A Mysterious World Revealed: Larval-Adult Matching of Deep-Sea Shrimps from the Gulf of Mexico

The identification of deep-sea (>200 m) pelagic larvae is extremely challenging due to the morphological diversity across ontogeny and duration of larval phases. Within Decapoda, developmental stages often differ conspicuously from their adult form, representing a bizarre and mysterious world still left to be discovered. The difficulties with sampling and rearing deep-sea larvae, combined with the lack of taxonomic expertise, argues for the use of molecular methods to aid in identification. Here, we use DNA barcoding combined with morphological methods, to match larval stages with their adult counterpart from the northern Gulf of Mexico and adjacent waters. For DNA barcoding, we targeted the mitochondrial ribosomal large subunit 16S (16S) and the protein coding cytochrome oxidase subunit 1 (COI). These data were combined with previous sequences to generate phylogenetic trees that were used to identify 12 unknown larval and two juvenile species from the infraorder Caridea and the suborder Dendrobranchiata. Once identified, we provide taxonomic descriptions and illustrations alongside the current state of knowledge for all families. For many groups, larval descriptions are missing or non-existent, so this study represents a first step of many to advance deep-sea larval diversity.


Introduction
In order to understand the evolution, distribution and ecology of marine organisms, as well as their impact on community and ecosystem processes, it is important to study their life history and developmental biology [1][2][3]. Decapod crustaceans, including shrimps, lobsters and crabs and are well-known due to their economic importance in the food, aquarium and pharmaceutical industries [4,5]. However, much less is known about their often-complex life histories. Decapods have numerous reproductive strategies, and those with sexual reproduction produce eggs which are either deposited directly in the bottom of the sea floor, remain attached to the parents, or are released as free moving organisms into the pelagic environment [6]. Many species progress through a series of larval stages (i.e., nauplius, mysis, zoea, phyllosoma), often representing bizarre forms unidentifiable from their adult counterpart [7] (Figures 1 and 2). The duration of the larval stages varies between and within taxonomic groups, sometimes lasting several months before settling as juveniles or benthic adults [8][9][10][11]. Due to the morphological disparity across ontogeny and duration of larval phases, the identification of planktonic decapod larvae, especially those in the deep sea (>200 m), is extremely challenging.
Descriptions of decapod larval stages are limited, with most of the preexisting literature focused on shallow-water species of economic interest because of their food and/or ornamental value [12][13][14]. For example, in the Gulf of Mexico, larvae stages are known from the shrimp family Penaeidae [15][16][17], the crab families Menippidae (stone crabs) and Portunidae (swimming crabs) [18][19][20][21] and the spiny lobster family Palinuridae [22]. In the  Our knowledge of pelagic or benthic deep-sea decapod larvae is inadequate or even non-existent and is further complicated by the technological demands and expense of sampling in deep oceanic waters. Extensive knowledge of taxonomy is required to achieve reliable larval identifications, and because this requires specialized training and years of practice, most researchers have difficulty recognizing larval stages in a plankton sample [26,27], especially those in the deep sea [28]. Those that have been identified come from larval-rearing experiments of females, and because males and females differ dramatically in larval morphology, several have been incorrectly identified [26,29]. Another factor that complicates identification is that the literature can be very old and difficult to access [7,29], however adequate library resources can aleviate this problem. Due to the abovementioned reasons, illustrated guides (based on external morphological characters that can be observed under a stereomicroscope) are necessary to aid future investigations and identifications, especially for those with limited taxonomic training.
Morphological descriptions can be given alongside molecular methods (DNA barcoding) to fully characterize and document larval-adult linkages. DNA barcoding is a molecular method for fast and accurate species identification and can be particularly useful in early life stages that differ conspicuously from their adult form [30,31]. Although rearing experiments have facilitated the taxonomic identification of larvae from plankton samples, most are difficult (or impossible) to breed and maintain in the laboratory. Molecular approaches, such as DNA barcoding, can be an excellent alternative or complementary method for larval identifications [32][33][34][35]. This method does require a reliable database of adult barcodes that are linked to vouchered museum specimens in zoological collections. When these adult datasets are available, larvae can be targeted from similar localities (or a species distributional range) and matched back to adults using DNA barcoding genes (ex. 16S and COI) and phylogenetic trees. A very recent barcoding study on adult deep-pelagic crustaceans was conducted in the Gulf of Mexico and adjacent waters [35], and we plan to use this dataset (alongside previously published datasets) to match unknown larvae collected on research expeditions into the northern Gulf of Mexico and adjacent waters over the past 5 years.
Adult-larval linkages are critical because they can enhance our basic biological understanding of the species under study. First, documenting and describing larval stages allows for the correct identification of a species during development. The correct identification of a species is arguably the most important first step to any scientific investigation. Secondly, larval-adult linkages have allowed for the description of complex life cycles and distributional ranges for many species [36][37][38]. An example is the deep-sea shrimp, Cerataspis monstrosus Gray, 1828, which can be found in the abyssal plains (up to 5000 m in the Gulf of Mexico) but has a larval form (Cerataspis-"monster" larvae) found in the mesopelagic (~500 m) [39]. Lastly, the correct identification and distribution of larvae is critical to understanding the food web dynamics in the Gulf of Mexico, as crustacean larvae are often the main food source for small and large migratory fishes, cephalopods and some marine mammals [40][41][42][43]. Overall, these adult-larval linkages do not only allow for advancements in taxonomy and systematics, but also provide fundamental information for studies in ecology and evolution.
In this paper we will use a molecular technique, namely DNA barcoding, to match early-life stages with their adult counterpart in an effort to better understand the life history and distribution of deep-sea (~200-1500 m) decapod crustaceans from the northern Gulf of Mexico and adjacent waters. We provide larval-adult matching for 14 species (12 larval, 2 juvenile) based on DNA barcoding and phylogenetic methods. For each species, detailed morphological illustrations and taxonomic descriptions of diagnostic characters are provided. Of the 14 species in this paper, only four have some previous larval knowledge: Heterocarpus ensifer, of which only the early four zoeal stages are known [44,45], Plesionika edwardsii, of which the seven first zoea stages are known [46], Funchalia villosa, of which some taxonomic data on its postlarva is known [47] and Cerataspis monstrosus of which some of the mysis stages are known [48]. We hope this research can guide future studies and aid in the identification of deep-sea crustacean larvae from the Gulf of Mexico.

Sample Collection
All material used in this study was collected during eight research expeditions totaling 126 days at sea (Supplementary Table S1). Six of the eight research cruises were in the Gulf of Mexico on the R/V Point Sur as part of the Deep Pelagic Nekton Dynamics of the Gulf of Mexico (DEEPEND) consortium (http://www.deependconsortium.org, accessed on 14 September 2021). The other two cruises were in the Florida Straits on the R/V Walton Smith as part of a National Science Foundation grant to study bioluminescence and vision in the deep sea. During the DEEPEND cruises, every collection site was sampled during the day (entire water column from the surface to 1500 m depth, sampled at noon) and at night (surface to 1500 m depth, sampled at midnight). Sampling occurred during the wet (August) and dry (May) seasons from 2015 to 2016 and one during the dry (May) season from 2017-2018. Gulf of Mexico samples were collected with a Multiple Opening/Closing Net and Environmental Sensing System (MOC-10) composed of six 3 mm mesh nets, allowing for collected specimens to be assigned to a depth bin (0-200 m, 200-600 m, 600-1000 m, 1000-1200 m, and 1200-1500 m; the sixth net sampled from 0 to 1500 m). Samples from all nets and depths were included as part of this study. More details on DEEPEND net sampling and methods can be found in [49]. Florida Straits samples were collected with a 9 m 2 . Tucker trawl fitted with a cod-end capable of closure at depth (for details see [50]), allowing for discrete depth sampling. All sampling was conducted in the midwater, from 0-800 m.
The contents of each net were placed in a large tray and crustacean larvae were sorted and preserved as whole-specimens, either in 80% EtOH or an RNA-stabilizing buffer (RNAlater) and stored at −20 • C onboard the vessel. Upon returning samples to the lab, all batch-stored individuals were transferred to the Florida International Crustacean Collection (FICC). All individuals selected for DNA barcoding were then given a unique voucher ID in the FICC database, including all relevant collection metadata. Metadata included collection date, time (day or night), collection locality and GPS coordinates, and depth. The unique voucher number ensured that the resulting DNA barcode matches to one and only one individual. Total genomic DNA was extracted from muscle tissue of the abdomen or the 3rd to 5th pleopod. Tissue collected from each vouchered specimen was stored in 80% EtOH at −20 • C and voucher specimens were preserved in 80% EtOH and deposited in the FICC.
We adopt the terminology of [51] for Dendrobranchiata and [52] for Caridea, to standardize the different life stages. The number of specimens examined per stage (N) is referred in each description. Measurements taken were Carapace length (CL), measured from the tip of rostrum to the posterior margin of the carapace and Total length (TL), corresponding to the distance from the tip of the rostrum to the posterior end of telson.

DNA Extraction, PCR and Sequencing
Total genomic DNA (gDNA) was extracted from muscle tissue of the abdomen or the 3rd to 5th pleopod using DNeasy ® Blood and Tissue Kits (Qiagen, Valencia, CA, USA). When the tissue did not completely digest, 10 µL of 10% DTT and an additional 10 µL Proteinase K were added, and samples were incubated until complete digestion was achieved. Visualization of total genomic DNA was performed using 2% agarose gels, run at 100 V for 90 min, and the DNA concentration was measured using a dsDNA HS Assay kit on the Qubit 2.0 Fluorometer (Invitrogen, Life Technologies, Carlsbad, CA, USA).
Two partial mitochondrial genes were selected due to their informativeness in decapod barcoding studies. These included the partial 16S large ribosomal subunit and cytochrome oxidase I (COI) gene, totalling~550 basepairs (bps) and~600 bps, respectively. All primers included M13 tails as a universal tag (Invitrogen, Carlsbad, CA, USA) ( Table 1).  Table 1), 1 min at 72 • C; final extension 2-3 min at 72 • C. Both forward and reverse strands were amplified, and all PCR products were sent to GENEWIZ (South Plainfield, NJ, USA) for sequencing. Consensus sequences were generated within Geneious 9.1.7 (Biomatters Ltd., Newark, NJ, USA) and primer regions and non-readable segments at the beginning of the sequences were manually removed prior to multiple sequence alignment. To check for pseudogenes, all six possible reading frames for the COI gene were translated to ensure stop codons were not present. On several occasions, several individuals of the same species were included to help identify contamination. All obtained sequences were deposited in the GenBank database (Supplementary Table S1).

Phylogenetic Tree Construction
Newly generated larval sequences were aligned with a subset of data generated in [35] alongside other sequences from previously published studies (Supplementary Table S1) to help identify the unknow larvae. The Multiple Sequence Alignment Tool (MAFFT) with the E-INS-i algorithm [57] was used to align the DNA sequences. ModelFinder [58] was used to determine the model of evolution that best fit each gene. Maximum Likelihood (ML) analyses were conducted using IQ_TREE 2.0.4 [59] and a search for the best-scoring tree with 1000 replicates [60] was performed. Ultrafast Bootstrapping (UFBoot) was used to assess confidence in the resulting topologies. Bayesian Inference (BI) analyses were performed using parameters identified by ModelFinder and conducted in MrBayes (v.3.2.6) [61]. Both single-gene trees (16S and COI) and concatenated trees (16S + COI) were constructed for each major group using ML and BI approaches. Trees were visualized in FigTree v.1.4.2 and topologies were compared across all phylogenies for congruence. All support values (UFBoot and posterior probabilities) are listed on the corresponding branch. High support is indicated by values >95.

Larval-Adult Identification using DNA-Barcoding
Phylogenetic trees were constructed to help in identification and evolutionary relationships should not be inferred based on these findings. In total, 28 larval individuals were included in this study. Our DNA barcoding efforts resulted in a total of 25 de novo 16S sequences and nine de novo COI sequences from these larvae. Using a subset of the dataset generated from [35] and previous studies, in combination with these newly generated larval sequences (Supplementary Table S1), the final tree (16S + COI) included 51 total species from the infraorder Caridea and suborder Dendrobranchiata ( Figure 3). Using this phylogeny, we were able to successfully match 14 larval and juvenile species (=16 developmental stages) with their adult counterparts. From the infraorder Caridea, the larvae represented six families, eight genera and eleven species. From the suborder Dendrobranchiata, the larvae represented two families, three genera and three species.  Figures S1 and S2). Carapace ( Figure 4A). Rostrum straight, reaching the end of the cornea, unarmed; epigastric spine present; eyes pedunculate.
Antennule ( Figure 17B). Peduncle 3-segmented, article 1 the longest armed with 5 simple setae, article 2 also with 3 simple setae and article 3 the smallest, with one simple setae and two flagella distally, flagella subequal in size.
Antenna ( Figure 19C). Protopod 3-segmented, article 1 and 2 unarmed, article 3 with 5 small spines and a flagellum; exopod flattened, subtriangular, with a slender and pointed projection on its distal region and 13 pointed projections on the superior margin and 64 plumose setae in the inferior margin.
Uropod ( Figure 26F). Endopod and exopod well developed, both missing setae. Telson ( Figure 26G) enlarged, subrectangular, with two pairs of lateral spines and posterior margin bearing row of 4 pairs of minute spinules and 2 pairs of spines on outer margin. Mysis. Size: 9 mm (Carapace length); 21 mm (Total length). N = 3. Carapace ( Figure 27A) with two lateral swollen process near the posterior margin, rostrum long, extend until the end of the article 1 of the antennule; orbital spine as a projected bump; antennal spine is a small bump; anteroventral margin bearing 1 strong and curved pterygostomial spine; eyes pedunculate. Pleon ( Figure 27A) with 6 somites, no spines or setae. Pleopods without setae. Antennule ( Figure 27B). Peduncle 3-segmented, article 1 the longest, slender, with 3 simple and 9-12 plumose setae, article 2 also with 6 plumose setae in the outer margins and article 3, the smallest with 3 lateral simple setae and two distal flagella, outer flagella unarmed and inner flagella with 4 lateral simple setae and 2 distal setae.
Telson ( Figure 30F). (Damaged in the specimen) Subrectangular, distal margin bearing row of 13 min spines and 3 pairs of spines on lateral margin, small simple setae between the lateral spines.  Carapace ( Figure 31A) with rostrum short, armed with 5-7 dorsal spines, epigastric tooth present.
Telson ( Figure 32G) enlarged, subtriangular, distal margin with a pointed projection, 3 pairs of spines near the distal margin, lateral margins with small simple setae.

Discussion
Here, we use DNA barcoding to successfully match 16 developmental stages and 14 larval and juvenile species with their adult counterpart. In the Section 3 we provide the phylogenetic evidence for the larval-adult identification accompanied by taxonomic descriptions and illustrations. Below, we summarize our main findings with a brief description of the current state of knowledge for deep-sea larval biology across each group. For many of these deep-sea shrimp species and some families, larval descriptions are scarce or non-existent.
It is important to note that many of these species likely have multiple larval stages and much more work is needed to fully describe the life history. Developmental plasticity in the number of larval stages is common for shrimps and several factors, including temperature, salinity and available food, can influence this variability [62][63][64][65][66][67]. These factors affect the molting cycle and can produce morphological differences across larvae stages [68]. Even at the population level, the same species can have a different number of larval stages and variation in the morphology (ex. the armature of thoracopods and pereopods [6,69].

Family Aristeidae
The family Aristeidae contains nine genera, of which only 6 are present in the Gulf of Mexico [39,70]. The species in this family predominantly occupy deep-sea benthic habitats, although there are species that inhabit the meso-and bathypelagic zones, where they play an important role in the oceanic food web [71,72]. For almost 180 years, the larval stages of some genera within this family including Plesiopenaeus (=Cerataspis) and Aristaeomorpha Wood-Mason, 1891 were called "Cerataspis". These "cerataspis-like" individuals were so morphologically distinct and bizarre they were considered a valid genus and their affinity to other groups was unknown [73]. However, in 2012, Bracken-Grissom et al., used molecular techniques to unravel the mystery surrounding one larval form called Cerataspis montrosus, identifying the adult counterpart to be Plesiopenaeus armatus within the family Aristeidae. Larval stages of these deep-sea shrimp are frequently found in the stomach contents of fish and collected in nekton nets in shallow water and deep-sea waters. In the Gulf of Mexico, the mysis stage of Cerataspis monstrosus Gray, 1828 is the only record from this family [74] and the larval stages of Aristeus antennatus and Aristeomorpha foliacea have been previously recorded [75][76][77][78].
In the present study, two mysis stages of Hemipenaeus carpenteri and an additional mysis stage of Cerataspis monstrosus are illustrated. Identifications were made using sequences obtained by [74]. In the case of the two zoea stages of H. carpenteri, we have found that both stages morphologically resemble the mysis II and mysis III stages described by [48] for Cerataspis monstrosus. This finding verifies that it is typical for multiple species within the family Aristeidae to present these bizarre "cerataspis-like" pelagic larval stages. In the case of the zoea Cerataspis monstrosus, our material appears to be an undescribed mysis stage and could be a more advanced developmental stage that the ones described by [48] due to the reduction of the exopods in the 1-5 pereopods. Nevertheless, more material is needed to confirm this result.

Family Penaeidae
The family Penaeidae consists of 27 genera, of which only eight are present in the Gulf of Mexico [39,70]. Many species within this family are considered valuable resources for fisheries and aquaculture, both in tropical and subtropical regions [79,80]. Many of the larval stages of species of commercial interest are known, such as the genera Penaeus Fabricius, 1798, Metapenaeopsis Bouvier, 1905, Rimapenaeus Perez-Farfante and Kensley, 1997 and Trachypenaeus Perez-Farfante, 1972, nevertheless, there are still problems in the identification of these larval stages [25,[81][82][83].
In this paper, we have a juvenile Funchalia villosa which was identified using sequences obtained by [35]. This species is pelagic, and it is known to perform diel vertical migrations, descending to 2608 m deep during the day and migrating to shallow water of around 50 m deep at night [84,85]. Our material does not present exopods on the pereopods and resembles an adult specimen according to [86].

Family Acanthephyridae
The family Acanthephyridae consists of seven genera, with six genera present in the Gulf of Mexico: Acanthephyra, Heterogenys, Hymenodora, Ephyrina, Meningodora and Notostomus [39,70]. This family inhabits only deep waters and meso-bathypelagic habitats, and many perform daily vertical migrations [87][88][89]. Past studies examining the larval biology of this family within the Gulf of Mexico is lacking, however some work does exist for species of Acanthephyra [90][91][92][93]. Egg size across the family varies drastically and much work is still to be done [67]. Past studies have divided the family into two major groups based on developmental characertistics. Group one consists of the genera Ephyrina and Hymenodora which have large lipid-filled eggs and five or fewer zoeal stages, whereas group two includes the genera Acanthephyra, Meningodora and Notostomus which have small eggs and nine or more planktotrophic stages [67,90].
In our study we identified one zoea stage of Meningodora longisulca and Ephyrina ombango, and one decapodite stage of M. vesca using sequences from [35]. In all cases, these are the first descriptions and illustrations of developmental stages belonging to these pelagic species. For Meningodora longisulca, the zoea is half the reported size for the adult and differs in several morphological characteristics. These include a zoea with (1) an unarmed rostrum in contrast to an armed adult rostrum with 7-10 dorsal spines, (2) the cornea wider than the peduncle in contrast to the adult cornea slightly narrowed than the eyestalk, and (3) underdeveloped mouthparts. For Meningodora vesca, the decapodite has characters very similar to those of the adult [87,94]. For Ephyrina ombango, the zoea differs from the adult in the shape of the rostrum. This includes the zoea possessing a blunt projection compared to the adult rostrum directed slightly anterodorsally [87][88][89].

Family Alvinocarididae
The family Alvinocarididae consists of 9 genera, but only the genus Alvinocaris is present in the Gulf of Mexico [39,70]. The members of this family are understudied and inhabit deep-sea cold seeps and hydrothermal vents areas around the world, with depths that vary from 250 to 4500 m [95][96][97]. This family is fraugth with taxonomic problems because larval stages have been erroneously described as new genera or species [98,99].
Across all alvinocaridids, only the morphology of the first zoea of four species is known including Alvinocaris muricola Williams, 1988, Mirocaris fortunata (Martin and Christiansen, 1995), Nautilocaris saintlaurentae Komai and Segonzac, 2004 and Rimicaris exoculata Williams and Rona, 1986 [98]. In this study we found a decapodite stage of Alvinocaris stactophila, and to identify this material, we used the sequences obtained by [99]. Our material is close to the adult size range; however, it still differs in some characteristics. This includes the shape of the decapodite carapace which is longer than wide, and the adult carapace is almost as long as wide. Differences also exist in mouthparts including the armature of the maxillipeds 2 and 3 lacking setae, which is a larval characteristic of this family. However, the remaining mouthparts such as the maxillula, maxilla and maxilliped 1 present an armature similar to that described for the adult [93,99]. As reported for several other species of alvinocaridids, the larval stages of Alvinocaris stactophila are pelagic [100,101]. This was confirmed with our material since the decapodite was captured using a MOCNESS trawl at depths of 600-1000 m. The adult of this species is benthic, inhabiting cold seeps at a depth of 534 m, making this a new depth record for this species. It is still unknown how the pelagic larval forms locate cold and hydrothermal seeps as they are presumably located 10 s to 100 s of meters from these ecosystems.

Family Eugonatonotidae
The family Eugonatonotidae consists of only one genus, Eugonatonus Schmitt, 1926, which is present in the Gulf of Mexico [39,70]. The collection of this deep-sea species has been considered rare or very unusual [102,103]. The lack of knowledge surrounding the larval stages of the species has led to the description of Galatheacaris abyssalis and the creation of the family Galatheacarididae (=Eugonatonotidae) and the superfamily Galatheacaridoidea (=Nematocarcinoidea) [104]. This mistake was later corrected by [105] which found the new discovery to be a larval stage of Eugonatonotus chacei Chan and Wu, 1991.
Our material contains a zoea stage of Eugonatonotus crassus, which was identified using the sequences of [106]. De Grave et al., [105], states that this genus of benthic shrimp possibly has several planktonic zoeal stages. This is the first time that illustrations for the zoeal stage of Eugonatonotus crassus have been documented.

Family Nematocarcinidae
The family Nematocarcinidae consists of five genera, of which only two are present in the Gulf of Mexico [39,70]. The members of this family represent a wide bathyal distribution and can be found associated with the benthic community [87,88,107]. Illustrations of larval stages have only been recorded for very few species within the genus Nematocarcinus [65].
In the present study, the zoea and decapodite of N. rotundus and the zoea of N. cursor are illustrated. To identify this material, we used the sequences of [108] and sequences obtained from adult specimen material found in the Florida International Crustacean Collection (FICC) that were identified using [88,107]. It appears that both zoeal stages of N. rotundus and N. cursor are advanced based on size [65]. As for the decapodite of N. rotundus, the specimen shows characters similar to those of the adult. These include a short rostrum (with dorsal teeth) that does not exceed the article 2 of the antennule and a telson that does not exceed the uropods. This is the first time that illustrations of these developmental stages have been recorded for N. cursor and N. rotundus.

Family Oplophoridae
The family Oplophoridae consists of three genera, all of which are present in the Gulf of Mexico [39,70]. The members of this family, like those of the family Acanthephyridae, inhabit deep waters in meso-bathypelagic habitats and perform daily vertical migrations [87,89]. For this family, larval stage illustrations have only been reported for two species, Oplophorus spinosus and Systellaspis debilis [67,90,91].
In this paper, information on the decapodite stage of Systellaspis braueri is provided for the first time and identifications were given using the sequences obtained by [35]. The complete larval development of S. debilis has four zoeal stages and one decapodite stage, which suggests that the species of this genus are lecithotrophic and have a short larval development with few stages. Lecithotrophy is considered an adaptation to the deep-sea environment where they live [67].

Family Pandalidae
The family Pandalidae consists of 19 genera, of which only three, Heterocarpus, Pantomus and Plesionika, are present in the Gulf of Mexico [39,70]. The representatives of this family are distributed world-wide, and many species inhabit deep waters [109]. In addition, due to their size, some species are of commercial interest [110][111][112][113]. The number of zoeal stages varies greatly among species within the family Pandalidae, where the complete life cycle of these species has been studied. For example, in the genus Pandalopsis (=Pandalus), the life cycle is completed in only 3-5 zoeal stages, while in the genus Pandalus Leach, 1814 (in Leach, 1813-1815), depending on the species, the life cycle is completed in 2-7 zoeal stages [114]. It is also known that species within the genus Plesionika have at least 7 to 8 zoeal stages [115].
In the present study, the juvenile stage of Plesionika ensis, decapodite stage of P. edwarsii and a zoea stage of Heterocarpus ensifer are presented. All material was identified using sequences obtained by [35,116]. Although the complete larval development of species belonging to the genus Plesionika are still unknown [114,115], past studies have reported the larval stages from seven species. This includes the following: the first zoeal stages for Plesionika acanthonotus (Smith, 1882), P. crosnieri Chan and Yu, 1991, P. ortmanni Doflein, 1902 and P. semilaevis Bate, 1888; the first to the seventh zoeal stages for P. edwardsii (Brandt, 1851); the first to the eighth zoeal stages for P. grandis Doflein, 1902; and the first five zoeal and the decapodite stages for P. narval (J. C. Fabricius, 1787) [10,46,116,117]. In the material presented here, the zoeal stages of the species in the genus Plesionika have the dorsal connection between carapace and abdomen at an almost 180 • angle, an eye peduncle narrowed at base, antennular peduncles strongly concave, a well-developed rostrum since the first stage and with dorsal spines in later stages, supraorbital spines present, and pereiopod 5 without an exopod [114]. The decapodite stages have a carapace with anterior and posterior dorsomedial tubercles, supraorbital spines present, a mandible without palp, the first four pereopods with exopods, and a carpus of pereiopod two not multi-articulated [114]. The material of Plesionika ensis represents a juvenile specimen, in which, the pereopods show reduction of the exopods, the mandibular palp is developed and the carpus of pereopod two is subdivided. Our material of P. edwarsii seems to be a decapodite stage due to the absence of the mandibular palp, the non-segmentation of the carpus in pereopod two, and the reduction in the pereopodal exopods. The reduction of exopods in the pereopods has also been recorded for the decapodite state of Plesionika narval [114].
Regarding Heterocarpus ensifer, only the first four zoeal stages of this species have been recorded [44]. Our material appears to be a more advanced zoea stage, presenting characters common to the zoea of the family Pandalidae, such as dorsal connection between carapace and abdomen at an almost 180-degree angle, the eye peduncle narrowed at base, well-developed rostrum, and supraorbital spines present. However, our material lacks a mandibular palp, subdivision of the carpus of pereopod two and has exopods on pereopods 1-4. These findings support our hypothesis that our Heterocarpus material is from a more advanced zoea stage. Our material represents the first illustrations of a juvenile of P. ensis, a decapodite stage of P. edwardsii and a zoea of H. ensifer.

Conclusions
This study represents the benefits of using DNA barcoding to help advance the field of larval biodiversity. More specifically, these methods can be used as a complementary approach alongside taxonomy to assist in species identification. This is especially useful for species where the larval morphology differs significantly from the adult and those that are difficult to rear in the laboratory [118,119]. Together, molecular and morphological methods hold great promise in the conservation of marine biodiversity [120] and should be used to reveal the unseen, bizarre and mysterious world that exists in the deep sea.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/d13100457/s1, Table S1. Taxonomy, voucher catalog numbers, localities and GenBank (GB) accession numbers for gene sequences used in the study.
An "N/A" = not available.