Diversity and life-cycle analysis of Pacific Ocean zooplankton by videomicroscopy and DNA barcoding: Crustacea

: Crustacea larvae and adults make up a large fraction of the biomass and number of organisms in both holoplankton (organisms that spend their entire lives in the plankton) and meroplankton (organisms that spend their larval stages in the plankton). The life cycles of these animals can be studied by raising individuals and studying them longitudinally in the laboratory, but this method can be very laborious. Here we show that DNA sequencing of a small element in the mitochondrial DNA (DNA barcoding) makes it possible to easily link life-cycle phases without the need for laboratory rearing. It can also be used to construct taxonomic trees, although it is not yet clear to what extent this barcode-based taxonomy reflects more traditional morphological or molecular taxonomy. Collections of zooplankton were made using conventional plankton nets in Newport Bay and the Pacific Ocean near Newport Beach, California, and individual crustacean specimens were documented by videomicroscopy. Adult crustaceans were collected from solid substrates in the same areas. Specimens were preserved in ethanol and sent to the Canadian Centre for DNA Barcoding at the University of Guelph, Ontario, Canada for sequencing of the COI DNA barcode. From 1042 specimens, 609 COI sequences were obtained falling into 169 Barcode Identification Numbers (BINs), of which 85 correspond to recognized species. The results show the utility of DNA barcoding for matching life-cycle stages as well as for documenting the diversity of this group of organisms.


Introduction
Crustacea is an Arthropod taxon that includes crabs, lobsters, crayfish, shrimps, prawns, krill, woodlice, and barnacles. Its members do not form a clade (a group defined by a single common ancestor), but rather form a paraphyletic group, meaning a group that is assumed to contain a common ancestor but contains only some of its descendants. In this case, crustacea includes animals in the clade Pancrustacea other than hexapods (insects and their relatives). As with other phyla in the zooplankton, crustacea include both holoplankton (animals that remain planktonic for their entire life cycle) and meroplankton (larval stages of benthic adults). Both types are included in the present study.
Like other arthropods, crustaceans have an exoskeleton, which is molted multiple times to allow growth and development. Most of them are free-living and aquatic, although a few (e.g. woodlice) are terrestrial, some are parasitic, and some (e.g. barnacles) are sessile. They are distinguished from other arthropods including insects, myriapods and spiders, by the possession of biramous (apparently branched) limbs, and by their aquatic larval forms. of interest was removed using a Pasteur Pipette, transferred to a depression slide, and recorded by video microscopy using a Zeiss microscope with a dark-field condenser, fitted with a phototube attached to a Nikon D5100 single-lens reflex camera. The most informative frames were taken from the videos and used in the figures for this paper. Most adults were photographed at the collection site, identified, and then released. Smaller specimens were removed and brought to the laboratory to be examined and photographed under a dissecting microscope. Tissue samples were removed using dissecting tools, then transferred to the microplates for DNA sequencing.
Filled plates were sent to the Canadian Centre for DNA Barcoding at the University of Guelph for sequencing of the standard 648-bp "DNA barcode" [6] in the COI mitochondrial gene, using the following primers: This usually produced a DNA barcode of 658 nucleotides, and only those containing >300 nucleotides were included in the sequence analysis. Groups of specimens with identical or almost identical DNA barcodes were assigned BIN numbers. "n=" indicates the number of DNA barcode sequences obtained from that group. The DNA sequences are in the public domain at the Canadian Centre for DNA Barcoding.

Results
From 1042 specimens, 609 COI sequences were obtained falling into 169 Barcode Identification Numbers (BINs), of which 85 correspond to recognized species. The remaining specimens, in apparently random taxonomic groups, failed to amplify for unknown reasons.
Class Branchiopoda [8] Our samples ( Figure 1) are all members of the order Cladocera (water fleas Class Copepoda [10] Copepods are a group of small crustaceans found in nearly every freshwater and saltwater habitat on earth. Most species are holoplanktonic and they are usually the dominant members of the zooplankton, but some are benthic and some are parasitic [11]. We have found members of all three types. The copepod egg hatches into a nauplius form, with a head and a tail but no true thorax or abdomen (Figures 2d, 5d). It then goes through several larval forms (Figure 4b,c) separated by molts, until at the final molt it becomes adult. Development from hatching to adulthood can take from a week to a year, depending on the species and environmental conditions. Mapping out this developmental process can be considerably simplified by using DNA barcoding to match specimens, rather than laboriously raising individual specimens in the laboratory.
Adult females often carry sacs of eggs or embryos (Figs. 5a, 5e, 6a, 6b, 6d 7b, 7f, 8a, 8b, 9e). In what follows, specimens are labeled as adults if they have the adult-like form, but some of those illustrated could be sub-adult stages.
Order Calanoida: Calanoid copepods are dominant in zooplankton samples from many parts of the world's oceans, making up 55%-95% of the total individuals. We identified the following:          Family Corycaeidae: three adults of Ditrichocorycaeus anglicus which, like other members of the family, have two large anterior eyes. Figure 6a, female carrying eggs.
Family Oithonidae: two female adults, both carrying eggs, of Oithona davisae. In the individual shown here (Figure 6b), developing embryos with red eye spots can be seen inside the egg shells.
Family Clausidiidae: Figure 6f: Clausidium californiense adult.     Order Harpacticoida. Most of these copepods are benthic, but some are planktonic so show up in our net samples.
Family Balanidae. Ivory barnacle, Amphibalanus eburneus, nauplius larva (Figure 11a). Native to the Western Atlantic, invasive to many other places. First population discovered in the US in 2000 [13], [14],  Family Caprellidae (skeleton shrimps). Although these animals are typically found attached to a substrate (usually floating algae) they often detach during capture and so appear in our plankton samples. Our collection (Figure 14) includes representatives of five different unidentified species, according to DNA barcodes. Order Decapoda [18].
Many decapods go through early planktonic stages called zoea and megalops. The following families are represented in the collection: Family Alpheidae: Individuals of two unidentified species were collected in Bahia de Los Angeles (Figure 16a,b).
Family Blepharopodidae: One zoea larva was collected and identified by DNA barcode, and an adult, recognized morphologically, was also collected (Figure 16c,d).
Family Callianassidae: One adult of Neotrypaea californiensis that was purchased from a dealer, and a juvenile of a different species, were identified by DNA barcode (Figure 16e,f).   (Figure 17). Surprisingly, some Mysid shrimp (Figure 17e,f) also gave a DNA barcode corresponding to Loxorhynchus grandis. We have to assume that this reflects gut contents, rather than body tissue of these specimens.   (Figure 18 a,b). One zoea larva and one adult of Pugettia dalli (Spined Kelp Crab) were matched by DNA barcode (Figure 18c,d), and one adult of Eriphia granulosa from Bahia de Los Angeles (Figure 18e) was barcoded. Order Mysida: [19], [21] Family Mysidae: Six unidentified species, distinguished by DNA barcode (Figure 40). Two specimens, morphologically mysids, gave DNA barcodes corresponding to the Sheep Crab Loxorhynchus grandis (Figure 17e,f). We assume this is due to their having consumed Loxorhynchus grandis larvae and that the PCR amplification amplified the Loxorhynchus grandis DNA barcode rather than the mysid sequence.

Discussion
DNA barcoding has often revealed unexpected species diversity in many taxa [2], [23] and this study leads to the same conclusion for marine crustacea, both in holoplankton and meroplankton. It shows the utility of this approach and the value of the COI Barcode for identifying species. Furthermore, our data show a clear "DNA barcode gap"; i.e. a much larger range of interspecific divergences versus intraspecific divergences in this DNA sequence for crustacea (Fig 42). Holoplankton refers to organisms that spend their entire life cycle in the plankton. The holoplanktonic taxa we have documented include Copepods (except for the parasitic species in the order Siphonostomatoida), as well as the Cumaceans, Euphausiids, Ostracods and some of the Amphipods and Mysids.
In contrast, meroplankton refers to those planktonic species that become benthic or even sessile as they mature. This group includes the barnacles and Decapods.
A third group is often collected accidentally in plankton nets but their lifestyle is benthic; this includes Caprellids, Isopods and Tanaids.
The DNA differences in the COI barcode are, of course, probably not responsible for the morphological differences we have observed between specimens in separate taxa. However, the DNA barcode differences that have evolved between morphologically distinct organisms can be used to examine the degree of relatedness between them. When the DNA sequence data are organized into a taxonomic tree, the results are generally consistent with the taxonomic tree according to conventional morphological methods. This can be examined most effectively by cladistic analysis, which in this context involves examination of the taxonomic tree for "DNA clades" -groups of species that are uniquely and exclusively related by DNA sequence. According to this analysis, all of the calanoid copepods constitute a DNA clade, although the cyclopoid and harpacticoid copepods are mixed. The Decapods apart from the Palaemonidae constitute a DNA clade while the one example of Palaemonidae appears to be more distantly related. The Ampeliscidae and some relatives constitute one clade of Amphipods, while the remainder of the Amphipods constitute another larger clade which includes two separate clades of Caprellids within it. The Tanaids; all of the barnacles; the Cladocerans including Penilia; the Euphausiids; the Isopods and the Ostracods present as distinct clades, while the Mysids group as several clades mixed with the Cumaceans and Decapods.
One of the major goals of this study was to use DNA barcoding to match life-cycle stages within a species, and this has been successful. We have identified, by DNA barcoding, all three major stages (zoea, megalops, adult) of a decapod species (Lined Shore Crab, Pachygrapsus crassipes), which is one of the commonest intertidal decapods in our area. We have also collected zoea and megalops of the Yellow Shore Crab Hemigrapsus oregonensis and the adult has been collected earlier at the Back Bay Science Center in Newport Bay.
In other cases, we have sequenced larval stages, which suggests the presence of adult populations but we have not yet found the adults, possibly because they are in subtidal areas where we have not been able to do enough sampling. For example, we have the zoea and megalops of the yellow rock crab Metacarcinus anthonyi and a megalops of the graceful rock crab M. gracilis, but no adults of these species. Of course, the finding of many larval specimens in different stages suggest the existence of nearby breeding adult populations. In the family Pinnotheridae (Pea crabs), we have found 19 zoeae but no adults of Pinnixa franciscana, and one zoea of Pinnixa tubicola. Adults of Pea crabs live in burrows of Callianassa (mud shrimp) and Urechis (Innkeeper worm), where we have not collected. We have found zoea of the Spiny Mole Crab Blepharipoda occidentalis, which is difficult to find but a common local resident.
The larval stages of meroplanktonic species can take advantage of ocean currents, drifting for dispersal and range expansion [24]. In addition to swimming, organisms can attach to floating objects to feed, rest, and travel great distances. For some, including crabs of the genus Planes, rafting is a way of life. Many species can extend their larval period for a time if they do not find a likely environment for settling and morphing. At some point, though, if a suitable environment is not found, the larvae die, fail to molt to the next stage, or are consumed as prey [25]. We find the larvae of the barnacle Chthamalus dalli in many Newport Bay samples and in the immediate areas north and south of the harbor entrance. We have found no adults but now have a good idea where to look, possibly on boat bottoms rather than rocks.
Amphibalanus eburneus, a Western Atlantic barnacle, was first found in the Pacific Ocean in Colorado Lagoon, Long Beach, California in 2000 [13] Colorado Lagoon is 10 miles NW of Newport Bay and 2 miles SE of the Port of Los Angeles. We have nauplia from two locations in Newport Bay, suggesting a range extension from Colorado Lagoon since 2000, consistent with the statement "Based on its broad tolerances, this species has the potential to expand its range on the Pacific coast" [13]. These larval catches will direct our search for adults in both of our sampling areas.
We also have two adult sequences of Amphibalanus amphitrite from the Salton Sea, an inland, brackish-water body, in Southern California, north of the upper reaches of the Gulf of California, without ocean contact either to the Pacific or to the Gulf of California. One nauplius larva of this species was found in Newport Bay.
Sometimes, a specimen of an exotic species may have arrived in ships ballast water, rather than representing a continuing presence in the ecosystem. For example, we identified one cypris larva of Megabalanus rosa, a Western Pacific barnacle species, in the entrance to Newport Harbor. We also found in Newport Bay one larva of the long-wristed hermit crab, Pagurus longicarpus, which is common along the Atlantic and Gulf coasts of the United States and the Atlantic coast of Canada, but at least one adult has been collected and sequenced by the Los Angeles County Museum of Natural History from the Port of Los Angeles (May 2014), 12 miles northwest of Newport Harbor.
Since there are apparently no other known records of these species in our area, they are unlikely to be from a local population and are more likely to have arrived from discharge of ballast water [26] possibly from the nearby and very busy Port of Long Beach. However, our finding of a zoea suggests a breeding population somewhere in Orange County between Newport Bay and the Port of Los Angeles. These larvae alert us to be watchful for adults as we sample other benthic species. However, water sampling for planktonic larvae is much simpler than searching for adults, and DNA barcoding is much more definitive than morphological identification, especially for early larval stages. Funding: This research received a grant of $1000 from the Newport Bay Conservancy