Chelarctus and Crenarctus (Crustacea: Scyllaridae) from Coral Sea waters, with molecular identification of their larvae

Abstract Chelarctus Holthuis, 2002 is widely distributed throughout the Indo-West Pacific, but its biogeographic patterns are unknown because Southern Hemisphere areas, such as the Coral Sea, remained poorly explored. Recent cruises organized by the Muséum national d'Histoire naturelle of Paris and the Australian Institute of Marine Science allowed the molecular identification of Crenarctus crenatus (Whitelegge, 1900), Chelarctus aureus (Holthuis, 1963) and Chelarctus crosnieri Holthuis, 2002 phyllosomae. The Coral Sea C. crenatus larvae are identical to stages IX and X of Scyllarus sp. Z, described in detail by Webber and Booth (2001). Descriptions of phyllosoma stages VI, IX and X of Ch. aureus and stages IX and X of Ch. crosnieri are also presented here. Morphological differences between Crenarctus and Chelarctus larvae are established for the first time and previous misidentifications in the literature are re-assessed.


Introduction
Although biogeographic barriers are well defined for terrestrial species (Lohman et al. 2011) comparatively fewer studies have addressed Indo-West Pacific (IWP) marine boundaries, which require clarification. Plate tectonics and sea level changes have been claimed to account for phylogeography patterns in the area (Hou & Li 2018), but historical factors and spatial boundaries may not equally affect shallow water (Lourie & Vincent 2004;Palero et al. 2016a) and deep-water taxa (Tsoi et al. 2011). Previous studies usually focused on limited areas or bathymetric ranges, however, further taxa should be analysed to better understand IWP marine biogeography (Lourie et al. 2005;Barber et al. 2006;Kochzius & Nuryanto 2008). The Indo-West Pacific biodiversity hotspot (Hall 2002) hosts representatives from almost every slipper lobster genus, a group of decapod crustaceans expanding from shallow waters to the continental slope (Holthuis 1991). Slipper lobsters (Scyllaridae Latreille, 1825) are a well-established monophyletic family characterized by their flattened distal antennal article (Holthuis 1985;Haug et al. 2016), but relationships between genera and species still remain unresolved Bracken-Grissom et al. 2014). The existence of slipper lobster species yet to be described is ensured by their cryptic coloration and small size, together with the fact that they occupy habitats such as coral reefs and underwater caves.
Chelarctus Holthuis, 2002 includes a total of four species and it is widely distributed throughout IWP waters, occupying hard and muddy bottoms at depths down to >300 m. The northernmost species, Ch. virgosus , inhabits shallow areas (<50 m) from Japan and northern Taiwan. Chelarctus cultrifer (Ortmann, 1897) and Ch. aureus (Holthuis, 1963) are mostly distributed along continental slopes (50-250 m) of the Coral Triangle and the Tropical Southwestern Pacific (TSWP) provinces, respectively (Holthuis 2002). Molecular data recently revealed two genetic clusters within Ch. cultrifer, but morphological analyses did not establish specific status to these groups . Furthermore, Ch. crosnieri Holthuis, 2002 occupies deeper waters (>250 m) from the southern hemisphere and it is only known from a few specimens (Figure 1). Another genus found in shallow waters of the IWP is Crenarctus Holthuis, 2002, which currently includes Cr. bicuspidatus (Ortmann, 1897), distributed from Japan to New Caledonia, and Cr. crenatus (Whitelegge, 1900) from southeast Australia (Holthuis 2002). Recent molecular results and adult morphology suggest that the eastern Pacific species Acantharctus delfini (Bouvier, 1909) should be assigned to Crenarctus (Genis-Armero et al. 2020). Campaigns carried out by the Muséum national d'Histoire naturelle, Paris (MNHN) in the Vanuatu archipelago (SANTO 2006), Papua New Guinea (BIOPAPUA 2010and PAPUA NIUGINI 2012 and New Caledonia (KANADEEP 2017) have now provided new regional data (Chan 2012;De Forges & Corbari 2012;Pante et al. 2012;Zaharias et al. 2020). In parallel, the Australian Institute of Marine Science (AIMS) launched several cruises to collect marine plankton from the Coral Sea area. These cruises have successfully collected hundreds of scyllarid phyllosoma (Palero et al. , 2016b, most of which await study. The phyllosoma is the larval form of slipper and spiny lobsters, particularly adapted to planktonic life and long-distance dispersal (Palero & Abello 2007). Difficulties distinguishing these larvae make generic identifications based on morphological highly unreliable. For example, Higa and Shokita (2004) have assigned putative Cr. bicuspidatus (De Man 1905) phyllosoma from previous works to Ch. cultrifer, but Wakabayashi et al. (2020) suggest that Ch. cultrifer phyllosoma described by Higa and Shokita (2004) and Inoue and Sekiguchi (2006) belong in fact to Ch. virgosus. Ueda et al. (2021) recently studied Chelarctus larvae from northern and central Pacific waters but, apparently due to the poor status of their specimens, illustrations and descriptions were deficient and their morphological results remain inconclusive.
The purpose of the present study is to identify Coral Sea phyllosoma samples collected during cruises 5441 and 5160 (AIMS) and the KANADEEP 2017 cruise (MNHN). Two mitochondrial (COI and 16S rDNA) and one nuclear (18S) marker are used to distinguish between larval stages of Chelarctus and Crenarctus species. Once identified, developmental stages of Chelarctus species are described, highlighting key morphological characters to discriminate taxa and revising larval identities from previous reports.

Material and methods
Phyllosomae used for molecular analysis and descriptions were obtained by AIMS during Cruise Figure  5160, from 24 th May to 10 th June 2011, and Cruise 5441, between 16 th and 26 th July 2012. Both cruises were carried out in the vicinity of Osprey Reef, a submerged atoll which rises from a depth of about 2,000 m in the Coral Sea. The reef is about 200 km off the eastern coast of northeast Queensland, with the nearest reefs approximately 60 km away. Specimens were stored directly in absolute ethanol at low temperature (−20°C), and later deposited in the Natural History Museum, London (NHM). In addition, during the KANADEEP 2007 cruise organized by the MNHN, larval specimens were collected from temperate waters south of the Coral Sea. These specimens were included in our molecular and morphological analyses, and they are kept in the MNHN collections (MNHN-IU-2017-2420and MNHN-IU-2017. Station number, latitude, longitude and sampling date, together with data from AIMS and KANADEEP, and previous campaigns available in the literature, are detailed in Table I and Supplementary Table.

Molecular analyses
Total genomic DNA extraction was performed using the Chelex-resin method (Palero et al. 2010) from a single pereiopod of each larva. One nuclear (18S) and two mitochondrial (COI and 16S) genes were used to identify the larvae and reconstruct phylogenetic relationships within Chelarctus, using standard universal primers previously tested in Achelata (Palero et al. 2008(Palero et al. , 2009Bracken-Grissom et al. 2014). After observing significant intraspecific variation for COI and considering  Ueda et al. (2021) reported difficulties using standard universal primers for DNA barcoding (Folmer et al. 1994), COI was also amplified using a new pair of primers proposed by Krehenwinkel et al. (2018), ArF1: 5' -GCNCCWGAYATRGCNTTYCCNCG -3' (Gibson et al. 2014) and Fol-degen-rev: 3' -TANACYTCNGGRTGNCCRAARAAYCA -5' (Yu et al. 2012). Amplifications were carried out using ~30 ng of genomic DNA in a reaction containing 1 U of Taq polymerase (Amersham), 1 × buffer (Amersham), 0.2 mM of each primer and 0.12 mM dNTPs. The polymerase chain reaction (PCR) thermal profile was 94°C for 4 min for initial denaturation, followed by 30 cycles of 94°C for 30s, 50°C for 30s, 72°C for 30s and a final extension at 72°C for 4 min. Sequences were obtained using the Big-Dye Ready-Reaction kit ver. 3.1 (Applied Biosystems) on an ABI Prism 3770 automated sequencer at the NHM sequencing facilities. Chromatograms for each DNA sequence were checked with BioEdit v7.2.5 (Hall 1999) and sequence alignment was conducted using the program Muscle v3.6 (Edgar 2004) with default parameters. Model selection was performed according to the BIC criterion as implemented in MEGA X (Kumar et al. 2018). The construction method of maximum-likelihood (ML) phylogenetic tree was applied as implemented in PhyML v.3.0 (Guindon et al. 2010). K2P genetic distances were also estimated for COI and 16S genes dataset using MEGA X (Kumar et al. 2018), in order to allow for comparison with previous values in the bibliography.

Morphological description
Drawings of whole larvae and appendages were made with a camera lucida attached to a Leica M165C high-performance stereo microscope (Leica Microsystems, Germany). Antennules, mouth appendages (including paragnaths and mandibles), maxillipeds and pereiopods were individually dissected for an accurate description and because they might convey information of taxonomic value (pers. obs). An Intuous-S graphic tablet (Wacom) and Adobe Illustrator (https:// adobe.com/products/illustrator) were used for digitalization of drawings following Coleman (2003Coleman ( , 2009). The sequence of larval descriptions was based on the malacostraca somite plan and described from anterior to posterior and proximal to distal (Clark et al. 1998). Boxshall (2004) has challenged the traditional description of the Malacostraca antennule developing from a uniramous appendage to a biramous structure with endopod and exopod. The terminology biramous is considered inappropriate for the antennule, and instead of exopod and endopod, the terms primary and accessory flagella should be used (see Boxshall et al. 2010 for review). Setae nomenclature follows Garm and Watling (2013). Stage division was made on the basis of morphological development and changes in total length (Genis-Armero et al. 2020). Body length (BL) was measured from anterior margin of cephalic shield between the eyes to posterior margin of telson; cephalic length (CL) from anterior to posterior margin of cephalic shield, cephalic width (CW) measured at widest part of cephalic shield, thorax length (TL) from anterior to posterior margin of thorax, thorax width (TW) measured at the widest part of thorax shield, pleon length (PL) from anterior to posterior margin of pleon, and pleon width (PW) measured as the distance between insertion points of fifth pereiopods (P5). Morphometric measurements were obtained using the software ImageJ (Schneider et al. 2012). Different morphological characters were used to define genera and species groups following previous studies (Maigret 1978;Phillips & McWilliam 1986;Webber & Booth 2001;Inoue & Sekiguchi 2006), cephalon posterior margin (CPM), cephalon shape (CS), articulation of fifth pereiopod (P5), thoracic dorsal spines (TDS), and CL/CW ratio. The new characters proposed here with taxonomic value for Chelarctus and Crenarctus were, the cephalon edge (CE), relative length of carpus and propodus of maxilliped 3 (Crp/Prd) and PL/PW and BL/CW ratios. The set of morphological characters are detailed in Table II.

Molecular analyses
New sequences obtained from the phyllosoma larvae have been deposited in GenBank under accession numbers: (16S rDNA) and MZ452441-MZ452444 (18S rDNA). Given the concerns raised by COI sequences obtained using Folmer universal primers (see below), only COI sequences obtained with the recent Krehenwinkel primer pair were used in the phylogenetic analyses. Total length of the concatenated alignment was 1699 bp, with 32.8% (555 bp) corresponding to the COI gene (Krehenwinkel primer pair), 23.2% (395 bp) to the 16S rDNA, and 44% (748 bp) corresponding to the 18S rDNA. The model selected for the COI alignment was the T92 + G model (lnL = −2308.3968), with Gamma parameter  Figure 2). In total, 11 phyllosomae have been identified using DNA barcoding as belonging to Ch. crosnieri (N = 2), Ch. aureus (N = 8) and Cr. crenatus (N = 1). For all Chelarctus larvae 18S genetic sequences were identical, 16S allowed to discriminate species but was identical between adults and larvae of both Ch. aureus and Ch. crosnieri, and only COI sequences showed intraspecific variation. Interestingly, COI genetic distances (K2P) between adult Ch. aureus from Taiwan and larvae from Coral Sea waters were much higher when using Folmer primers (0.165 ± 0.020) than the distance observed using the recent Krehenwinkel primer pair (0.020 ± 0.006), but this was not the case for Ch. crosnieri (0.090 ± 0.013 in both cases).

Discussion
Final larval stages of Chelarctus crosnieri and Crenarctus crenatus are identified by DNA barcoding for the first time, as well as larval stages VI, IX and X of Chelarctus aureus. These results, together with a thorough revision of the previous literature, allowed Chelarctus and Crenarctus subfinal and final stages to be distinguished based on cephalic shield shape, relative length of carpus and propodus of maxilliped 3 (Crp/Prd ratio) and P5 articulation in the final larval stage. Chelarctus phyllosomae present a kidney-shape cephalon with a convex posterior margin, while Crenarctus show a rectangular cephalon shape with straight margin. Similarly, Chelarctus phyllosomae show a significantly larger Crp/Prd ratio (≥ 0.9) than Crenarctus specimens (≤ 0.7). Regarding P5 articulation in the last stage, Crenarctus larvae have 4-articled P5, whereas P5 in Chelarctus has only three articles. The presence of 4-articles mentioned by some authors (Higa & Shokita 2004;Inoue & Sekiguchi 2006;Ueda et al. 2021) is contradicted by previous works on North Pacific larvae (Johnson 1971(Johnson , 1979Sekiguchi 1990) and our results. This oversight is probably due to the apparent swelling, but lack of segmentation, of the P5 of Chelarctus final stage phyllosomae. The number of antennular sensory setae or spines on maxillipeds and pereiopods also seem to be useful characters to distinguish Ch. aureus and Ch. crosnieri larvae. The characters suggested to distinguish Ch. aureus larvae by Ueda et al. (2021) were based on single specimens for each stage and do not hold when the new Coral Sea phyllosomae are considered. The larval description of Chelarctus sp., assigned by the same authors to a putative Ch. crosnieri subspecies, is limited and should be examined accurately to discard the presence of pseudogenes and gather further morphological evidence.
Crenarctus crenatus and Ch. virgosus larvae have been confused in the literature until recently (Webber & Booth 2001;Ueda et al. 2021 Tsoi et al. 2011), identifying new species with one gene can be misleading (Ballard & Whitlock 2004;Galtier et al. 2009) and might overestimate biodiversity (Song et al. 2008). Despite COI genetic distances were higher, a second mitochondrial gene (16S) and the nuclear marker (18S) did not show any intraspecific variation between Coral Sea Chelarctus larvae and their putative adults (Ch. aureus/Ch. crosnieri). Moreover, the significantly higher intraspecific variation observed for Ch. aureus when using COI primers designed by Folmer et al. (1994) suggest the presence of nuclear mitochondrial pseudogenes, which have already been found in several crustaceans (Buhay 2009) and particularly in decapods (Williams & Knowlton 2001;Nguyen et al. 2002;Schubart 2009). The new pair of primers proposed by Krehenwinkel et al. (2018), designed specifically to amplify arthropod DNA, seem to be a better option when analyzing crustacean taxa. Although COI has been popularized as the main DNA barcoding gene, it may provide misleading results and it should be complemented with evidence from other mitochondrial and nuclear genes. The new evidence presented here highlight the value of integrative studies, combining comprehensive molecular data with detailed morphological analyses from adults and larvae. A particular focus should be given to increase the number of markers and to further explore Southern Hemisphere populations to better understand phylogeography and diversity of lobsters along the IWP.

Acknowledgements
This paper is dedicated to the late Alain Crosnier, for his invaluable work in advancing carcinology. Thanks are due to Dr. Hall for shipping the larvae and the crew of the Research Vessel Cape Ferguson and staff and volunteers on the collection trips and the Australian Institute of Marine Science for ship time. We also thank Laure Corbari and Paula Martin-Lefevre (Muséum national d'Histoire naturelle, Paris) and Romana Capaccioni and her team (Marine Biology Lab, University of Valencia) for encouraging the completion of this work. FP acknowledges the projects "CIDEGENT/2019/028 -BIOdiversity PAtterns of Crustacea from Karstic Systems (BIOPACKS): molecular, morphological, and functional adaptations" funded by the Conselleria d'Innovació, Universitats, Ciència i Societat Digital and "PRO2020-S02-PALERO -Fauna aquàtica en coves anquihalines del País Valencià: un mon encara per descriure" funded by the Institut d'Estudis Catalans.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Sampling and field studies
All necessary permits for sampling and observational field studies have been obtained by the authors from the competent authorities and are mentioned in the acknowledgements.