinlan & Bayly (2017) updated the key of Australasian *Boeckella* by including *B. spinogibba* at couplet 18, using characters that are similar in *B. sibleti sp. nov.* as well. To
incorporate the new species in the key, couplet 18 is left unchanged but it now points towards a newly created couplet 24 for the two New Caledonian species, not just to *B. spinogibba*.

‘18. Left Ri small, reduced to a triangular plate and hidden by a rounded process covered in thick spiniform hairs; left B1 noticeably longer than right B1’.... 24

24. Right Re3 with acute claw-like, rounded shape with bulge at mid-length disto-lateral spine of Re1 approximately two-thirds as long as Re2 .... *B. sibleti* sp. nov.

Right Re3 slightly curved; disto-lateral spine of Re1 less than one-third as long as Re2
.... *B. spinogibba* Defaye, 1998

Impact of Goro mine

The pond that was the type locality of *B. spinogibba* was destroyed and filled in between 2007 and 2014 as a result of expansion of the Goro mine (Fig. 11). This event exemplifies the threat that mines pose for ponds and other freshwaters inside and outside their enclosures. Along with the destruction of the environment to build roads and other infrastructure and to extract minerals, mines in New Caledonia produce clouds of fine tailings that fill ponds outside the exploited perimeter. The open-pit complex of the Goro mine is very close to the present populations of *B. spinogibba* and the southern border of the RAMSAR area. According to Jeanpert *et al*. (2016), about 8,500 ponds occupied the 148 km² area around the future mining site in 2006 before the construction of the mine. Supplementary studies are needed in such areas, in particular in the RAMSAR area, where ponds and many other habitats are threatened by mining activities (Pascal *et al.*, 2008).

Perspectives and limitations

New Caledonia has been extensively sampled, but ultramafic soils are often less accessible.
Our discovery of a new species in Mont-Dore suggests that other still unknown species may
be found in unexplored parts of the Plaine des Lacs and elsewhere in New Caledonia.

The concatenated phylogeny computed in this study was mainly constructed from
sequences concatenated by other workers, and the sources did not specify which individual
copepods provided the sequences. Hence, there is a possibility that COI and 28S sequences of
different individuals were concatenated for some portion of the Australian, New Zealand,
Antarctic, and South American species. Because the mitochondrial marker (COI) we used
only provides information about the maternal lineage and only two nuclear (28S) sequences
were retrieved from shotgun sequencing, further exploration of the genetic structure of
*Boeckella* populations in New Caledonia, notably using other genes, is desirable.
Microsatellite sequences of populations and species could also be used to test the hypothesis
of isolation by distance and to help assess the dispersal potential of *Boeckella* in New
Caledonia (Teske *et al.*, 2018).

SUPPLEMENTARY MATERIAL

S1 Figure. Table of present *Boeckella* species and associated regions.

S2 Figure. Distribution of amplified *Boeckella sibleti* sp. nov. and *B. spinogibba*.

S3 Figure. Accession numbers for sequences obtained for *Boeckella sibleti* sp. nov. and *B. spinogibba*.

S4 Figure. Accession numbers for sequences used in producing the 28S+COI phylogeny after
Maturana *et al.* (2021).

S5 Figure. COI phylogeny of *Boeckella* in New Caledonia, other *Boeckella* species, and the
outgroup *Calamoecia ampulla*.

S6 Figure. Raw results from bPTP analyses.

S7 Figure. Haplotype network with all alternative links and median vectors of 74 individuals
of *B. spinogibba*.

ACKNOWLEDGEMENTS

The New Caledonia Hydrobiological expeditions 2016–2018 were conducted under the ‘La Planète Revisitée’ program implemented by the Muséum national d’Histoire naturelle (MNHN), Paris in partnership with the Conservatoire d’Espaces naturels (CEN). We deeply thank Pascale Joannot, head of the expeditions program at MNHN, and Phillippe Bouchet, the expedition leader. These expeditions were operated under permits issued by New Caledonia’s Province Sud (under APA_NCPS_2017_028) and Province Nord, and the organizers thank Emmanuel Coutures and Isabelle Jurquet (Province Sud) and Jean-Jérôme Cassan and Yannick Monlouis (Province Nord) for their help in obtaining these permits. We thank Sébastien Faninoz and Alice Leblond. We also thank Céline Bonillo for her help with DNA extractions and PCR, and Marion Mennesson for the idea of using a haplotype network and AMOVA to represent the genetic diversity of *Boeckella spinogibba*. We are grateful to Géraldine Toutirais (Plateau technique de Microscopie Électronique (PTME) du Muséum national d’Histoire naturelle) for her help in the acquisition of SEM images. We also thank Valentin de Mazancourt for sharing photographs of the dry Mont-Dore pond and Claudia Maturana for sharing genetic concatenation data and for providing much advice about her methodology. Finally, we thank the editors and anonymous reviewers for improvements to the manuscript. Author contributions: CR (text, laboratory manipulations, data cleaning and
analysis), N.R (sampling, phylogeny, SEM images, some field photography, and detailed
review of the text); NC (sampling and information and photos of habitats). Funding for
molecular biology manipulations were granted to NR by Sorbonne Université through the
Emergence (2017-2018) call for projects. Funding for ‘La Planète Revisitée’ expeditions
were provided by the Gouvernement de la Nouvelle-Calédonie, Province Sud, Province Nord,
Office des Postes et Télécommunications (OPT), Maison de la Nouvelle-Calédonie, and the
Ministère Français des Outre-mer.

Data availability statement

AB1 files from sequencing and datasets generated during and/or analysed during the current
study are available in the Data.InDoRes repository
[https://doi.org/10.48579/PRO/DGVWPW].

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