Novel molecular resources for single-specimen barcoding of enigmatic crustacean y-larvae.

Despite discovery more than 100years ago and documented global occurrence from shallow waters to the deep sea, the life cycle of the enigmatic crustacean y-larvae isincompletely understood and adult forms remain unknown. To date, only 2 of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach. This approach provided descriptions of the morphology of the naupliar and cyprid stages, and made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the evolutionary history and ecological importance of y-larvae, we developed a novel protocol that maximises the amount of morpho-ecological and molecular data that can be harvested from single larval specimens. This includes single-specimen DNA barcoding and daily imaging of y-nauplii reared in culture dishes, mounting of the last naupliar exuviae on a slide as a reference voucher, live imaging of the y-cyprid instar that follows, and fixation, DNA extraction, amplification and sequencing of the y-cyprid specimen. Through development and testing of a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we showcase how new sequence data can be used to estimate the phylogeny of Facetotecta. We expect that our novel procedure will help to unravel the complex systematics of y-larvae and show how these fascinating larval forms have evolved. Moreover, we posit that our protocols should work on larval specimens from a diverse array of moulting marine invertebrate taxa.

Tightening the requirements for species diagnoses would help integrate DNA-based descriptions in taxonomic practice.

Modern advances in DNA sequencing hold the promise of facilitating descriptions of new organisms at ever finer precision but have come with challenges as the major Codes of bionomenclature contain poorly defined requirements for species and subspecies diagnoses (henceforth, species diagnoses), which is particularly problematic for DNA-based taxonomy. We, the commissioners of the International Commission on Zoological Nomenclature, advocate a tightening of the definition of "species diagnosis" in future editions of Codes of bionomenclature, for example, through the introduction of requirements for specific information on the character states of differentiating traits in comparison with similar species. Such new provisions would enhance taxonomic standards and ensure that all diagnoses, including DNA-based ones, contain adequate taxonomic context. Our recommendations are intended to spur discussion among biologists, as broad community consensus is critical ahead of the implementation of new editions of the International Code of Zoological Nomenclature and other Codes of bionomenclature.

Single-specimen systematics resolves the phylogeny and diversity conundrum of enigmatic crustacean y-larvae.

Resolving the evolutionary history of organisms is a major goal in biology. Yet for some taxa the diversity, phylogeny, and even adult stages remain unknown. The enigmatic crustacean "y-larvae" (Facetotecta) are one particularly striking example. Here, we use extensive video-imaging and single-specimen molecular sequencing of >200 y-larval specimens to comprehensively explore for the first time their evolutionary history and diversity. This integrative approach revealed five major clades of Facetotecta, four of which encompass a considerable larval diversity. Whereas morphological analyses recognized 35 y-naupliar "morphospecies", molecular species-delimitation analyses suggested the existence of between 88 and 127 species. The phenotypic and genetic diversity between the morphospecies suggests that a more elaborate classification than the current one-genus approach is needed. Morphology and molecular data were highly congruent at shallower phylogenetic levels, but no morphological synapomorphies could be unambiguously identified for major clades, which mostly comprise both planktotrophic and lecithotrophic y-nauplii. We argue that lecithotrophy arose several times independently whereas planktotrophic y-nauplii, which are structurally more similar across clades, most likely display the ancestral feeding mode of Facetotecta. We document a remarkably complex and highly diverse phylogenetic backbone for a taxon of larval marine crustaceans, the full life cycle of which remains a mystery.

Three nuclear protein-coding genes corroborate a recent phylogenomic model of the Branchiopoda (Crustacea) and provide estimates of the divergence times of the major branchiopodan taxa.

The class Branchiopoda (Crustacea) shows great diversity in morphology and lifestyle among its constituent higher-level taxa: Anostraca, Notostraca, Laevicaudata, Spinicaudata, Cyclestherida and Cladocera. The phylogenetic relationships among these taxa have long been controversial. We sequenced three orthologous nuclear genes that encode the catalytic subunit of DNA polymerase delta and the largest and second-largest subunits of RNA polymerase II in the expectation that the amino acid sequences encoded by these genes might be effective in clarifying branchiopod phylogeny and estimating the times of divergence of the major branchiopodan taxa. The results of phylogenetic analyses based on these amino acid sequences support the monophyly of Branchiopoda and provide strong molecular evidence in support of the following phylogenetic relationships: (Anostraca, (Notostraca, (Laevicaudata, (Spinicaudata, (Cyclestherida, Cladocera))))). Within Cladocera, comparison of the nucleotide sequences of these same genes shows Ctenopoda to be the sister group of Haplopoda + Anomopoda. Three statistical tests based on the present amino acid sequence data-the approximately unbiased test, Kishino-Hasegawa test and weighted Shimodaira-Hasegawa test-tend to refute most of the previous molecular phylogenetic studies on Branchiopoda, which have placed Notostraca differently than here; however, our results corroborate those of one recent phylogenomic study, thus confirming the effectiveness of these three genes to investigate relationships among branchiopod higher taxa. Divergence time estimates calibrated on the basis of fossil evidence suggest that the first divergence of extant branchiopods occurred about 534 Ma during the early Cambrian period and that diversification within the extant branchiopod lineages started in or after the late Permian.

A new internal structure of nauplius larvae: A "ghostly" support sling for cypris y left within the exuviae of nauplius y after metamorphosis (Crustacea: Thecostraca: Facetotecta).

Facetotecta, or crustacean "y-larvae," occur in all the world's oceans although the adult forms remain completely unknown. At the metamorphic molt from the last naupliar instar to the terminal cypris larval stage a free carapace, six pairs of natatory thoracopods, and a segmented thorax and abdomen all develop anew. Unlike in earlier molts, the cephalic shield and the so-called "faciotruncal integument" usually remain together at this last naupliar molt, and the posterior "trunk" portion of the exuviae, while hollow, is not empty. In mounted preparations examined by phase contrast or differential interference contrast (DIC) microscopy, a ghost-like image of part of the cypris thorax, particularly the thoracopods and even their setae, is commonly visible inside the naupliar exuviae, and may be universally present in the Facetotecta. To investigate this "ghost," we used DIC and digital photographic stacking, and also scanning electron microscopy, on slide or stub-mounted final naupliar exuviae of an assortment of undescribed species of Facetotecta that had been reared from planktonic lecithotropic nauplii to the cypris stage at Sesoko Island, Okinawa, Japan, and at Keelung and Green Island, Taiwan. These techniques showed that the "ghost" is a delicate, three-dimensional, fibrous structure, essentially a sling-like mold or matrix with struts attached to the outer cuticle and pairs of deep pockets that previously held the thoracopods of the developing cypris y. Whether it is endoskeletal in nature, the (partial) exuvia of an additional instar, remnants of apoptosis, or something else is currently unknown. Nothing similar has been reported in other thecostracans, or in other crustaceans that undergo a similarly abrupt metamorphosis at the last naupliar molt.

Recognition and partial solution of nomenclatural issues involving copepods of the family Monstrillidae (Crustacea: Copepoda: Monstrilloida).

This work seeks to expose and clear up nomenclatural irregularities involving copepods of the order Monstrilloida, family Monstrillidae. The diagnostic text related to Monstrilla minuta Isaac, 1974 and four nominal species of Thaumaleus Krøyer, 1849 (now Cymbasoma Thompson, 1888) proposed by Isaac in 1974 is sufficient for all names to be available from their original description except for Thaumaleus similirostratus, which was proposed conditionally in 1974 and was first made available by Isaac in 1975; "similirostris" as used by Grygier in 1995 is an incorrect subsequent spelling. Four other specific names proposed in 1975 by Isaac, but disclaimed by him as nomina nuda (an action permitted retroactively by the Fourth Edition of the International Code of Zoological Nomenclature) have never been made available. By quoting the necessary information from Isaac's doctoral dissertation, two of them are validated herein under the names Thaumaleus frondipes Isaac in Grygier Suárez-Morales, sp. nov., and Strilloma scotti Isaac in Grygier Suárez-Morales, sp. nov., and are immediately reassigned as new combinations to Cymbasoma and Monstrilla Dana, 1849, respectively. A fifth such name, Thaumaleus tumorifrons, has already been made available under the authorship of Suárez-Morales, 1999, but its females are excluded from the type series; the spelling of the specific name of the new species recently proposed for those females, Cymbasoma mediterranea Suárez-Morales, Goruppi, Olazabal Tirelli, 2017, is emended to mediterraneum to match the gender of the genus. For Cymbasoma bowmani Suárez-Morales Gasca, 1998, the "Form B" female mentioned in the original description is excluded from the type series. The authorship and date of availability of Haemocera (currently Cymbasoma) morii depends on which language version of Article 13.1.1 of the Code is followed; a ruling by the International Commission on Zoological Nomenclature under Article 87 of the Code is necessary to resolve the matter. The composition of the type series of Cymbasoma bullatum (Scott, 1909) in terms of both number and sex has become unclear; its type locality is restricted herein to the vicinity of Obi Island in the Moluccas. Despite a published statement to the contrary, the syntype series of Cymbasoma germanicum (Timm, 1893) included specimens from other localities than just Helgoland. The type series of Cymbasoma guerrerense Suárez-Morales Morales-Ramírez, 2009 consists only of the holotype, which was mistakenly reported under the wrong registration number. The supposed invalidity of Monstrilla capitellicola Hartman, 1961 is discussed. Monstrilla javensis Isaac, 1974, nomen nudum, has remained unavailable owing to lack of adherence to Article 16.1 of the Code by later authors; the specific name is made available herein, under Suárez-Morales' authorship, in the combination Cymbasoma javense sp. nov. The taxonomic (and eventual nomenclatural) question of the status of M. mariaeugeniae Suárez-Morales Islas-Landeros, 1993 vis à vis M. wandelii Stephensen, 1913, i.e. as a separate species or a subspecies of the latter, remains unsettled. Cymbasoma lenticula Suárez-Morales McKinnon, 2014 and Monstrillopsis boonwurrungorum Suárez-Morales McKinnon, 2014 are fixed herein as the correct original spellings of those two specific names. Resolution of the problem posed by assignment of the specific name reticulata to supposedly non-conspecific males and females in the genus Monstrillopsis Sars, 1921 requires the designation of a neotype by the International Commission on Zoological Nomenclature.

Rediscovered syntypes of Procrangonyx japonicus, with nomenclatural consideration of some crangonyctoidean subterranean amphipods (Crustacea: Amphipoda: Allocrangonyctidae, Niphargidae, Pseudocrangonyctidae).

Two missing syntypes of the Japanese subterranean amphipod Procrangonyx japonicus (Uéno, 1930), the type species of Procrangonyx Schellenberg, 1934, were rediscovered in the collections of the Kyoto University Museum. The morphology of uropod 3, which has been considered the principal diagnostic character of the genus, is redescribed on the basis of one of the syntypes, and the nomenclatural history of the generic names Procrangonyx and Eocrangonyx Schellenberg, 1937 (corrected from 1936) for some Far-Eastern subterranean amphipod species is reviewed. Owing to confusion between the terms "type fixation" and "type designation"-the latter being just one means of accomplishing the former-the view that Procrangonyx is unavailable and invalid has prevailed in recent literature. Procrangonyx was indeed proposed after 1930 with no type species "designation", but under Articles 67.2.1 and 68.3 of the International Code of Zoological Nomenclature, Eucrangonyx japonicus Uéno, 1930 was "fixed" as its type species by monotypy in the original publication. Since a diagnosis of the genus was also provided in the same work, Procrangonyx is available under Article 13.3 of the Code. However, because endopodal segmentation of uropod 3 proves to be variable in P. japonicus, doubt is thrown on the taxonomic distinctness of Procrangonyx vis à vis Pseudocrangonyx Akatsuka Komai, 1922. Additionally, the publication dates of Allocrangonyx Schellenberg, 1937 and Niphargus foreli speziae Schellenberg, 1937 are corrected from 1936.

Description of a new species of Sternomoera (Crustacea: Amphipoda: Pontogeneiidae) from Japan, with an analysis of the phylogenetic relationships among the Japanese species based on the 28S rRNA gene.

A new species of amphipod, Sternomoera morinoi Tomikawa and Ishimaru, is described from subterranean aquatic habitats in Shiga Prefecture, Japan. In addition, Relictomoera tsushimana (Uéno, 1971) from a well on the island of Tsushima in Japan is transferred to Sternomoera and redescribed based on the holotype. Sternomoera morinoi sp. nov. is most similar to S. tsushimana (Uéno, 1971) comb, nov., but is distinguished by having many fine setae on the body, a shorter antenna 1, fewer C-setae on mandibular palp article 3, a shorter mandibular palp article 3, sparsely setose anterior margins of coxae 1-4, a different armature of the palmar margins of gnathopods 1 and 2, and no long setae on the posterior margin of the basis in gnathopod 2 and pereopods 3 and 4. The phylogenetic relationships among the Japanese species of Sternomoera are also estimated, based on partial DNA sequences of the nuclear 28S rRNA gene.

Endemism of subterranean Diacyclops in Korea and Japan, with descriptions of seven new species of the languidoides-group and redescriptions of D. brevifurcus Ishida, 2006 and D. suoensis Ito, 1954 (Crustacea, Copepoda, Cyclopoida).

Copepods have been poorly studied in subterranean habitats in Korea. Previous records have indicated mostly the presence of species already described from Japan, with very few endemic elements. This commonality has usually been explained by repeated dispersal across the land bridges that connected the two countries several times during the Pleistocene glacial cycles. However, the Korean Peninsula is known for pockets of Cambrian and Ordovician carbonate rocks, with more than 1,000 caves already having been explored. The relative isolation of these carbonate pockets makes for an enormous speciation potential, and the development of a high level of short-range endemism of subterranean copepods should be expected. Representatives of the genus Diacyclops Kiefer, 1927 are here investigated from a range of subterranean habitats in South Korea, with comparative material sampled from central Honshu in Japan. Morphological analyses of microcharacters, many of which are used in cyclopoid taxonomy for the first time herein, reveal high diversity in both countries. No subterranean species is found in common, although the existence of four sibling species pairs in Korea and Japan may be indicative of relatively recent speciation. We describe seven new stygobiotic species, including three from Korea (Diacyclops hanguk sp. n., Diacyclops leeae sp. n., and Diacyclops parasuoensis sp. n.) and four from Japan (Diacyclops hisuta sp. n., Diacyclops ishidai sp. n., Diacyclops parahanguk sp. n., and Diacyclops pseudosuoensis sp. n.). Diacyclops hanguk, Diacyclops parasuoensis, Diacyclops ishidai, and Diacyclops parahanguk are described from newly collected material, while the other three new species are proposed for specimens previously identified as other, widely distributed species. Diacyclops brevifurcus Ishida, 2006 is redescribed from the holotype female, and Diacyclops suoensis Ito, 1954 is redescribed from material newly collected near the ancient Lake Biwa in Japan. This research provides evidence for the importance of subterranean habitats as reservoirs of biodiversity, and also demonstrates the inadequacy of current morphological methods of identifying closely related species of copepods. The disproportionately high diversity discovered around Lake Biwa provides further evidence in support of the hypothesis about the role of ancient lakes as biodiversity pumps for subterranean habitats. A key to the East Asian species of the languidoides-group is provided.

Larval development of Japanese "conchostracans": Part 3, larval development of Lynceus biformis (crustacea, branchiopoda, laevicaudata) based on scanning electron microscopy and fluorescence microscopy.

For comparison with the remarkable larvae of the laevicaudatan (clam shrimp) Lynceus brachyurus, a basic description of the larval sequence of another laevicaudatan branchiopod, the Japanese Lynceus biformis, is provided. Four larval stages have been identified, ranging in size from 258 to 560 µm in length. The first stage has no flattened dorsal shield, in contrast to the three following stages, in which such a shield is present. During development, the only significant changes to the naupliar appendages occur in the antenna at the molt from stage 1 to 2, with the addition of a fourth apical seta to the endopod and a change in the form of the naupliar process, used for food manipulation, from a long, unbranched, pointed spine to a bifid structure. In addition, buds of trunk limbs (five pairs) first appear externally in stage 4 but can be recognized through the cuticle in the previous stage. The larval sequence and larval morphology of L. biformis differ from those of L. brachyurus in at least two respects. L. brachyurus has a dorsal shield in the earliest known stages, but such a shield is lacking in the first stage of L. biformis. Another difference is that L. brachyurus has a huge, flattened, kidney-shaped labrum, whereas that of L. biformis is smaller and bears four robust, denticulate spines on the distal margin. Based on out-group comparison, the morphology of L. biformis, at least in these respects, is likely to represent the ancestral morphology. Despite the partly peculiar morphology of the larvae of Lynceus species, they share many similarities with other branchiopod larvae, at least two of which, the naupliar swimming/feeding apparatus and the mode of development of the trunk limbs, could be considered synapomorphies for the Branchiopoda.

Induced metamorphosis in crustacean y-larvae: towards a solution to a 100-year-old riddle.

BACKGROUND: The y-larva, a crustacean larval type first identified more than 100 years ago, has been found in marine plankton samples collected in the arctic, temperate and tropical regions of all oceans. The great species diversity found among y-larvae (we have identified more than 40 species at our study site alone) indicates that the adult organism may play a significant ecological role. However, despite intense efforts, the adult y-organism has never been identified, and nothing is therefore known about its biology. RESULTS: We have successfully and repeatedly induced metamorphosis of y-larvae into a novel, highly reduced juvenile stage by applying the crustacean molting hormone 20-HE. The new stage is slug-like, unsegmented and lacks both limbs and almost all other traits normally characterizing arthropods, but it is capable of vigorous peristaltic motions. CONCLUSION: From our observations on live and preserved material we conclude that adult Facetotecta are endoparasitic in still to be identified marine hosts and with a juvenile stage that represents a remarkable convergence to that seen in parasitic barnacles (Crustacea Cirripedia Rhizocephala). From the distribution and abundance of facetotectan y-larvae in the world's oceans we furthermore suggest that these parasites are widespread and could play an important role in the marine environment.

A taxonomic study of species of Bothriocephalus Rudolphi, 1808 (Cestoda: Pseudophyllidea) from eels in Japan: morphological and molecular evidence for the occurrence of B. claviceps (Goeze, 1782) and confirmation of the validity of B. Japonicus Yamaguti, 1934.

A taxonomic study of specimens of Bothriocephalus from eels ( Anguilla spp.) in Japan has demonstrated the occurrence of two species, B. claviceps (Goeze, 1782) and B. japonicus Yamaguti, 1934. The former species is a parasite of eels ( A. anguilla and A. rostrata ) in the Holarctic Region and was recently reported from A. marmorata in Japan. The conspecificity of tapeworms newly found in an eel ( A. ? japonica ) from Lake Biwa, central Japan, with B. claviceps has been confirmed by the great similarity of their ITS-2 gene sequences (similarity 95.3% and 95.2%). However, the sequences of worms identified as B. claviceps from A. marmorata differed considerably from those of B. claviceps from two populations of A. anguilla from Europe and the above-mentioned one from Japan (similarity 66.3%, 67.1% and 65.1 %, respectively), thus indicating that the former cestodes may have been misidentified. This assumption was confirmed by morphological evaluation of a voucher specimen from A. marmorata. The morphology of this cestode, as well as those from A. japonica from two localities in Japan (Lakes Biwa and Suwa), indicates their conspecificity with B. japonicus. The validity of this taxon has been confirmed on the basis of a re-examination of the type-specimens. The two taxa, B. japonicus and B. claviceps, differ from each other in the shape and length of the scolex (619-730 microm in B. japonicus versus 1,180-2,100 microm in B. claviceps ), the relative position of the cirro-vaginal and uterine pores (opposite each other in relation to the median line of the body in B. japonicus versus tandem or slightly offset along the median line in the latter species), and the size of the eggs (41-52 x 28-35 microm in B. japonicus versus 50-70 x 31-43 microm in B. claviceps ).

Larval development of Japanese 'conchostracans': part 2, larval development of Caenestheriella gifuensis (Crustacea, Branchiopoda, Spinicaudata, Cyzicidae), with notes on homologies and evolution of certain naupliar appendages within the Branchiopoda.

As part of a larger project examining and comparing the ontogeny of all major taxa of the Branchiopoda in a phylogenetic context, the larval development of Caenestheriella gifuensis (Ishikawa, 1895), a Japanese spinicaudatan 'conchostracan', is described by scanning electron microscopy. Seven different larval stages are recognised, in most cases based on significant morphological differences. They range in length from about 200 to 850mum. Nauplius 1 has a plumb and lecithotrophic appearance with a rounded hind body and a labrum with an incipient medial spine. Limb segmentation is mostly unclear but the second antennae have more putative segments delineated than are expressed in the later stages. Feeding structures such as the mandibular coxal process and antennal coxal spine are only weakly developed. Nauplius 2 is very different from nauplius 1 and has three large spines on the labral margin and two long caudal spines. Feeding structures such as the mandibular coxal process and various spines and setae are developed, but whether feeding begins at this stage was not determined. The mandible has developed an 'extra' seta on endopod segment 1, absent in Nauplius 1. The segmentation of the second antenna has changed significantly due to fusions of various early segments. Nauplius 3 is like nauplius 2 in morphological detail, but larger and more elongate. Nauplius 4 has developed a pair of small anlagen of the carapace and rudiments of the first five pairs of trunk limbs, and the coxal spine of the antenna has become distally bifid. Nauplius 5 has a larger carapace anlage, externally visible enditic portions of the elongate trunk limbs, and a pair of primordial dorsal telson setae. Nauplius 6 has a larger and partly free carapace and better-developed, partly free trunk limbs with incipient enditic, endopodal, and exopodal setation. A pair of caudal spines, dorsal to the large caudal spines, has appeared. Nauplius 7 is quite similar to nauplius 6 but is larger and has slightly longer caudal and labral spines; also, the setation of the most anterior trunks limbs is better developed. The larval development is largely similar to that of other spinicaudatans. The larval mandible, which is evolutionarily conservative within the Branchiopoda, reveals a setation pattern similar to that of the Anostraca and Notostraca (two setae on mandibular endopod segment 1). Most other spinicaudatans and all examined laevicaudatans share another setal pattern (one seta on mandibular endopod segment 1), which could indicate a close relationship among these taxa. The second antenna undergoes a special development, which provides an insight into the evolution of this limb within the Branchiopoda. In nauplius 1 the basipod, endopod, and exopod are all superficially divided into a relatively high number of segments. In later nauplii some of these have fused, forming fewer but larger segments. We suggest that this ontogeny reflects the evolution of antennae in the conchostracans. Various aspects of the morphology of the antennae are discussed as possible synapormorphies for either the Diplostraca or subgroups of the Conchostraca.