A new genus for Pericera septemspinosa Stimpson, 1871 and Pericera heptacantha Bell, 1836 (Crustacea, Brachyura, Majoidea), based on morphology and molecular data

A new genus of majoid spider crab, Pohleus gen. nov. is established for Pericera septemspinosa Stimpson, 1871 and Pericera heptacantha Bell, 1836, based on morphology and molecular data from the partial sequences of the 12S and 16S mitochondrial genes and the 18S small subunit rRNA nuclear locus. The species are re-described and illustrated, based on material from several localities of the western Atlantic and eastern Pacific oceans. The carapace, antennal and pterygostomial spines, male thoracic sternum and first gonopods are distinctive characters, distinguishing Pohleus gen. nov. from species assigned to Macrocoeloma Miers, 1879, where P. septemspinosus and P. heptacanthus are currently included.


Introduction
Macrocoeloma heptacanthum (Bell, 1836) and M. septemspinosum (Stimpson, 1871) were originally described as Pericera Latreille, 1825 and transferred by Miers (1886) to Macrocoeloma Miers, 1879 (see Colavite et al. 2016 for review). As part of an ongoing revision of Macrocoeloma, we identified the necessity to establish a new genus for the above two species. The description of Pericera heptacantha was based on two syntypes, one male and one female. This type material was treated as missing in literature for more than 50 years (see Rathbun 1925;Garth 1958) until the female specimen was re-discovered by Di Mauro (1982) in the dry crustacean collection in the University Museum, Oxford (OUM). Pericera septemspinosa, from the western Atlantic, was described by Stimpson (1871) as a geographical analogue to Pericera heptacantha from the eastern Pacific and, unfortunately, the type material of P. septemspinosa was most likely destroyed in the Great Chicago Fire in 1871 (Evans 1967;Deiss and Manning 1981;Manning 1993;Vasile et al. 2005;Manning and Reed 2006).
Pohleus gen. nov., is proposed herein to receive Pericera heptacantha and P. septemspinosa and a lectotype and a neotype are designated for each species, respectively. The species are re-described, illustrated and the morphological differences between them are detailed below. A phylogenetic framework for Pohleus gen. nov. and allied genera is proposed, based on partial sequences of the 12S and 16S mitochondrial genes and the 18S small subunit rRNA nuclear locus. DNA extraction, PCR, and sequencing Total genomic DNA was extracted from muscle tissue using either the Qiagen DNeasy Blood and Tissue extraction kit or Omega Bio-tek EZNA Tissue DNA Kit. Partial sequences of the 12S, 16S mitochondrial genes were amplified with the following primers, respectively: 12SF (Mokady et al. 1994) and 12S1R (Shull et al. 2005), 16SF/16SR (Hultgren and Stachowicz 2008). The nuclear loci of the small subunit 18S rRNA were amplified with the primers A and B (Medlin et al. 1988) and C, Y, O and L of Apakupakul et al. (1999) or D18s1R, D18s2F/R, D18s3F/R, D18s4F/R and D18s5F of Bracken et al. (2009). Annealing temperatures for PCRs were 58 °C and 54 °C for 12S/18S and 16S, respectively. Reagent volumes and concentrations followed manufacturer's instructions; primer concentrations were 10 µM. Sequencing reactions were performed using 1 µl of purified PCR product in a 10 µl reaction containing 0.5 µl primer, 1.75 µl BigDye buffer and 0.5 µl BigDye (Life Technologies). Sequencing reactions were purified using Millipore Sephadex plates , according to the manufacturer's instructions and sequenced on the ABI 3730XL automated DNA sequencer. Sequences were assembled, trimmed of primers and checked for quality using Geneious 8.0.5 and 9.1.8. DNA extraction and sequencing were carried out at the Smithsonian Institution's Laboratories of Analytical Biology.

Molecular data analysis
Sequences, generated for this study, were combined with other sequences available from GenBank and previously unpublished sequences generated by AMW, in order to place the target taxa within the context of the superfamily Majoidea. Locality information and GenBank accession numbers for taxa included in the molecular analyses are provided in Suppl. material 1: Table S1. All individual sequences for each species were initially analysed and assembled using the software Geneious 8.0.5 (http://www. geneious.com; Kearse et al. 2012). The alignment amongst all sequences was generated with the algorithm Muscle (Edgar 2004) under default parameters and the perl script PartitionFinder (Lanfear et al. 2017) on CIPRES Portal (Miller et al. 2010) was run to determine the appropriate model of evolution and partitioning scheme. To determine whether the sequence data was appropriate for phylogenetic studies, the occurrence of substitution saturation was evaluated in the molecular data and identical sequences were excluded, resulting in 23 unique sequences. Numbers of transition and transversions versus ML-Compos-iteTN93 distance were plotted using the software DAM-BE 7.2.7 (Xia 2013(Xia , 2017, with the options of pairwise deletion and genetic distance model F84. The individual datasets were concatenated in RAxML, where we used the '-f ae' option with 1000 bootstrap replicates. Likelihood parameters followed the General Time Reversible (GTR) model with a gamma distribution on the partitioned dataset and RAxML estimated all free parameters. The resulting best tree was used to reflect phylogeny ( Fig. 1). To establish the phylogenetic context for Pohleus septemspinosus gen. nov. et comb. nov., we followed the previous phylogenies of Majoidea Stachowicz 2008, 2009;Windsor and Felder 2014) to choose the genera used in this study and rooted the tree with the hymenosomatid crab Elamena producta Kirk, 1879. Only nodes with maximum likelihood bootstrap support greater than 50% are shown on the maximum likelihood phylogram (Fig. 1). All analyses were run on the Hydra computing cluster at USNM or CIPRES Portal (Miller et al. 2010). Nodes, where maximum likelihood bootstrap support is greater than 50%, are shown on the maximum likelihood phylogram (Fig. 1).
The ongoing revision and phylogenetic study of Macrocoeloma by the authors (unpubl. data), based on both morphological and molecular data, revealed this genus to be a paraphyletic group. Pohleus gen. nov. therefore, needs to be established to accommodate P. septemspinosus gen. nov. et comb. nov. and P. heptacanthus gen. nov. et comb. nov.

Results and discussion
All three genes (12S, 16S and 18S) were successfully amplified and sequenced only for Pohleus septemspinosus gen. nov. et comb. nov. The final alignment for this combined analysis of nuclear and mitochondrial genes included a total of 2659 bp (1868 bp 18S, 417 bp 16S and 372 bp 12S). The data were not saturated, considering the R 2 value for transitions R² (s) = 0.8665 and R² (v) = 0.9856 transversions, demonstrating that the sequences are appropriate for phylogenetic studies at this level.
Pohleus septemspinosus gen. nov. et comb. nov. has a high support value (84%) as sister to a clade containing Thersandrus compressus and Macrocoeloma spp. Thersandrus, presently assigned to the subfamily Majinae sensu Ng et al. (2008), is supported 89% as basal to Macrocoeloma and nested between Macrocoeloma and Pohleus gen. nov. in all scenarios analysed and with high support (Fig. 1). Thus, we believe that Thersandrus could be better fitted in Pisinae, based on the molecular results. Pohleus gen. nov. nested amongst other Pisinae as expected (Fig. 1 Diagnosis. Carapace subglobose, covered by short velvet pubescence interspaced by dense rows of long, hooked and simple setae in all carapace regions. Carapace armed with strong spines, including seven sharp spines on posterior half: one short mesial metagastric, four long, strong, conical lateral spines (two in each branchial region) aligned with one cardiac spine and one intestinal spine; lateral spines longest, slightly directed upwards. Pterygostomial region with strong spines visible in dorsal view. Rostrum bifurcated, base elongated, fused, diverging abruptly forming a Y-shape, ending in acute tips. Pre-orbital spine strong, acute, directed upwards; post-orbital spine short, acute. Basal article of antenna with three spines, one tubercle, not visible in dorsal view. Cheliped long, merus armed with short spines or tubercles, granulated. P2 shorter than cheliped, dactylus much shorter than propodus. Thoracic sternal somite IV with lateral margins straight. Sternite VIII concealed by pleon in males. Male and female pleon with six somites not fused plus telson. Male telson tight-fitting into sterno-pleonal cavity, distinctly triangular. G1 slender, straight, with bilobed apex. G2 slender, straight, tapering distally, short about one fifth of G1 length.

Remarks. Macrocoeloma
Miers, 1879 s. str. is an amphi-American genus with 12 species. This genus is characterised by the pyriform or triangular carapace, densely covered by short, velvet-like setae; with well-developed bifurcated or parallel rostral spines; the eyes completely protected by the orbits when retracted; orbits composed by the pre-orbital and the post-orbital spines and one or two projections of the basal article of antenna forming a functional, laterally projected protective hood. Although some of these characters can be observed in Pohleus gen. nov., the new genus can be easily distinguished from Macrocoeloma s. str. by a unique combination of characters, which include: (i) carapace relatively more globose in Pohleus gen. nov. ( In Macrocoeloma, the gonopods are highly variable amongst species, but it is possible to recognise a general pattern with G1 being longer than the thoracic sternal suture IV/V, parallel and usually with a bilobed apex ( Thersandrus Rathbun, 1897, is a monotypic genus exhibiting extremely efficient camouflage behaviour as Macrocoeloma and Pohleus gen. nov. However, Thersandrus does not actively decorate itself, presenting crypsis behaviour consisting of matching the body to the environment in shape and colour, being morphologically adapted to live on green algae fronds. For instance: (i) Thersandrus has a carapace covered by long setae giving a felt-like texture (vs. velvet-like and hooked setae in Macrocoeloma and Pohleus gen. nov.); (ii) the carapace and pereopods are flattened in Thersandrus (vs. carapace subtriangular or piriform, not flattened and with cylindrical pereopods in Macrocoeloma and subglobose carapace and cylindrical pereopods in Pohleus gen. nov.); (iii) the orbital spines are reduced, not forming a hood in Thersandrus (vs. orbital spines long, blunt, forming a hood in Macrocoeloma and long, acute and forming a hood in Pohleus gen. nov.); all characters that prevented us from synonymising Thersandrus to Macrocoeloma. However, it is important to note that, based on the molecular results, Thersandrus should be transferred from Majidae to Pisinae as mentioned above.
Etymology. Generic name in honour of the renowned marine biologist and carcinologist Gerhard Werner Pohle (Atlantic Reference Centre, Huntsman Marine Science Centre). Gender masculine.  Diagnosis. Rostrum width half of interorbital length bifurcated, base elongated, fused, diverging abruptly forming a Y-shape. Pleonal somite II smooth; merus of second pereopod armed with a spine.

Pohleus septemspinosus
Description. Cephalothorax and appendages sparsely covered with short, velvet-like pubescence. Carapace subglobose wider than long, convex, with long lines of hooked and simple setae in all carapace regions, denser in rostral and lateral spines. Rostrum width half of interorbital length bifurcated, base elongated and fused, abruptly diverging forming a Y-shape, ending in acute tips. Interorbital region slightly depressed medially. Hepatic region broad. One metagastric spine. Four long, strong, conical lateral spines (two in each branchial region), in line with the cardiac spine. One short intestinal spine. Orbital region very prominent, eyes completely protected in orbit when retracted, ocular peduncle visible when not retracted. Pre-orbital spine acute, directed upwards, tip curved, longer than post-orbital spine, ventral margin of pre-orbital spine with a small crenulation; post-orbital spine curved upwards. Antennular fossae wider than long, margins smooth. Interantennular septum elongated, compressed laterally, forming distinct, ventrally-directed lobe. First and second antennal articles fused to epistome, suture between an-tenna and epistome visible, antennal gland opening near suture line. Basal article of antenna with three spines, one tubercle, not visible in dorsal view. Antennal flagella longer than rostral spines, behind rostrum in dorsal view.
Epistome anterior margin narrower than antennular fossae, smooth. Buccal field sub-quadrate, narrower at posterior edge with acute spine at anterolateral angle in line with antennal spines. Third maxillipeds completely covering buccal field. Exopod long, nearly reaching distal margin of merus. Pterygostomial region subtriangular, slightly inflated, separated from subhepatic region by marked groove, one long, strong spine slightly curved upwards on medial margin, visible in dorsal view.
Chelipeds equal, longer than pereopods in adults, more robust in adult males; females chelipeds shorter than males, slender. In males, ischium, merus, carpus and propodus segments granulate. Ischium with one prominent tubercle laterodistally. Merus with one spine on proximal half, one on distal margin. Carpus with four tubercles sparsely distributed. Dactylus arched in adult males, a small gap between fingers, distinctly shorter than palm. Cutting edges with sub-equal teeth in distal half, one distinct proximal tooth in larger males; distal half with dark brown colour in fixed specimens. Female ischium, merus, carpus and propodus with smaller tubercles than males, dactylus slightly arched, without gap between fingers.
Male thoracic sternites I-IV fused, broadly triangular, smooth, anterior half declivous in ventral view, forming a carina along the sterno-pleonal cavity margin. Thoracic sternal somite IV with lateral margins straight. Telson fully fitted to cavity, anterior margin smooth. Sternite VIII Pleonal somites I-VI, telson free in males and females, slightly raised medially forming low longitudinal ridge. Male telson sub-triangular, apex rounded. Female pleon markedly arched, with row of setae marginally. Female telson transversally ovate.
G1 longer than thoracic sternal suture IV−V, stout, straight, parallel, with torsion in distal half, apex bilobed; mesial lobe short, with tip curved upwards; lateral lobe long, slightly arched, ending in an acute tip; lateral margin smooth. G2 slender, straight, very short (one fifth of G1 length), with disto-medial process.
Description based on female lectotype (male characters modified from Bell 1836b). Cephalothorax and appendages slightly covered with short, velvet-like pubescence. Carapace subglobose wider than long, convex, with long lines of hooked and simple setae in all regions. Rostrum short, less than one-third of interorbital length, bifurcated, base elongated, fused, diverging abruptly forming a Y-shape, ending in acute tips. Interorbital region slightly depressed medially. Hepatic region broad. One metagastric spine. Four long, strong, conical lateral spines (two in each branchial region), in line with cardiac spine. One short intestinal spine. Orbital region very prominent, eyes completely protected when retracted, ocular peduncle visible when not retracted. Pre-orbital spine directed upwards, slightly curved on tip, longer than post-orbital spine, ventral margin of pre-orbital spine with small crenulation; post-orbital spine curved upwards. Antennular fossae wider than long, margins smooth. Interantennular septum elongate, laterally compressed, forming distinct ventrally-directed lobe. First and second antennal articles fused to epistome, with suture between antenna and epistome visible, antennal gland opening near suture line. Basal article of antenna with two spines, not visible in dorsal view: proximal spine smallest. An- tennal flagella longer than rostral spines, behind rostrum in dorsal view.
Epistome anterior margin narrower than antennular fossae, smooth, posterior margin slightly depressed. Buccal field sub-rectangular, narrower at posterior edge with one acute spine in anterolateral angle aligned with antennal spines. Third maxillipeds covering buccal frame when closed, leaving a small gap between ischia. Exopod long, nearly reaching distal margin of merus. Pterygostomial region subtriangular, slightly inflated, separated from subhepatic region by marked groove, with one long, strong spine slightly curved upwards on medial margin, visible in dorsal view.
Male chelipeds equal, longer than pereopods; covered with sparse granulation, unarmed. Dactylus arched in adult males, leaving small gap between fingers, distinctly shorter than palm. Cutting edges with sub-equal teeth in distal half, one distinct proximal tooth in larger males; distal half with light brown colour in fixed specimens. Pereopods short, slender, cylindrical. P2 longest; P3-P5 progressively decreasing in length. Dactylus slightly curved, covered with short setae.
Male thoracic sternites I-IV fused, broadly triangular, smooth, anterior half declivous in ventral view. Telson fully fitted to cavity, anterior margin smooth.
Female pleonal somites I-VI markedly arched, telson free, transversally oval, with a row of setae on margin and one small spine in first somite. Male pleon rather prominent, pleonal somites I-VI, telson free, somite II with a mesial tubercle. Somite III with slight elevations. Somite VI longest, with a mesial tubercle and a small projection each side.

Colour in life.
Light brown, covered with darker hair, first pair of pereopods reddish (Bell 1936b). Type locality. Costa Rica, Central America, Puerto Potrero, in sand at a depth of 23.7 m.
Remarks. Bell (1836) described Pericera heptacantha, based on two specimens as syntypes, one male and one female. The male syntype is considered lost and the female is deposited in the dry crustacean collection of the Oxford University Museum (OUM 13764) (Di Mauro 1982). Thus, the female syntype (OUM 13764) is here designated as the lectotype of Pericera heptacantha since it is the only specimen from the type series remaining (Fig. 5C, D). The locality Puerto Potrero in Central America probably refers to the Puerto Potrero, Guanacaste, in Costa Rica. Rathbun (1937: 136) examining Lithadia cumingii Bell, 1855, a species described from the same locality by Bell (1855)