Morphology of planktonic zoeal stages of Palicus caronii (Decapoda, Brachyura), identified by DNA barcoding, provides novelties to Palicoidea larval systematics

The zoeal development of the brachyuran crab, Palicus caronii, comprises two zoeal stages and the morphology is described and illustrated in detail. The zoeae were collected in plankton samples from the Southern Ligurian Sea (Western Mediterranean). Although the morphology of the larval stages of this species was unknown, a combination of characters allowed the zoeae to initially be assigned to the Palicidae, based on the previous unique known first zoeal description of one species of this family. Later, the identification of the larvae as Palicus caronii was confirmed through molecular analysis. The morphological features of the zoeae that characterize the Palicidae and separate them from the Crossotonotidae are confirmed. Also, the larval development comprising only two zoeal stages observed in Palicus caronii, the peculiar and uncommon carapace surface setation, and the presence of anterodorsal and posterodorsal sensory dorsal organs suggest that these characters could be common to the Palicoidea.

The systematic placement of Cymopolia Roux, 1830 (now Palicus) has been an issue especially since larval descriptions were involved. Cano 13 described zoeae he attributed to Cymopolia and he assigned the genus to the Dorippidae. Later, Rathbun 14 included Cymopolia within Grapsoidea. Gurney 15 however, quoted both authors, stating that the zoea described by Cano 13 is "unmistakably a Dorippid". Later, Bourdillon-Casanova 16 , using the descriptions of Cano 13 , retained this genus within the Dorippidae, including it within her key to the brachyuran zoeae.
As the authors of the present study conclude that the zoeal description of Cano 13 was based on misidentification, currently the larval development of P. caronii maintains the status as undescribed. Consequently, the aim of the present work is to provide the first morphological description and illustrations of plankton caught zoeal stages of P. caronii from the Western Mediterranean Sea, identified through DNA barcoding of the mitochondrial cytochrome oxidase subunit 1 gene (COI).

Results
All the individuals were found all the year round in horizontal samples, showing a high peak in August 2014 with value of abundance of 0.27 ind. m −3 (Table 1), whereas no larvae were found in vertical samples. The larval stages identified were zoea I and II (24 and 17 specimens respectively), however, no megalops were collected. Five specimens (3 zoeae I and 2 zoeae II) were collected afterwards, exclusively for molecular analysis. Molecular analysis. The COI sequence obtained from the collected zoeae did not match with any in GenBank or BOLD Systems databases. Then, the zoeal sequence was compared with that from an adult P. caronii specimen collected from Cartagena, Spain (that is now deposited at the CBR of ICM-CSIC, code CMHR4, and in GenBank under the accession code MN782322) by the MEGALOPADN project, showing a 99% match, with only 4 nucleotide substitutions out of 663 bp being observed.
The zoeal sequence was edited and uploaded to BOLD Systems database under the project "BMZ -Barcoding Mediterranean Zooplankton", assigning it the Barcode Index Number ADL4122.
Larval description. The first zoeal stage is described in detail, whereas for the second stage only morphological differences (e.g., number and/or type and position of setae) are noted.

Discussion
Collection of ovigerous females in good condition for identification and viable laboratory larval cultures is the traditional method for obtaining zoeae and megalopa material for morphological description. Netting plankton and identifying brachyuran larvae to species based on morphology has proved extremely difficult, if not misleading. Capturing egg bearing specimens of P. caronii has proved problematical. The present study, however, managed to collect plankton zoeae of this Mediterranean palicid species and confirmed its identification by DNA www.nature.com/scientificreports www.nature.com/scientificreports/ barcoding. This technique represents a valuable and faster method for the description of brachyuran larvae and additional characters for the appraisal of current systematics based on adult characters 17 .
Clark et al. 1 were the first to describe the zoeal stages of Palicoidea species, namely Crossotonotus spinipes and Pseudopalicus serripes. Based on their descriptions of the first zoeae only, they proposed characters that allowed them to support the classification of palicoids into the Crossotonotidae and Palicidae respectively. Although the Palicoidea is a relatively small superfamily with 69 assigned species 3 , no further larval data have been published. The first zoeal stages described by Clark et al. 1 were included in Table II by Clark and Cuesta 18 of larval characters defining brachyuran families. From their table, it is apparent that the main shared familial characters included antenna type and mouthpart setation patterns and these could be considered as features at the superfamilial level. Characters that currently distinguish the Crossotonotidae and Palicidae are presence/absence of lateral spines on the cephalothorax, fourth pleonite with or without dorsolateral processes, and presence/absence of one additional small lateral spine in telson furcae. From the present study of P. caronii, the presence of lateral spines in the cephalothorax, the absence of both dorsolateral processes on the fourth pleonite, and the small additional lateral spine on furcae confirm that these characters are consistent within the Palicidae and distinguish them from crossotonotids.
The carapace (Fig. 5a-d) and pleon in dorsal view (Fig. 5e) of P. caronii zoeae have an unusual surface morphology as highlighted in the photos obtained from scanning electron microscopy, although clearly visible at optical microscopy. They are covered with mushroom shaped globular outgrowths that appear to be unique to this species. This feature is in contrast to the carapace and pleon surface morphology of C. spinipes and P. serripes which are reticulated and highly setose 1 . Such unusual surface morphology of the carapace and pleon may be considered as an additional common character of palicoid superfamily.
The zoeae of P. caronii present two sensory dorsal organs (SDO), one on the anterodorsal and another one on the posterodorsal regions of the carapace (Figs. 1a, 3a,b and 5a). These protuberances, although not described, are also present in the zoea I of C. spinipes and P. serripes (Fig. 1a,b and 2a,b, respectively 1 ). Therefore, SDOs could be considered as another typical feature of palicoid zoeae. SEM scanning provided details of posterior SDO ultrastructure with a central pore and five or probably six surrounding sensory plates (Fig. 5c,d). This arrangement is similar to that described for other brachyuran larvae (Fig. 5b-e,k-m 19 , 20 ) as a "cuticular organ complex".
Clark et al. 1 only described the zoea I morphology of two species, but the presence of biramous third maxillipeds and pereiopods buds in both cases suggested that these zoeae hatched in an advanced stage of development. The authors, however, were not able to specify the exact number of zoeal stages in palicoids larval development.
The present study appears to confirm a zoeal phase with only two zoeal stages. The zoea II of P. caronii has all the features developed prior to the metamorphosis to megalopa (i.e. antennal endopod, third maxilliped, pereiopods, and pleopods buds well-developed). Despite of being the terminal stage, the zoea II of P. caronii does not possess a mandibular palp bud, in common with the same stage of Inachus and Macropodia species 18 . More descriptions of palicoid zoeal development are required to confirm whether this is also a familial character, or if it is just related to the short zoeal development with only two zoeal stages.
The description of the zoeal phase of P. caronii sheds some light on palicid development. Nevertheless, only the finding of the megalopa will give a complete image of larval morphology of this group. The use of DNA barcoding on plankton samples, focusing on megalopae, may be the key approach to achieve such a goal.

Materials and Methods
Fieldwork. Larvae were collected in the Southern Ligurian Sea (Western Mediterranean Sea) 12.5 NM off the Tuscan coast (Italy). The sampling area is characterized by a peculiar extension of the continental shelf and shallow waters (about 100 m). The three sampling stations S1 (43°29′40″ N, 10°01′45″ E), S2 (43°28′10″ N, 10°01′55″ E) and S3 (43°27′10″ N, 10°03′00″ E) were aligned along a transect parallel to the coast as shown in Fig. 6, above bottom depths ranging from 109 to 114 m. The area was investigated for one year through seasonal sampling,

Molecular analysis.
To recover the minimal amount of DNA required for COI amplification, total DNA was extracted from whole individuals. DNA extraction, amplification and sequencing were carried out by CCDB www.nature.com/scientificreports www.nature.com/scientificreports/ (Canadian Centre for DNA Barcoding), one of the main analytical nodes for the International Barcode of Life Project (iBOL), using standard procedures 22 .
The COI sequence of the zoeae was amplified using the primers ZplankF1_t1 (tgtaaaacgacggccagtTCTAS-WAATCATAARGATATTGG) and ZplankR1_t1 (caggaaacagctatgacTTCAGGRTGRCCRAARAATCA), a specific set of primers developed for the zooplankton, which significantly increase the average amplification success to barcode micro crustaceans 23 . The sequence will be uploaded on the Bold Systems database and a Barcode Index Number (BIN) will be assigned 24,25 . Morphological description. Ten specimens for each zoeal stage were measured, using a B500TPL microscope with ocular micrometer. Measurements include: rostro-dorsal length (RDL) from the tip of the rostral spine to the tip of the dorsal spine; cephalothorax length (CL) measured laterally from the frontal margin (between the eyes) to the posterior margin of the cephalothorax; rostral spine length (RL) from the base to the tip of the rostral spine; dorsal spine length (DL) from the base to the tip of the cephalothoracic dorsal spine; carapace width (CW) from tip to tip of lateral spines; antennal length (AL) from the base of the eye to the tip of the spinous process.
Following Clark and Cuesta 16 , 5 specimens for each zoeal stage were dissected under an Optika SZM2 stereo microscope and mounted in glycerine on semi-permanent slides. Drawings were made using a Leitz Dialux 22 microscope equipped with camera lucida.
Samples of zoea I and II have been deposited in the Invertebrate Collection of the Museo Regionale di Scienze Naturali of Torino (Italy), under the accession code MRSN Inv74.
Larval description and figures were carried out according to Clark et al. 26 and Clark and Cuesta 18 . The description of the setae follows the definition and classification of Garm 27 , except for the new undescribed outgrowth found covering the surface of the carapace and the dorsal part of the pleon.
To carry out high definition images of the external and superficial morphology of the zoeae, scanning electron microscope in Low Vacuum mode was used (SEM Jeol JSM IT-300 LV), after pre-treatment of the specimens through a graded ethanol series and critical point dehydration.
Ethical approval. This article does not contain any studies with human participants performed by any of the authors. All applicable international, national, and institutional guidelines for the care and use of animals were followed.