DNA barcoding allows identification of undescribed crab megalopas from the open sea

Megalopas of 15 brachyuran crab species collected in the open sea plankton, and unknown until now, were identified using DNA barcodes (COI and 16S rRNA). Specimens belonging to the families Portunidae, Pseudorhombilidae and Xanthidae (Crustacea, Decapoda, Brachyura), and corresponding to the species Achelous floridanus, Arenaeus mexicanus, Callinectes amnicola, C. arcuatus, C. ornatus, C. toxones, Charybdis (Charybdis) hellerii, Portunus hastatus, Thalamita admete, Scopolius nuttingi, Etisus odhneri, Liomera cinctimanus, Neoliomera cerasinus, Pseudoliomera variolosa, and Williamstimpsonia stimpsoni, are described and illustrated, and compared with other congeneric species previously described. We also provide a new geographical record for N. cerasinus and the most remarkable features for each species.

Pleon (Fig. 2a Cephalothorax (Figs. 5a, 7a) Longer than broad; frontal margin with a pair of frontal submedian horns with a rostrum directed obliquely downwards; prominent tubercle on mesogastric, and a pair of lobes on protogastric and mesobranchial region; small tubercle on cardiac region; hepatic region inflated; dorsal organ present; eyes stalked.

Remarks.
Family Portunidae Rafinesque, 1815 The megalopas of Portunidae can be distinguished from those of other brachyurans for the following diagnostic combination of characters: presence of rostral spine projecting almost horizontally, the dactyl of the 5 th pereiopods paddle-like, pair of spines projecting posteriorly on 4 th sternite, and 5 th segment of pleon with lateral spines. Therefore, such features can also be observed in the megalopas of the following genera analyzed in this study:  29,30,47,48 the identification of the megalopa stage of portunids at specific level is a difficult task, because to the close similarity of its morphologies that makes all larvae remarkably similar. This task is more complicated when the larvae are from planktonic samples. Next, we highlight the most distinctive morphological features for each studied species.
Achelous floridanus (Figs. 2a, 3a-m, 4a, g, 8a) Mantelatto et al. 38 proposed, and recently corroborated by Mantelatto et al. 8 , the resurrection of genus Achelous De Haan 1833, a reassignment of nine American species and eleven (of twelve) eastern Pacific species respectively, formerly treated as Portunus Weber, 1795. Achelous now contains a total of 21 American species, between them: Achelous spinicarpus Stimpson, 1871 and Achelous spinimanus (Latreille, 1819), the only ones  www.nature.com/scientificreports/ the pair of spines projecting posteriorly on 4 th sternite on the sternum is much longer in A. floridanus, reaching the 3 rd pleonite but it is smaller in both remaining species; A. floridanus shows a characteristic rostrum curved upwards with numerous minute setae (Fig. 2a), and the orbital region presents 7 plumose setae (Fig. 4g), both characters are not present in A. spinicarpus and A. spinimanus. Finally, other important character that usually remains constant within the genus, such as the number of antennal segments and setation of endopod of the maxillule, is different between these species: A. floridanus shows an antenna 8-segmented while 7-segmented in A. spinicarpus and A. spinimanus.
Similar to the Achelous genus, the species included in Portunus show a high variability in certain morphological characters that have been considered key to characterize the genus. Therefore, there is not a set of morphological characters common to all species of the genus Portunus that allow distinguishing its megalopas from those of other genera of portunids. However, there are some characters that can be used to distinguish the megalopas already described, such as: presence of carpal spine in P. hastatus, P. pelagicus and P. gibbesii and absence in P. trituberculatus; ischial hook on cheliped present in P. pelagicus and P. trituberculatus, but absence in P. hastatus and P. gibbesi; and the 8-segmentation of the antennal segment in P. hastatus and P. pelagicus while it is 7-segmented in P. gibbesii. (Figs. 2d, 4d, j, 8d). Dineen et al. 59 described the larval development of C. hellerii but no provided detailed morphological description of the megalopa stage, only one photograph. Kurata and Nishina 52 described the megalopas of C. acuta and C. japonica, collected in Japan, but they are too brief to allow detailed comparison with other species of Charybdis (see Table 4). www.nature.com/scientificreports/ Although, the earlier published larval descriptions lack enough detail, it is noted that these species share the same external characters, such as: antennal flagellum 8-segmented, absent of carpal spine and ischial hook on cheliped (absent in C. feriata), and a coxal spine on 2 nd pereiopod (absent in C. feriata).
We compared them and found that all species share the absence of spines on cheliped and pereiopods. These characters combined with an 8-segmented antennal flagellum and setation of uropods (1,11) characterize the genus. Thalamita admete can be differentiated from the three species by antennal setation (Table 4).
Callinectes spp. (Figs. 2f-I, 4f, l-o, q, r, 8f-i) The megalopa stage known of Callinectes are: C. sapidus Rathbun, 1896 by Costlow and Bookhout 63 , and C. similis Williams, 1966 by Bookhout and Costlow 64 . The authors differenced the megalopa stage of these two species by size and examination of minute characteristics, but larval descriptions lack enough detail.
In this study megalopas of four species of the Callinectes are described: C. amnicola, C. arcuatus, C. ornatus, and C. toxotes as shown in Fig. 2f-i. The megalopas of Callinectes genus are strongly similar, being difficult to differentiate them only based on external characters. These six species share the most important external characters like the antennal flagellum 8-segmented, carpal spine on cheliped absent, ischial hook on cheliped present and coxal spine on 2 nd pereiopod absent. Even the carapace with tubercle on protogastric region with a row of 8-10 minute setae is the same for the four Callinectes species of this study.
The four species can be differentiated only by a thorough examination of the mouthparts (see Tables 2 and 4).

Family Xanthidae MacLeay, 1838
We have identified the megalopa stage of 5 species of xanthids. Xanthidae is a large and heterogeneous family containing about 124 genera and around 640 species 47,58 . The intergeneric variation in xanthids megalopas appears to be too significant to find constants group characters, as in adults 40,65 .
Family Pseudorhombilidae Alcock, 1900 Pseudorhombilidae includes 19 genera and 50 species 67 , but larval data are only known for 3 species. In the present study only one megalopa of the monospecific genus Scopolius have been identified, S. nuttingi (Figs. 5e, 7e, j, 8n), that lack of previous larval descriptions.

Discussion
DNA barcoding is a useful tool for the identification of crustaceans by assigning indeterminate specimens to known species 11,68,69 and faster method for the descriptions of brachyuran larvae 13 . As more research uses these genetic markers begin to be addressed questions relating to the biodiversity, ecology, and evolution of natural systems 70,71 .
But, although this molecular technique is widely used and popular, identifying unknown specimens through DNA barcodes requires a reference library containing morphologically-identified barcoded specimens against which unknowns can be compared 72 , highlighting the reliability of the database with adequate validation and detection of erroneous sequences 73,74 . While the use of DNA barcode databases for the identification of many marine species is an increasingly used technique in taxonomy, there are still large numbers of unexplored taxa, with little or no DNA barcode coverage or, sometimes, several species lack of sequence in the database is correctly assigned to higher taxa 75,76 . Thus, the results provide useful information to estimate species numbers, regardless of their formal taxonomic state, distribution and ecology as well as a framework for future taxonomic work 77,78 .
The availability of this information, especially for the family Portunidae, is of great importance not only to understand the life history of these species that are of commercial and therefore economic interest, but also because they have invasive potential. Species belonging to this family has a high dispersal potential because the adults are swimmers, have long larval periods and all stages of the life cycle can actively migrate long distances and, therefore, are dispersal agents both within the region of origin and to new environments 79 , which can turn them into invasive species, as is the case of the blue crab, Callinectes sapidus, in the Mediterranean. www.nature.com/scientificreports/ In the present work, Charybdis hellerii is the only one portunid that has been reported as an invasive crab 80 where it was collected, and this species continues to expand its range 81 in Caribbean Sea. However, although all the other Portunidae megalopas were collected within their known range, it is interesting to note that they were collected several miles from the coast, in the open sea, which highlights the potential to expand their range of distribution.This work focused mainly on the taxonomic applications of DNA barcoding to increase the knowledge of unknown brachyuran megalopa stages. This study provides a valuable larval morphology information about 9 portunids, 5 xanthids and 1 pseudorhombilid that will support future systematic, ecological, and biological studies about these families.  Table 5). The MALASPINA Circumnavigation Expedition was carried out with the general objectives of assessing the impact of global change on the oceans and exploring its biodiversity. The research cruise was conducted between December 2010 and July 2011, involved two oceanographic research vessels, the Hespérides and the Sarmiento de Gamboa, which covering a total of 42,000 nautical miles through the tropical and subtropical regions of the Atlantic, Indian, and Pacific oceans, sampling in a total of 147 stations. MAF research cruise was held to assess the carbon vertical active flux in the open sea due to zooplankton and micronekton and main responsible species. A total of 13 stations were sampled between 3 rd and 29 th April 2015 on board of the RV Hespérides, which crossed the tropical and subtropical Atlantic regions from Salvador de Bahia, Brazil, to Las Palmas, Canary Islands, Spain. (Fig. 1).

Methods
Sample processing. In both research expeditions, megalopas were collected from the superficial layer with a neuston net with a mesh size of 200 microns, hauled from 10 to 15 min at 2-3 knots. Samples were immediately fixed and preserved in 95% ethanol. All brachyuran megalopa stages were counted and sorted from the zooplankton samples using a stereomicroscope. Prior to DNA extraction, all larvae were examined morphologically and sorted into morphotypes according to the external characters.

Megalopas morphological descriptions.
For easier observation of larvae structures and setation under microscope, megalopas were first placed for 5-10 min in a watch glass with 2 ml of warm lactic acid before proceeding with the dissection of the body parts 82 .
Drawings and measurements of megalopa stage were made using a Leica MZ6 and microscope Nikkon Eclipse 90i with integrated camera lucida. All measurements were made using an ocular micrometer. Descriptions were based on all the collected megalopas of each species identified by DNA barcoding (see Table 1). The following measurements were taken for the megalopa: cephalothorax length (CL), measured from the rostrum (tip of www.nature.com/scientificreports/ rostrum in portunids) to posterior margin of cephalothorax; and cephalothorax width (CW), measured as the cephalothorax maximum width (mesobranchial regions). For the megalopas dorsal view, only the left pereiopods were drawn since one of the right pereiopods was used for molecular analyzes.
Descriptions were arranged according to the standards proposed by Clark et al. 83 and Clark & Cuesta 84 , and setal terminology follows the classification by Landeira et al. 85 . A detailed description of Achelous floridanus is provided while the others portunids descriptions are summarized in the Table 2. For Xanthidae and Pseudorhombilidae families, the specie of Neoliomera cerasinus is described in detail and the others xanthoids descriptions are summarized in the Table 3.

Molecular analysis.
The identification of the megalopas was based on partial sequences of the 16S rRNA and COI mitochondrial genes. Total genomic DNA of the megalopas from MALASPINA Expedition was extracted from muscle tissue from one pereiopod and incubated for 1-24 h in 300 µl lysis buffer (5 ml of 1 M Tris-HCl (pH 8), 1 ml 0.5 M EDTA, and 5 ml of 10% SDS solution to 400 ml of distilled water) at 65º C. Protein was precipitated by addition of 100 µl of 7.5 M ammonium acetate and subsequent centrifugation, and DNA precipitation was obtained by addition of 300 µl of isopropanol and posterior centrifugation. The resulting pellet was washed with ethanol (70%), dried, and finally resuspended in Milli-Q distilled water 82 . In the megalopas from MAF Expedition, total genomic DNA was also extracted from muscle tissue from one pereiopod, but the extraction process followed a modified Chelex 10% protocol by Estoup et al. 86 . Target mitochondrial DNA from the 16S rRNA and COI genes was amplified with the primers and the cycling conditions of the polymerase chain reaction (PCR) listed in Table 6. PCR products were sent to New Biotechnic, CISA-INIA, and Stab Vida companies to be purified and then bidirectionally sequenced.
Sequences were edited using the software Chromas version 2.0. With the obtained final DNA sequences were performed a BLAST (Basic Local Alignment Search Tool) on NCBI (National Center for Biotechnology Information) web facility on GenBank sequences database (http:// www. ncbi. nlm. nih. gov/ genba nk/) to get the best matches for identification. The COI sequences were also searched in the official Barcode of Life database (BOLD) (http:// v3. bolds ystems. org/ index. php/ IDS_ OpenI dEngi ne). Identifications were considered as positive when retrieved sequences showed similarity values greater than 99%, only differed in 1-3 or 1-7 mutations in 16S or COI, respectively, a more conservative limit than other previous works identifying decapod larvae considering > 98% 96 . Larval sequences for both genes are deposited in Genbank (see Table 1).

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. www.nature.com/scientificreports/