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.

Keywords:

• Decapoda
• DNA barcoding
• slipper lobster
• Indo-West Pacific

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 (Yang et al. 2012; 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 Yang & Chan, 2012, 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 (Yang & Chan 2012). 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 2010 and PAPUA NIUGINI 2012–2013) 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. 2014, 2016b), most of which await study.

Figure 1. Localities where Chelarctus and Crenarctus larvae (triangles) and adults (circles) have been reported from the Indo-West Pacific. Green: Chelarctus aureus; orange: Ch. crosnieri; yellow: Ch. cultrifer; red: Ch. virgosus; black: Chelarctus sp.; purple: Crenarctus crenatus and blue: Crenarctus sp.

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 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-2017-2420 and MNHN-IU-2017-10,460). 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.

Table I. Sampling locations from Indo-West Pacific waters where Chelarctus and Crenarctus larvae have been reported. Sampling information includes cruise, station, date, coordinates and original reference

Cruise	Station	Date	Longitude	Latitude	Reference
C5160	15	27/5/12	13°46’ S	146°27ʹE	Present study
	26	26/5/12	13°44’ S	146°25’ E
	50	28/5/12	14°06’ S	146°42’ E
	413	6/6/11	-	-
C5441	68	20/7/12	13°47’ S	146°33’ E
	142	21/7/12	13°48’ S	146°36’ E
	159	21/7/12	13°47’ S	146°35’ E
	175	22/7/12	13°47’ S	146°32’ E
	181	22/7/12	13°47’ S	146°34’ E
	205	22/7/12	13°50’ S	146°38’ E
C4922	-	10–19/5/2010	14°00’ S	146°40’ E
KANADEEP	CP4937	4/9/17	25°26’ S	159°47’ E
	CP4947	5/9/17	24°06’ S	159°37’ E
RV SHUNYO-MARU	2a	23/6/09	25°33’ N	127°02’ E	Ueda et al. 2021
	7	8/6/09	25°30’ N	126°05’ E
	20a	19/6/09	25°16’ N	124°68’ E
	22	9/6/09	23°19’ N	126°09’ E
	23	10/6/09	26°09’ N	126°01’ E
RV KOYO-MARU	-	30/1/15	27°07’ N	142°05’ E
	-	31/1/15	27°02’ N	142°05’ E
RV HAKUHO-MARU	226	11/9/16	19°00’ S	147°30’ W
-	-	31/5/84	25°39’ N	128°31’ E	Inoue & Sekiguchi 2006
	-	16/6/84	26°43’ N	133°19’ E
	-	5/8/84	30°15’ N	132°34’ E
	-	5/11/86	31°29’ N	129°59’ E
	-	16/6/84	26°43’ N	133°19’ E
NAGASAKI MARU	Site N-1	23/11/89	24°58’ N	123°29’ E	Higa & Shokita 2004
	Site N-1	23/11/98	24°58’ N	123°29’ E
	Site N-1	23/11/98	24°58’ N	123°29’ E
KEITEN MARU	-	11/11/98	26°51’ N	134°47’ E
	-	15/6/98	15°59’ N	136°18’ E
-	-	1969–92	36°52’ S	178°27’ E	Webber & Booth 2001
83-R-10	-	25/9-8/1/83	32°59’ N	135°45’ E	Sekiguchi 1986
	-		34°27’ N	137°56’ E
	-	13–21/9/84	32°20’ N	135°45’ E
	-		34°45’ N	138°45’ E

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, 2009; Bracken-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 (2003, 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.

Table II. Phyllosoma larvae (final stage) assigned to Chelarctus and Crenarctus based on our morphological and molecular results. Original and true identification, cephalon posterior margin (CPM), cephalon edge (CE), carpus length/propodus length ratio of maxilliped 3 (Crp/Prd), P5 articulation, cephalon shape (CS), thoracic dorsal spines (TDS), body length (BL), morphometrics (CL/CW, BL/CW, PL/PW), region where the larvae were collected and original references

Original identity	New identification	CPM	CE	Crp/Prd	P5	CS	TDS	BL (mm)	CL/CW	BL/CW	PL/PW	Region	References
Chelarctus aureus	Ch. aureus *	convex	P2	1.1	3	kidney-shape	P2–P4	23–24.6	0.6	1	0.7	Coral Sea	Present study
Chelarctus aureus	Ch. aureus *	convex	P2	-	4?	kidney-shape	-	23.0	0.7	1.1	0.8	Japan	Ueda et al. (2021)
Scyllarus sp. A	Ch. aureus	convex	P2	1.1	3	kidney-shape	P1–P4?	22.0	0.7	1.1	0.7	South China	Johnson (1971)
Chelarctus crosnieri	Ch. crosnieri *	convex	P2	1.0	3	kidney-shape	P2–P4	18.4–19.5	0.7	1.1	0.8	Coral Sea	Present study
Chelarctus sp. 1	Chelarctus sp.*	convex	P2	2.0	4?	kidney-shape?	-	28.4	0.8	1.3	0.9	Japan	Ueda et al. (2021)
Chelarctus virgosus	Ch. virgosus	convex	P2	1.0	3	pentagonal	P1–P4	22.0	0.8	1.3	1.1	Taiwan	Present study
Chelarctus virgosus	Ch. virgosus *	concave?	P2	1.0	4?	pentagonal	-	21.4	0.8	1.3	1.0	Japan	Ueda et al. (2021)
Chelarctus cultrifer	Ch. virgosus	convex	P2	-	4?	pentagonal	P1–P4	20.7	0.9	1.4	1.1	Japan	Inoue & Sekiguchi (2006)
Chelarctus cultrifer	Ch. virgosus	convex	P1?	1.1	4?	pentagonal	P1–P4	20.7	0.9	1.4	1.1	Taiwan	Higa & Shokita (2004)
Scyllarus bicuspidatus	Ch. virgosus	-	P2	1.0	3	pentagonal	P1–P4	25.0	0.8	1.3	1.0	Mariana	Sekiguchi (1990)
Scyllarus sp.	Ch. virgosus	convex	P2	0.9	3	pentagonal	P1–P4	21.0	0.8	1.3	1.2	Japan	Johnson (1979)
Scyllarus sp. A	Ch. cultrifer ?	convex	P2	-	3	pentagonal	P1–P4	21.0	0.8	1.3	1.0	South China	Johnson (1971)
Scyllarus delfini	Cr. delfini	straight	P1	0.5	4	rectangular	P1–P4	25.0	0.8	1.4	1.2	Juan Fernández Is.	Baez (1973)
Scyllarus sp. Z	Cr. crenatus	straight	P1	0.7	4	rectangular	P1–P4	18.6–30.5	0.8	1.3	1.2	New Zealand	Webber & Booth (2001)
Crenarctus crenatus	Cr. crenatus *	straight	P1	0.7	4	rectangular	P1–P4	21.6	0.8	1.5	1.1	New Caledonia	Present study

(*) DNA barcoded

Results

Molecular analyses

New sequences obtained from the phyllosoma larvae have been deposited in GenBank under accession numbers: MZ452434-MZ452440 (COI Folmer), OM534650-OM534652 (COI Krehenwinkel), MZ460954-MZ460961 (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 (G = 0.1940), for 16S was HKY+G (lnL = −1198.6509), with Gamma parameter (G = 0.1547) and JC model for 18S. Bootstrap results on the Maximum Likelihood tree strongly supported the species-level assignment of Coral Sea and New Caledonian larvae to Ch. aureus, Ch. crosnieri, and Cr. crenatus (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).

Figure 2. Maximum likelihood phylogenetic tree obtained from the concatenated alignment using 18S, 16S and COI genes of Chelarctus and Crenarctus specimens. Samples are detailed in the supplementary table except for the RCL larvae (see Palma et al. 2011). Larval images adapted from Johnson (1971), Baez (1973), Webber and Booth (2001) and Ueda et al. (2021). Only significant bootstrap values (>70) are shown.

Morphological analyses

A list of the larvae used for molecular and morphological analysis, as well as previous larval records for Chelarctus, are shown in the Supplementary Table. Chelarctus aureus and Ch. crosnieri phyllosomae are reported from Western Coral Sea waters for the first time, and Chelarctus adults were never recorded here before (Figure 2). Intermediate (stage VI), subfinal (IX) and final (X) stages of Ch. aureus and subfinal (IX) and final (X) stages for Ch. crosnieri are described in detail (Figures 3–12). The subfinal stage of Ch. crosnieri was identified by the number of spines in maxillae and maxillipeds, number of aesthetascs and BL. Specimens identified as Cr. crenatus (MNHN-IU-2017-2420 and MNHN-IU-2017-10,460) or Ch. virgosus (NMNS-004987-00006) are not redescribed here because they are identical to those accurately drawn by Webber and Booth (2001) and Johnson (1979), but Chelarctus and Crenarctus larval morphology is compared in Table II. Cephalon shape, Crp/Prd ratio of maxilliped 3 and P5 articulation allow to differentiate both genera, while morphometrics (CL/CW, BL/CW, PL/PW) of final stage phyllosoma define two groups: Ch. aureus/Ch. crosnieri and Ch. virgosus/Chelarctus sp.1/Crenarctus.

Figure 3. Chelarctus aureus (Holthuis, 1963), stage VI. A, ventral (right) and dorsal (left) view; B, dactylus of second pereiopod; C, dactylus of third pereiopod. Scale bars: A = 1 mm; B and C = 500 µm.

Figure 4. Chelarctus aureus (Holthuis, 1963), stage VI. A, antenna and antennule; B, C, left and right paragnaths (ventral view); D, E, left and right mandibles (dorsal view); F, maxillule; G, maxilla and first maxilliped; H, second maxilliped; I, third maxilliped (distal part); J, pleon and fifth pereiopod (ventral view). Scale bars: A, J and I = 1 m; B–F = 100 µm; G, H = 200 µm.

Figure 5. Chelarctus aureus (Holthuis, 1963), subfinal stage. A, ventral (right) and dorsal (left) view; B, dactylus of first pereiopod; C, dactylus of second pereiopod; D, dactylus of third pereiopod; E, dactylus of fourth pereiopod. Scale bars: A = 2 mm; B–E = 400 µm.

Figure 6. Chelarctus aureus (Holthuis, 1963), subfinal stage. A, antenna and antennule; B, C, left and right paragnaths (ventral view); D, E, left and right mandibles (dorsal view); F, maxillule; G, maxilla and first maxilliped; H, second maxilliped; I, third maxilliped (distal part); J, pleon and fifth pereiopod (ventral view). Scale bars: A, H and I = 500 µm; B, C and G = 200 µm; D–F = 100 µm; J = 2 mm.

Figure 7. Chelarctus aureus (Holthuis, 1963), final stage. A, ventral (right) and dorsal (left) view; B, dactylus of first pereiopod; C, dactylus of second pereiopod; D, dactylus of third pereiopod; E, dactylus of fourth pereiopod. Scale bars: A = 5 mm; B–E = 500 µm.

Figure 8. Chelarctus aureus (Holthuis, 1963), final stage. A, antenna and antennule; B, C, right and left paragnaths (ventral view); D, E, left and right mandibles (dorsal view); F, maxillule; G, maxilla and first maxilliped; H, second maxilliped; I, third maxilliped (distal part); J, pleon and fifth pereiopod (ventral view). Scale bars: A = 2 mm; C, D, H and I = 500 µm; D–F = 200 µm; G = 1 mm; J = 5 mm.

Figure 9. Chelarctus crosnieri Holthuis, 2002, subfinal stage. A, ventral (right) and dorsal (left) view; B, dactylus of first pereiopod (P1); C, dactylus of second pereiopod; D, dactylus of third pereiopod (P3); E, dactylus of fourth pereiopod (P4). Scale bars: A = 5 mm; B–E = 200 µm.

Figure 10. Chelarctus crosnieri Holthuis, 2002, subfinal stage. A, antenna and antennule; B, C, left and right paragnaths (ventral view); D, E, left and right mandibles (dorsal view); F, maxillule; G, maxilla and first maxilliped; H, second maxilliped; I, third maxilliped (distal part); J, pleon and fifth pereiopod (ventral view). Scale bars: A and J = 1 mm; B–D = 100 µm; F = 250 µm; G–I = 500 µm.

Figure 11. Chelarctus crosnieri Holthuis, 2002, final stage. A, ventral (right) and dorsal (left) view; B, dactylus of first pereiopod (P1); C, dactylus of second pereiopod (P2); D, dactylus of third pereiopod (P3); E, dactylus of fourth pereiopod (P4). Scale bars: A = 2 mm; B–E = 400 µm.

Figure 12. Chelarctus crosnieri Holthuis, 2002, final stage. A, antenna and antennule; B, C, right and left paragnaths (ventral view); D, E, left and right mandibles (dorsal view); F, maxillule; G, maxilla and first maxilliped; H, second maxilliped; I, third maxilliped (distal part); J, pleon and fifth pereiopod (ventral view). Scale bars: A, G–I = 1 mm; B and C = 200 µm; D–F = 100 µm.

Chelarctus aureus (Holthuis, 1963)

Stage VI (samples from cruise C5160: 26_03, 15_02, 50_02, 50_03). Morphometrics: N = 6, BL = 8.9–10.5 mm, CL = 6.7–7.2 mm, CW = 8.8–9.7 mm, TL = 3.3–3.7 mm, TW = 3.6–3.8 mm, PL = 0.7–0.8 mm, PW = 1.2–1.3 mm, CL/CW = 0.8–0.7. Cephalic shield (Figure 3A): Rectangular shape, wider than longer. Antennule (Figures 3A, 4A) equal in length than antenna, with 3-articulated peduncle. Primary flagellum with 10–11 rows of aesthetascs; accessory flagellum shorter than primary. Antenna (Figures 3A, 4A): Biramous, unarticulated, shorter than antennule; endopod longer than exopod. Paragnaths (Figure 4B, C): Asymmetrical. Both fringed marginally with setules and denticulettes. Mandibles (Figure 4D, E): Asymmetrical dentition. Both mandibles with abundant small teeth distributed over surface and molar process crowned with many denticles. Left mandible with 3 elongated teeth on incisor process; right mandible with 4 teeth curved towards molar process. Palp absent. Maxillule (Figure 4F): Coxal endite with 2 simple setae and 4 serrate setae (2 long and strong); basial endite with 4 simple setae and 3 cuspidate setae with denticles, long and strong. Endopod and exopod absent. Maxilla (Figures 3A, 4G): Unarticulated and underdeveloped. First maxilliped (Figures 3A, 4G): Present as minute bud. Second maxilliped (Figures 3A, 4H): Uniramous. Coxa without setae; basis delimited by distal seta; endopod with 4 articles, ischium-merus (undifferentiated), carpus, propodus and dactylus with 1, 0, 7 (2 serrate setae) and 2 setae, respectively. Exopod absent. Third maxilliped (Figures 3A, 4I): Uniramous. Coxa with ventral distal spine; basis and endopod undifferentiated; endopod with 4 articles, ischium-merus (undifferentiated), carpus, propodus and dactylus with 5, 6, 23 (2 distal serrate) and ~30 simple setae, respectively. Setae on inner margin longer than outer margin. Exopod absent. Pereiopods (Figures 4A–C, 4J): P1–4 biramous. Coxa without setae, long distal ventral spine; basis delimited by distal spine, P1–3 basis with medial seta, P4 basis with 2 medial setae; endopod 4-articulated, ischium-merus (undifferentiated) with 2 distal spines, carpus with distal spine. Dactylus of P2 and P3 with 5 and 7 simple setae, respectively. P1–4 exopods with 19, 19–21, 18–19, 17 annulations respectively, each annulation with 2 long plumose setae. P5 (Figure 4J) uniramous and unarticulated with minute seta. Exopod absent. Thorax (Figure 3A): Sternites 5–7 with dorsal distal spine. Sternite 4 and 8 without spine. Pleon (Figures 3A, 4J): Pleopod 1 absent. Pleopods 2–5 uniramous, not developed, endopod present, exopod absent. Uropods biramous. Telson (Figures 3A, 4J): Margin concave, 2–3 pairs of dorsal setae, 2 long terminal processes with proximal seta each.

Stage IX (C5441: 142_01, 142_02, 205_02, 175_01)Morphometrics: N = 4, BL = 16.9–19.7 mm, CL = 11.1–12.9 mm, CW = 16.6–19.8 mm, TL = 6.0–9.8 mm, TW = 6.8–8.2 mm, PL = 1.9–2.6 mm, PW = 3.6–4.4 mm, CL/CW = 0.65–0.67. Cephalic shield (Figure 5A): Rectangular shape, wider than longer. Antennule (Figures 5A, 6A): Primary flagellum with 16–17 rows of aesthetascs. Simple setae in both margins of antennular peduncle. Antenna (Figures 5A, 6A): equal in length than antennule. Paragnaths (Figure 6B, C): Both with more setae than previous stage. Otherwise unchanged. Mandibles (Figure 6D, E): Both mandibles with more teeth than previous stage. Otherwise unchanged. Maxillule (Figures 5A, 6F): Coxal endite with 3 simple setae and 5 serrate setae (2 long and strong); basial endite with 5 simple setae and 3 cuspidate setae with denticles, long and strong. Otherwise unchanged. Maxilla (Figures 5A, 6G): Uniramous and unarticulated. Endites and endopod not differentiated with 4–5 setae on superior margin; scaphognathite (exopod) present, slightly developed and rectangular, without marginal setae. First maxilliped (Figures 5A, 6G): Uniramous. Endites undifferentiated; endopod present and unarticulated. Exopod absent. Second maxilliped (Figures 5A, 6H): Endopod 4-articled, ischium-merus (undifferentiated), carpus, propodus, dactylus with 0, 1, 12 (1 serrate seta) and 4 simple setae respectively. Otherwise unchanged. Third maxilliped (Figures 5A, 6I): Endopod 4-articled, ischium-merus (undifferentiated), carpus, propodus and dactylus with ~8, ~10, ~40 (2 distal serrated) and ~70 simple setae respectively. Setae on inner margin longer than outer margin. Otherwise unchanged. Pereiopods (Figures 5A-E, 6J): P1–4 biramous. Coxa without setae, distal ventral spine present; basis delimited by distal spine, P1–P2 without medial spines, P3 with medial spine, P4 with 2 medial spines; endopod 4-articled, ischium-merus (undifferentiated) with 2 distal spines, carpus withdistal spine, propodus with 75, 50, 50 and 25 small simple setae scattered over article surface, dactylus with 10, 6, 5, 2 setae respectively. Exopod with 25, 25–27, (> 21)–26, (> 20)–26 annulations respectively. P5 (Figure 6J) Uniramous. Coxa without setae, ventral distal spine present; basis delimited by medial spine; endopod 2-articled, ischium-merus (undifferentiated) with 2 distal spines, proximal articles not differentiated. Exopod absent. Thorax (Figure 5A): Dorsal distal spine larger than previous. Otherwise unchanged. Pleon (Figures 5A, 6J) Pleopod 1 absent. Pleopods 2–5 and uropods biramous. Telson (Figures 5A, 6J): Fork margin slightly concave, proximal setae on inner margin probably missing, 6–8 pairs of dorsal setae.

Stage X (C5160: 413_01; C5441: 68_01, 205_04, 214_01, 214_04) Morphometrics: N = 5, BL = 21.6–24.3 mm, CL = 13.2–14.7 mm, CW = 20.5–22.8 mm, TL = 7.4–8.5 mm, TW = 8.9–10.3 mm, PL = 3.9–4.5 mm, PW = 5.4–6.3 mm, CL/CW = 0.63–0.65. Cephalic shield (Figure 7A): Unchanged. Antennule (Figures 7A, 8A): Slightly shorter than antenna. Primary flagellum with 17–18 rows of sensory setae, accessory flagellum unarticled with minute setae in both margins. Antenna (Figures 7A, 8A): Slightly longer than antennule, with perceptible lobular articulation. Paragnaths (Figure 8B, C): Unchanged. Mandibles (Figure 8D, E): Both mandibles with more teeth than previous stage. Left mandible with 4 teeth on incisor process. Palp absent. Maxillule (Figures 7A, 8F): Coxal endite with 6 serrate setae and 3 simple setae; basial endite with 3 cuspidate setae with denticles and 7 simple setae. Otherwise unchanged. Maxilla (Figures 7A, 8G): Uniramous. Endite and endopod undifferentiated with 5–6 setae on superior margin of lateral process; scaphognathite (exopod) flattened and expanded, without marginal setae. First maxilliped (Figures 7A, 8G): Unarticulated and bilobed; outer lobe (endite) flattened and round; inner lobe (endopod) elongated and unarticulated. Exopod absent. Second maxilliped (Figures 7A, 8H): Biramous. Coxa without setae; basis delimited by distal seta; endopod with 4 articles, ischium-merus (undifferentiated), carpus, propodus, dactylus with 0, 1, 13 (2 serrate setae) and simple 5 setae respectively. Exopod present as minute bud. Third maxilliped (Figures 7A, 8I): Biramous. Gills buds present; 1 pleurobranch, 1 arthrobranch and 2 podobranchs. More densely setose than previous stage. Basis delimited by exopod (minute bud); endopod with 4 articles, ischium-merus (undifferentiated), carpus, propodus and dactylus with 10, 8, 50 (2 distally serrated) and ~100 simple setae respectively. Otherwise unchanged. Pereiopods (Figures 7A–E, 8J): P1–4 biramous. P1–P4 basis with 2 medial setae, P2–P3 setae probably missing; propodus with 85, 65, 40 and 25 small simple setae scattered over the surface, dactylus with 8, 10, 4, 2 simple setae respectively. Exopods with 23–27, 23–26, 25–27 and 23–25 annulations respectively. Otherwise unchanged. P5 longer than previous stage. Gills (Figure 7A, F): Gill buds present. P1 with 1 pleurobranch, 1 arthrobranch and 2 podobranchs. P2–P4 with 2 pleurobranchs, 1 arthrobranch, 2 podobranchs. P5 with 1 pleurobranch.Thorax (Figure 7A, F): Unchanged. Pleon (Figures 7A, 8J): Pleopods and uropods well-developed.Telson (Figures 7A, 8J): 9–11 paired dorsal setae. 2 long terminal processes with 1 pair of setae each in inner margin.

Chelarctus crosnieri Holthuis, 2002

Stage IX (C5441: 159_02, 205_08, 205_10, 205_11) Morphometrics: N = 5, BL = 13.1–17.5 mm, CL = 10.3–11.7 mm, CW = 16.4–16.7 mm, TL = 5.7–6.1 mm, TW = 6.6–7.0 mm, PL = 1.9–2.3 mm, PW = 3.5–4.0 mm, CL/CW = 0.67–0.70.Cephalic shield (Figure 9A): Rectangular shape, wider than longer. Antennule (Figures 9A, 10A): Slightly longer than antenna with 3-articled peduncle. Primary flagellum with 11 rows of aesthetascs; accessory flagellum equal in length than primary.Antenna (Figures 9A, 10A): Biramous. Not articulated, slightly shorter than antennule. Paragnaths (Figure 10B, C): Asymmetrical. Both with fringed marginally with setules and denticulettes. Mandibles (Figure 10D, E): Asymmetrical dentition. Both mandibles with abundant small teeth distributed over surface and molar process crowned with many different denticles. Left mandible with multiple teeth on incisor process, right mandible larger and with 3 teeth on incisor process; right mandible teeth curved towards molar process while teeth of right mandible are elongated. Palp absent. Maxillule (Figures 8A, 10F): Coxal endite with 5 serrate setae (2 long and strong) and 2 simple setae; basial endite with 3 cuspidate setae with denticles, long and strong and 5 simple setae. Endopod and exopod absent. Maxilla (Figures 9A, 10G): Uniramous and unarticulated. Endites and endopod undifferentiated with 5 simple setae on superior margin; scaphognathite (exopod) slightly developed and rectangular, without marginal setae. First maxilliped (Figures 9A, 10G): Present and undifferentiated.Second maxilliped (Figures 9A, 10H): Uniramous. Coxa without setae; basis delimited by distal simple seta; endopod with 4 articles, ischium-merus (undifferentiated), carpus, propodus (elongated) and dactylus with 0, 0, 11 (2 serrate setae) and 3 simple setae respectively. Exopod absent. Third maxilliped (Figures 9A, 10I): Uniramous. Coxa without setae, ventral spine present; basis not differentiated, without setae; endopod 4-articled, ischium-merus (undifferentiated), carpus, propodus and dactylus with 3, 0, 33 (2 distal serrate setae) and 70 simple setae respectively. Setae on inner margin longer than outer margin. Exopod absent. Pereiopods (Figures 9A–E, 10J): P1–4 biramous. Coxa without setae and with distal ventral spine; basis elongated and demarcated distally by single spine; endopod 4-articled, ischium-merus (undifferentiated) with two distal spines, carpus with one distal spine, propodus with 60, 55, 40 and 25 simple setae scattered over article surface, dactylus with 8, 7, 5 and 2 simple setae respectively. Exopod with (>20) –25, (>14)–26, 24–25, 21–25 annulations respectively, each annulation with two long plumose setae. P5 (Figure 10J) Uniramous. Coxa with ventral distal spine; basis demarcated by single spine; endopod with article, proximal articles undifferentiated. Exopod absent. Thorax (Figure 9A): Sternites 5–7 with dorsal distal spine. Sternite 4 and 8 without spine. Pleon (Figures 9A, 10J): Pleopod 1 absent, pleopods 2–5 and uropods biramous. Telson (Figures 9A, 10J): Margin slightly concave with 2 terminal processes proximally.

Stage X (C5441: 159_01, 181_01, 181_02, 181_03, 205_01, 205_06) Morphometrics: N = 6, BL = 17.7–19.2 mm, CL = 10.7–11.7 mm, CW = 14.6–16.3 mm, TL = 5.5–5.9 mm, TW = 6.8–7.4 mm, PL = 3.7–4.0 mm, PW = 4.5–5.0 mm, CL/CW = 0.70–0.74. Cephalic shield (Figure 11A): Unchanged. Antennule (Figures 11A, 12A): Slightly shorter than antenna. Accessory flagellum unarticulated; primary flagellum slightly shorter than accessory with 15–16 rows of sensory setae. Antenna (Figures 11A, 12A): With perceptible lobular articulation. Paragnaths (Figure 12B, C): Unchanged (right paragnath damaged). Mandibles (Figure 12D, E): More teeth in both mandibles than previous stage. Otherwise unchanged. Maxillule (Figure 12F): Coxal endite with 5 serrate and 3 simple setae; basial endite with 3 cuspidate setae with denticles and 6 simple setae. Otherwise unchanged. Maxilla (Figures 11A, 12G): Uniramous. Endites and endopod not differentiated with 3–4 simple setae on lateral process; scaphognathite (exopod) flattened and expanded, without marginal setae. First maxilliped (Figures 11A, 12G): Uniramous and bilobed. Endite flattened, rounded, not differentiated. Endopod elongated and unarticulated. Exopod absent. Second maxilliped (Figures 11A, 12H): Biramous. Coxa without setae; basis delimited by distal setae; endopod present with 4 articles, ischium-merus (undifferentiated), carpus, propodus and dactylus with 0, 1, 10 (3 serrate setae) and 3 simple setae, respectively. Exopod present as minute bud. Third maxilliped (Figures 11A, 12I): Biramous. Gill buds present; 1 pleurobranch, 1 arthrobranch and 2 podobranchs. More densely setose than previous stage. Basis delimited by exopod (minute bud); endopod, propodus and dactylus with 35 (2 distal serrate setae) and > 60 simple setae, respectively. Otherwise unchanged. Pereiopods (Figures 11A–E, 12J): P1–4 biramous. Propodus with 75, 60, 40 and 20 small simple setae scattered over the surface, dactylus with 8, 10, 4 and 4 respectively. Exopods with 23–25, 23–26, 25–27 and 23–25 annulations respectively. Otherwise unchanged. P5 (Figure 12J) basis and ischium-merus not differentiated (probably seta on basis missing); endopod with 2 articles, ischium-merus with two distal spines, proximal articles undifferentiated. Longer than previous stage. Gills (Figure 11A, F): Gill buds present; P1 with 1 pleurobranch, 1 arthrobranch and 2 podobranchs; P2–P4 with 2 pleurobranchs, 1 arthrobranch, 2 podobranchs; P5 with 1 pleurobranch. Thorax (Figure 11A, F): Unchanged. Pleon (Figures 11A, 12J): Pleopods and uropods biramous and well-developed. Telson (Figures 11A, 12J): 9–11 paired setae dorsally. Otherwise unchanged.

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, 1979; Sekiguchi 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) because both share morphological characters such as BL or narrow cephalon and pleon. New molecular results and detailed morphological analyses using trait differences that had previously passed unnoticed (e.g., length of carpus and propodus of maxilliped 3), have allowed us to re-assess inferences made by previous authors. Our results support that the larval series Scyllarus sp. Z, tentatively assigned to S. aoteanus by Webber and Booth (2001), belongs to Cr. crenatus. Indeed, S. aoteanus was recently synonymized with Cr. crenatus by Chan et al. (2013). Likewise, the morphology of S. delfini phyllosomae (see Baez 1973) is very similar to our Cr. crenatus larvae (Table II) and specimens from Juan Fernández island (Palma et al. 2011) cluster with Cr. crenatus and Cr. bicuspidatus. These results are congruent with previous observations of adult morphology and confirm that Acantharctus delfini should be considered as Cr. delfini (Genis-Armero et al. 2020). The taxonomy of Acantharctus and, more generally, the Scyllarinae, need a thorough revision using both morphological and molecular data.

Previous phylogeographic studies have uncovered complexes of cryptic species on shallow-water taxa from West Pacific (shrimps: Tsoi et al. 2007; cephalopods: Cheng et al. 2014; bivalves: DeBoer et al. 2014). While COI has been generally used as supporting evidence for establishing new taxa (Poore & Andreakis 2011; 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.

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.

Supplemntal material

Supplemental data for this article can be accessed here

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).

Additional information

Funding

This work was supported by the EU-Synthesys grant GB-TAF-2737: DNA barcoding identification of phyllosoma larvae from Coral Sea waters; 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 “PRO2021-S02-PALERO - Fauna aquàtica en coves anquihalines del País Valencià: un mon encara per descriure” funded by the Institut d'Estudis Catalans. The research was financed by NCN OPUS BIOPASS 2018/31/B/NZ8/03198.”