Exceptional preservation of reproductive organs and giant sperm in Cretaceous ostracods.

The bivalved crustacean ostracods have the richest fossil record of any arthropod group and display complex reproductive strategies contributing to their evolutionary success. Sexual reproduction involving giant sperm, shared by three superfamilies of living ostracod crustaceans, is among the most fascinating behaviours. However, the origin and evolution of this reproductive mechanism has remained largely unexplored because fossil preservation of such features is extremely rare. Here, we report exceptionally preserved ostracods with soft parts (appendages and reproductive organs) in a single piece of mid-Cretaceous Kachin amber (approximately 100 Myr old). The ostracod assemblage is composed of 39 individuals. Thirty-one individuals belong to a new species and genus, Myanmarcypris hui gen. et sp. nov., exhibiting an ontogenetic sequence from juveniles to adults (male and female). Seven individuals are assigned to Thalassocypria sp. (Cypridoidea, Candonidae, Paracypridinae) and one to Sanyuania sp. (Cytheroidea, Loxoconchidae). Our micro-CT reconstruction provides direct evidence of the male clasper, sperm pumps (Zenker organs), hemipenes, eggs and female seminal receptacles with giant sperm. Our results reveal that the reproduction behavioural repertoire, which is associated with considerable morphological adaptations, has remained unchanged over at least 100 million years-a paramount example of evolutionary stasis. These results also double the age of the oldest unequivocal fossil animal sperm. This discovery highlights the capacity of amber to document invertebrate soft parts that are rarely recorded by other depositional environments.

Removal of extracellular coat from giant sperm in female receptacle induces sperm motility in Mytilocypris mytiloides (Cyprididae, Ostracoda, Crustacea).

Previous studies of cypridoidean ostracods have noted that (1) their giant spermatozoa are immotile inside the male, (2) these spermatozoa are motile in the female seminal receptacle and (3) these receptacles are often filled with empty sperm coats. Such findings have led previous authors to hypothesize that sperm must shed their coats in the female receptacle to become motile. We present light and electron microscopy results and video recordings of mating experiments with virgin specimens of Mytilocypris mytiloides. We show that the empty sperm coats frequently found in the female receptacles are not the result of sperm molting but are the resistant inner coats of exhausted sperm not used for egg fertilization. In contrast, we show that an outer granular coating material is successively removed from the sperm while resident inside the female receptacles before first oviposition occurs. During this period, previously immotile sperm gain motility, showing strong movement shortly before first oviposition takes place. By correlation of these phenomena, we suggest that dissolution of the outer coat material is required for motility to develop.

Sperm length variations in five species of cypridoidean non-marine ostracods (Crustacea).

Spermatozoa of the ostracod superfamily Cypridoidea include some of the longest in the animal kingdom, but unlike other so-called giant spermatozoa, they are aflagellate, probably evolved only once, and represent an exceptionally old trait. Sperm length variations within cypridoidean species remain poorly known, a lack that hinders the development of hypotheses to explain their length and variation. For this study, the lengths of 500 spermatozoa from each of five species of freshwater cypridoidean ostracods, Candonopsis tenuis (Brady, 1886), Fabaeformiscandona subacuta (Yang, 1982), Heterocypris rotundata (Bronshtein, 1928), Ilyocypris japonica Okubo, 1990, and Notodromas trulla Smith and Kamiya, 2014, were measured, including the lengths of the posterior and anterior regions. No overall pattern in sperm variation was discernible. Length variations between species, between males of the same species, and within individual males varied from low (Candonopsis tenuis) to extraordinarily large (Notodromas trulla and Fabaeformiscandona subacuta). Sperm competition, cryptic female choice, sperm heteromorphism, and testis size are unlikely to explain all of the variations observed. Age structures of the populations sampled might play a role in explaining some intraspecific variation. The differing amounts of variation in sperm characters revealed in this study suggest that multiple evolutionary trends and pressures shape sperm lengths in this superfamily.

Subcellular preservation in giant ostracod sperm from an early Miocene cave deposit in Australia.

Cypridoidean ostracods are one of a number of animal taxa that reproduce with giant sperm, up to 10 000 µm in length, but they are the only group to have aflagellate, filamentous giant sperm. The evolution and function of this highly unusual feature of reproduction with giant sperm are currently unknown. The hypothesis of long-term evolutionary persistence of this kind of reproduction has never been tested. We here report giant sperm discovered by propagation phase contrast X-ray synchrotron micro- and nanotomography, preserved in five Miocene ostracod specimens from Queensland, Australia. The specimens belong to the species Heterocypris collaris Matzke-Karasz et al. 2013 (one male and three females) and Newnhamia mckenziana Matzke-Karasz et al. 2013 (one female). The sperm are not only the oldest petrified gametes on record, but include three-dimensional subcellular preservation. We provide direct evidence that giant sperm have been a feature of this taxon for at least 16 Myr and provide an additional criterion (i.e. longevity) to test hypotheses relating to origin and function of giant sperm in the animal kingdom. We further argue that the highly resistant, most probably chitinous coats of giant ostracod sperm may play a role in delaying decay processes, favouring early mineralization of soft tissue.

How is a giant sperm ejaculated? Anatomy and function of the sperm pump, or "Zenker organ," in Pseudocandona marchica (Crustacea, Ostracoda, Candonidae).

'Giant sperm', in terms of exceptionally long spermatozoa, occur in a variety of taxa in the animal kingdom, predominantly in arthropod groups, but also in flatworms, mollusks, and others. In some freshwater ostracods (Cypridoidea), filamentous sperm cells reach up to ten times the animal's body length; nonetheless, during a single copulation several dozen sperm cells can be transferred to the female's seminal receptacle. This highly effective ejaculation has traditionally been credited to a chitinous-muscular structure within the seminal duct, which has been interpreted as a sperm pump. We investigated this organ, also known as the Zenker organ, of a cypridoid ostracod, Pseudocandona marchica, utilizing light and electron microscope techniques and produced a three-dimensional reconstruction based on serial semi-thin histological sections. This paper shows that numerous muscle fibers surround the central tube of the Zenker organ, running in parallel with the central tube and that a thin cellular layer underlies the muscular layer. A cellular inner tube exists inside the central tube. A chitinous-cellular structure at the entrance of the organ has been recognized as an ejaculatory valve. In male specimens during copulation, we confirmed a small hole derived from the passage of a single spermatozoon through the valve. The new data allowed for proposing a detailed course of operation of the Zenker organ during giant sperm ejaculation.

Karyotype Variability and Inter-Population Genomic Differences in Freshwater Ostracods (Crustacea) Showing Geographical Parthenogenesis.

Transitions from sexual to asexual reproduction are often associated with polyploidy and increased chromosomal plasticity in asexuals. We investigated chromosomes in the freshwater ostracod species Eucypris virens (Jurine, 1820), where sexual, asexual and mixed populations can be found. Our initial karyotyping of multiple populations from Europe and North Africa, both sexual and asexual, revealed a striking variability in chromosome numbers. This would suggest that chromosomal changes are likely to be accelerated in asexuals because the constraints of meiosis are removed. Hence, we employed comparative genomic hybridization (CGH) within and among sexual and asexual populations to get insights into E. virens genome arrangements. CGH disclosed substantial genomic imbalances among the populations analyzed, and three patterns of genome arrangement between these populations: 1. Only putative ribosomal DNA (rDNA)-bearing regions were conserved in the two populations compared indicating a high sequence divergence between these populations. This pattern is comparable with our findings at the interspecies level of comparison; 2. Chromosomal regions were shared by both populations to a varying extent with a distinct copy number variation in pericentromeric and presumable rDNA-bearing regions. This indicates a different rate of evolution in repetitive sequences; 3. A mosaic pattern of distribution of genomic material that can be explained as non-reciprocal genetic introgression and evidence of a hybrid origin of these individuals. We show an overall increased chromosomal dynamics in E. virens that is complementary with available phylogenetic and population genetic data reporting highly differentiated diploid sexual and asexual lineages with a wide variety of genetic backgrounds.

Revision of the genus Tanycypris (Ostracoda, Cypricercinae) with the description of Tanycypris alfonsi n. sp., and an identification key to the genus.

Specimens of a new species of the non-marine ostracod genus, Tanycypris Triebel, 1959 were found in samples from water plant containers, displayed in a greenhouse of the botanical garden in Munich, Germany. Beside the ubiquitous species Cypridopsis vidua O.F. Müller, 1776, the samples contained four alien species of the subfamily Cypricercinae, namely Chlamydotheca arcuata Sars, 1901, Strandesia bicuspis Claus, 1892, Tanycypris centa Chang et al., 2012, and Tanycypris alfonsi n. sp.. The genus Tanycypris has mainly been reported (native) from Asia, and (invasive) from Italian rice fields. The Cypricercinae unite all species possessing a Triebel loop, a character of the caudal rami attachment. The subfamily is split into the tribes Cypricercini McKenzie, 1971, Bradleystrandesiini Savatenalinton & Martens, 2009 and Nealecypridini Savatenalinton & Martens, 2009, the latter of which comprises the genera Tanycypris Triebel, 1959, Astenocypris G.W. Müller, 1912, Diaphanocypris Würdig & Pinto, 1990 and Nealecypris Savatenalinton & Martens, 2009. During the process of describing the new species, a number of taxonomic uncertainties were detected within the genus Tanycypris, leading to a revision of the nine species currently ascribed to it: Tanycypris camaguinensis (Tressler, 1937), Tanycypris centa Chang et al., 2012 Tanycypris clavigera (G.W. Müller, 1898) (now: Nealecypris clavigera nov. comb.), Tanycypris madagascarensis (G.W. Müller, 1898), Tanycypris marina (Hartmann, 1965) (now: Dolerocypris marina nov. comb.), Tanycypris pedroensis (Tressler, 1950) (now: Diaphanocypris pedroensis nov. comb.), Tanycypris pellucida (Klie, 1932), Tanycypris siamensis Savatenalinton & Martens, 2009a, and Tanycypris telavivensis (Krampner, 1928) (now: Herpetocypris telavivensis). An identification key has been developed to the species of the genus Tanycypris.

Skeleto-musculature of the mandible and its function in podocopid ostracodes exemplified by Loxoconcha pulchra (Cytheroidea: Loxoconchidae) and Fabaeformiscandona tyrolensis (Cypridoidea: Candonidae).

A new anatomical interpretation of the skeleto-musculature of the mandible in podocopid ostracodes is proposed based on ultrastructural observations of Loxoconcha pulchra Ishizaki, 1968 and Fabaeformiscandona tyrolensis (Löffler, 1963). Attachment cells with their numerous microfibers anchor the sclerotized lamella cuticle (chitinous rod) to the outer lamella cuticle via intracuticular fibers. A pan-shaped structure develops at the attachment area in the outer lamella cuticle and is responsible for the mandibular scar. The sclerotized lamella cuticle is continuous with the dorsal apex of the mandibular coxa, which touches the fulcral point directly without intermediate epidermis. The calcification of the fulcral point starts immediately after ecdysis and this rapid calcification suggests that the fulcral point must play a significant role in functional morphology of podocopid ostracodes. After 3D-reconstruction of the set of mandibular extrinsic muscles in a podocopid ostracode, we suggest that the fulcral point is a key character for carapace opening by transmitting the force from the mandibular coxa to the valve and at the same time functions as the stable fulcrum for mandibular movement during mastication.

Giant spermatozoon coiled in small egg: fertilization mechanisms and their implications for evolutionary studies on Ostracoda (Crustacea).

Ostracods of the superfamily Cypridoidea have giant spermatozoa. However, little data exist on the sperm-egg interaction in this group: only two publications have so far given the most ambiguous indication that the entire sperm enters the egg on fertilization. These assumptions have not yet been tested with modern techniques, nor has their putative value for developmental and evolutionary investigations been realized. The present paper gives the first, clear, light- and scanning electron microscopical evidence of the entire giant ostracod spermatozoon being incorporated into the egg. Coiling of the sperm underneath the egg shell is shown in the early zygotes of the species Mytilocypris praenuncia and Pseudocandona marchica. Additionally, data on the morphology of female and male reproductive tracts are given for M. praenuncia. Hypotheses on the evolution of giant filiform sperm in the Animal Kingdom are reviewed, and their applicability to ostracods is discussed. The demonstrated ingression of the entire sperm implies the entry of the two giant paternal mitochondrial derivates into the zygote in Cypridoidea and potentially casts doubt upon the dogma of strict maternal inheritance of mitochondrial DNA. Evidence of paternal inheritance of mtDNA in several organisms has recently given rise to a controversial debate on this issue; the possible significance of this phenomenon for molecular studies on ostracod phylogeny and evolution is discussed.