A new species of Orobdella (Hirudinida, Arhynchobdellida, Orobdellidae) from Taipei, Taiwan

A new quadrannulate species of Orobdella, Orobdella ketagalansp. n., from Taipei, Taiwan, is described. This is the first record of Orobdella and the family Orobdellidae from Taiwan. This new species possesses small, paired sperm duct bulbs in the male reproductive system. In addition to these bulbs, the following combination of characters distinguishes this new species from other quadrannulate species: somite IV uniannulate, male gonopore at XI b6, female gonopore at XIII a1, 1/2 + 4 + 1/2 between gonopores, simple tubular gastroporal duct, lacking epididymides, and undeveloped atrial cornua. Phylogenetic analyses using nuclear 18S rDNA and histone H3 as well as mitochondrial COI, 12S rDNA, tRNA(Val), and 16S rDNA markers showed that Orobdella ketagalan is related to the two Ryukyu Archipelago species Orobdella dolichopharynx Nakano, 2011 and Orobdella shimadae Nakano, 2011.     

Evolutionary morphology of the male reproductive system,spermatozoa and seminal fluid of spiders (Araneae,Arachnida) – Current knowledge and future directions

The male reproductive system and spermatozoa of spiders are known for their high structural diversity. Spider spermatozoa are flagellate and males transfer them to females in a coiled and encapsulated state using their modified pedipalps. Here, we provide a detailed overview of the present state of knowledge of the primary male reproductive system, sperm morphology and the structural diversity of seminal fluids with a focus on functional and evolutionary implications. Secondly, we conceptualized characters for the male genital system, spermiogenesis and spermatozoa for the first time based on published and new data. In total, we scored 40 characters for 129 species from 56 families representing all main spider clades. We obtained synapomorphies for several taxa including Opisthothelae, Araneomorphae, Dysderoidea, Scytodoidea, Telemidae, Linyphioidea, Mimetidae, Synotaxidae and the Divided Cribellum Clade. Furthermore, we recovered synspermia as a synapomorphy for ecribellate Haplogynae and thus propose Synspermiata as new name for this clade. We hope that these data will not only contribute to future phylogenetic studies but will also stimulate much needed evolutionary studies of reproductive systems in spiders.     

Phylogeny of the New World medicinal leech family Macrobdellidae (Oligochaeta: Hirudinida: Arhynchobdellida)

Phylogenetic analyses of the leech family Macrobdellidae were accomplished with all nominal species in the family save one. A total of 17 specimens in nine ingroup species were analysed, along with four outgroup taxa. Twenty-two morphological characters based on jaw dentition, sexual anatomy, and external morphology failed to provide a resolution for many of the relationships in the family. DNA sequence data from nuclear 18S rDNA, nuclear 28S rDNA, mitochondrial 12S rDNA, and mitochondrial cytochrome c oxidase subunit I were examined separately and in combination with morphological characters. The resulting combined analysis strongly corroborated the placement of the genus Philobdella within the family Macrobdellidae and as sister to a monophyletic genus Macrobdella , the typical North American medicinal leeches. Furthermore, sequence divergences among these taxa confirmed the existence of two species, Philobdella gracilis and P. floridana , readily distinguishable on the basis of jaw dentition .     

Evolution of sperm morphology in potamid freshwater crabs (Crustacea: Brachyura: Potamoidea)

We investigated sperm cells and spermatophores of four species of Old World freshwater crabs belonging to three different genera of the subfamily Potaminae (family Potamidae). Characters previously believed to be apomorphic for the potamid subfamily Potamiscinae were also found to occur in the Potaminae. To infer the morphological ancestral character state combination of the Potamidae, ancestral character state analysis of four different sperm traits was performed, based on a 16S rDNA phylogeny of the investigated species. Comparing molecular phylogeny and character state distribution, several cases of convergent evolution could be identified. The densely packed, coenospermic spermatophores and the occurrence of a ‘tongue‐and‐groove’ connection between operculum and acrosomal zones are probably apomorphies for the whole Potamidae. The spermatozoa of Socotrapotamon socotrense show several unique characters. We also analysed the evolution of acrosome size. The sperm cells of the Potamidae and their sister‐group Gecarcinucidae only slightly overlap in acrosome size. Within the investigated species, the ‘East Asia’ subclade (subfamily Potamiscinae) developed significantly larger acrosomes than the subfamily Potaminae. Our results suggest that the use of brachyuran acrosome morphology for phylogenetic inference at the family level is strongly affected by small sample size, and by convergent character evolution. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010.     

Ordered arrangement and rearrangement of chromosomes during spermatogenesis in two species of planarians (Plathelminthes)

The pattern of distribution of telomeric DNA (TTAGGG), 28S rDNA, and 5S rDNA has been studied using fluorescence in situ hybridization (FISH) and primed in situ labelling during spermatogenesis and sperm formation in the filiform spermatozoa of two species of planarians, Dendrocoelum lacteum and Polycelis tenuis (Turbellaria, Plathelminthes). In both species, the positions of FISH signals found with each probe sequence are constant from cell to cell in the nuclei of mature sperm. Chromosome regions containing 5S and 28S rDNA genes are gathered in distinct bundles of spiral form. In early spermatids with roundish nuclei, the sites of a given sequence on different chromosomes remain separate. Centromeres (marked by 5S rDNA) gather into a single cluster in the central region of the slightly elongated sperm nucleus. During spermatid maturation, this cluster migrates to the distal pole of the nucleus. In Polycelis, telomeric sites gather into three distinct clusters at both ends and in the middle of the moderately elongated nucleus. These clusters retain their relative positions as the spermatid matures. All the chromosome ends bearing 28S rDNA gather only into the proximal cluster. Our data suggest that structures in the nucleus selectively recognise chromosome regions containing specific DNA sequences, which helps these regions to find their regular places in the mature sperm nucleus and causes clustering of the sites of these sequences located on different chromosomes. This hypothesis is supported by observations on elongated sperm of other animals in which a correlation exists between ordered arrangement of chromosomes in the mature sperm nucleus and clustering of sites of the same sequence from different chromosomes during spermiogenesis. Received: 15 December 1997; in revised form: 24 March 1998 / Accepted: 14 April 1998     

A comparative study of sperm morphology, cytology and activation in Caenorhabditis elegans, Caenorhabditis remanei and Caenorhabditis briggsae

Studies of sterile mutants in Caenorhabditis elegans have uncovered new insights into fundamental aspects of gamete cell biology, development, and function at fertilization. The genome sequences of C. elegans, Caenorhabditis briggsae and Caenorhabditis remanei allow for informative comparative studies among these three species. Towards that end, we have examined wild-type sperm morphology and activation (spermiogenesis) in each. Light and electron microscopy studies reveal that general sperm morphology, organization, and ultrastructure are similar in all three species, and activation techniques developed for C. elegans were found to work well in both C. briggsae and C. remanei. Despite important differences in the reproductive mode between C. remanei and the other two species, most genes required for spermiogenesis are conserved in all three. Finally, we have also examined the subcellular distribution of sperm epitopes in C. briggsae and C. remanei that cross-react with anti-sera directed against C. elegans sperm proteins. The baseline data in this study will prove useful for the future analysis and interpretation of sperm gene function across nematode species.     

Morphology of the female reproductive system of three dorippid crabs (Crustacea: Decapoda: Brachyura: Dorippidae) and the role of accessory cuticle structures associated with seminal receptacles

The reproductive systems of crabs reveal characters of considerable importance for the understanding of brachyuran phylogeny and evolution. The Dorippoidea show several plesiomorphic characters within Eubrachyura and similarities to podotreme crabs. Hence, they are often considered as an early diverging lineage, sometimes even as the sister group to all remaining eubrachyurans. Due to their role as prime candidates for putative plesiomorphic characters of the reproductive system of the Eubrachyura, we compared the morphology of the vaginae, seminal receptacles, and ovaries of three dorippid species using histological methods, micro‐computed tomography, and 3‐D reconstructions. Despite the putative phylogenetic position of dorippids, the female reproductive system shows features that are regarded as derived characters in eubrachyurans, including a concave vagina and a ventral‐type seminal receptacle. In contrast to other eubrachyurans, the oviduct does not enter the seminal receptacle directly but through specific cuticular valves. The female reproductive systems of Dorippe sinica and Dorippe quadridens are remarkable in further aspects. The seminal receptacles of both species are completely cuticle‐lined and have accessory sperm storage structures, the bursae. Our findings on the morphology of the female reproductive system of dorippids with its unique combination of basal, derived, and new characters challenges the prevailing hypothesis on the evolution of sperm storage organs in Eubrachyura.     

Revision of the Childiidae (Acoela), a total evidence approach in reconstructing the phylogeny of acoels with reversed muscle layers

The Childiidae sensu Dörjes 1968 comprises the acoel worms characterized by a cone‐shaped penis with muscular or sclerotized elements. Based on differences in body‐wall musculature arrangement, Hooge (2001) recently restricted the family to the genus Childia Graff, 1910 and placed the remaining genera to his new family Actinoposthiidae Hooge 2001 . This rearrangement has been questioned ( Raikova et al. 2004 ). We reconstructed the phylogeny of the Childiidae sensu Dörjes 1968 by means of a total evidence analysis including Histone H3, 28S rDNA and new 18S rDNA sequences, as well as 50 morphological characters. New characters of the muscular system and copulatory organs discovered through confocal laser scanning microscopy of phalloidin‐stained specimens are included in the phylogenetic analysis. A total of 12 taxa (nine ingroup and three outgroup) were used in the parsimony analysis of the 18S data set, which was aligned with different parameters for a sensitivity analysis, and the combined data set (18S + 28S + H3 + morphology). Incongruence in the node support of the groups among the four partitions was very low in the total evidence tree; except for the H3 partition. The conflict observed in the H3 partition is likely due to large homoplasy observed in the synonymous alternatives at both first and third codon positions. All data partitions demonstrated that Actinoposthia beklemischevi Mamkaev 1965 , and the newly defined taxon Childiidae (comprising Childia and Paraphanostoma Westblad 1942 ) are not close relatives. The monophyly of Childia and Paraphanostoma is strongly supported by both the 18S and 28S data partitions. Our study also reveals additional apomorphies uniting Childia with Paraphanostoma from body‐wall musculature, statocyst muscles and male copulatory organ. Muscular system, statocyst muscles, male copulatory organ and nervous system characters proved to be the best characters for taxonomic delimitations of subtaxa within the Childiidae, whereas the seminal bursa (a frequently used character in the taxonomy of Acoela) was highly homoplastic. We also described the body‐wall musculature of six Paraphanostoma species, which is characterized by the reversed arrangement of the longitudinal and circular muscle layers, and by the absence of diagonal muscles on the ventral side of the body and the presence of two types of diagonal muscles on the dorsal side. Childia groenlandica (Levinsen, 1879) is nested among the Paraphanostoma species in our total evidence tree, so we synonymize Paraphanostoma with Childia; all former members of Paraphanostoma are transferred to Childia.     

28S rDNA haplotypes of males are distinct from those of androgenetic hermaphrodites in the clam Corbicula leana

The clam Corbicula leana exists in two forms, hermaphrodites and males. Our previous study on mitochondrial DNA suggested that the male nuclear DNA might have derived from hermaphrodite C. leana relatively recently. To clarify the origin of males in the clam, sequences of the nuclear 28S rDNA divergent domain (which is 441-444 bp long) in androgenetic hermaphrodites and males and dioecious (bisexual) species were analyzed. Unexpectedly, the nuclear 28S rDNA haplotypes of males and hermaphrodites were distinct. Haplotype network analysis indicated that males and hermaphrodites are reproductively isolated from each other without sharing the same nuclear haplotype. These results support a hypothesis that the egg nuclear genome of androgenetic hermaphrodites is replaced by the male sperm genome, and only males develop after fertilization by a male spermatozoon.     

Multiple origins of anural development in ascidians inferred from rDNA sequences

Ascidians exhibit two different modes of development. A tadpole larva is formed during urodele development, whereas the larval phase is modified or absent during anural development. Anural development is restricted to a small number of species in one or possibly two ascidian families and is probably derived from ancestors with urodele development. Anural and urodele ascidians constitute a model system in which to study the evolution of development, but the phylogeny of anural development has not been resolved. Classification based on larval characters suggests that anural species are monophyletic, whereas classification according to adult morphology suggests they are polyphyletic. In the present study, we have inferred the origin of anural development using rDNA sequences. The central region of 18S rDNA and the hypervariable D2 loop of 28S rDNA were amplified from the genomic DNA of anural and urodele ascidian species by the polymerase chain reaction and sequenced. Phylogenetic trees inferred from 18S rDNA sequences of 21 species placed anural developers into two discrete groups corresponding to the Styelidae and Molgulidae, suggesting that anural development evolved independently in these families. Furthermore, the 18S rDNA trees inferred at least four independent origins of anural development in the family Molgulidae. Phylogenetic trees inferred from the D2 loop sequences of 13 molgulid species confirmed the 18S rDNA phylogeny. Anural development appears to have evolved rapidly because some anural species are placed as closely related sister groups to urodele species. The phylogeny inferred from rDNA sequences is consistent with molgulid systematics according to adult morphology and supports the polyphyletic origin of anural development in ascidians. Correspondence to: W.R. Jeffery     

Spermatozoa and spermiogenesis of the wolf spider Schizocosa malitiosa (Lycosidae, Araneae) and its functional and phylogenetic implications

The wolf spider Schizocosa malitiosa is a well-known model system for studies on sexual selection in spiders. Despite this, little is known about the morphology of the reproductive system and spermatozoa in this species. In the present study, we investigate the male genital system and sperm cells of S. malitiosa using electron microscopy and provide a computer-based 3D reconstruction of the spermatozoa for the first time for arthropods. In general, the male genital system consists of two long, tube-like testes that lead into convoluted deferent ducts. The ejaculatory duct is enlarged and contains a large quantity of sperm and secretion. As revealed by transmission electron microscopy, only one type of secretion droplet is present in the seminal fluid. The spermatozoa of S. malitiosa resemble an organization known for members of the RTA clade, i.e., with an arrow-shaped acrosomal vacuole partly sunk into the nucleus and a chambered centriolar adjunct (a newly introduced character). This organization provides further support for these characters as potential synapomorphies for the RTA clade. By the end of the spermiogenesis, the nucleus and axoneme coils within the cell and a multi-layered secretion sheath are formed representing cleistospermia. The function of the thick secretion sheath is still unknown, but might be correlated either with the residency time in the female (insemination until oviposition) since female S. malitiosa do not lay eggs before the fourth month after copulation or with the receptivity-inhibiting substances suggested for this species.     

Reproductive system and spermatozoa of <Emphasis Type="Italic">Paraturbanella teissieri</Emphasis> (Gastrotricha,Macrodasyida): implications for sperm transfer modality in Turbanellidae

The morphology of the reproductive apparatus of several species of Turbanellidae, which are sequential hermaphrodites, has been studied for a comparison with that of other Gastrotricha Macrodasyida, which are simultaneous hermaphrodites. The common structural plan of the genital system of Turbanellidae includes two testes which extend into two sperm ducts turning anteriorly and fusing in a midventral pore, two ovaries with oocytes maturing in a cephalic direction and only one accessory organ, a seminal receptacle, provided with an external pore. A possible sperm transfer modality alternative to that described in the literature is advanced. Spermatological characters of Paraturbanella teissieri have been compared with those of the two Turbanella species studied up to date. Turbanellidae share with other Macrodasyida the general model of spermatozoon, but are the only representatives of this taxon known so far which have sperm devoid of the striated cylinder around the axoneme. Both the structure of the reproductive apparatus and the fine morphology of the spermatozoa of Turbanellidae species agree with the evolutionary view, recently supported by morphological and molecular data, which puts this taxon on a separate clade, early divergent from the stem lineage of Macrodasyida.     

Is Sperm Ultrastructure Useful in Polychaete Systematics? An Example Using 20 Species of the Fabriciinae (Polychaeta: Sabellidae)

The utility of sperm morphology in the systematics of polychaetes has been questioned in the past. This doubt stems from a problem with methodology, not with sperm morphology. It is argued that sperm characters used in combination with other morphological features have utility at various hierarchical levels. As a test of this proposition males in species from each of the following 10 fabriciin genera: Augeneriella, Fabricia, Fabriciola, Fahricinuda, Manayunkia, Novafabricia, Parafabricia, Pseudofabricia, Pseudofabriciola and an undescribed new Genus A (Fitzhugh et al., in preparation), were examined in order to provide new characters for phylogenetic systematic studies. All species were found to have a dorsal sperm duct running beneath the faecal groove of the thoracic region. No sabellin sabellids or serpulids have this duct. The above Fabriciinae have spermatids developing in large clusters of several hundred cells. Serpulids and sabellins nearly always have sperm developing in tetrads or small groups. Fabriciins studied also have a sperm structure distinct from the Sabellinae and the Serpulidae. Proposed synapomorphies for the members of the Fabriciinae, based on sperm structure, include (1) a thick glycocalyx over the plasma membrane; (2) a distinctive nuclear projection, with an anterior thickening of the nuclear membrane; (3) a thickened. spiraling ridge of nuclear membrane; (4) an extra-axonemal sheath and (5) a unique sheath of mitochondrial material in the midpiece. Within the sub-family there were differences among species in sperm structure. This variability involves acrosome morphology; the structure of the sperm nucleus and nuclear projection; the spiral of thickened nuclear membrane; the structure of the extra-axonemal sheath; the mitochondrial sheath. The monophyly of the Fabriciinae (sensu Fitzhugh, 1991, 1993) is well supported by this analysis. The status of Caobangia is still unresolved and a close examination of this genus is warranted.     

A new species of Euglena (Euglenozoa: Euglenales) isolated from extreme environments in "boiling mudflats" of Rincón de la Vieja volcano, Costa Rica

A new species of euglena isolated from a hot and acid mud pool located in Las Pailas de Barro, Volcán Rincón de la Vieja, Costa Rica is described. This species inhabits hot and acid environments. Euglena pailasensis sp. nov. main features are: the absence of flagella, the presence filaments like "pilis", the presence of chloroplasts with pyrenoids crossed by several tylakoids, and acid and heat tolerance. Molecular phylogeny studies using 18S rDNA and Gap C genes indicated that the new species is related to E. mutabilis. Its taxonomic characters based on morphology, biology and sequence of the 18S rDNA and Gap C genes are discussed and compared with other closely related species of the genus.     

Ultrastructural and chemical study of the chromatin during spermiogenesis of the fish Scyliorhinus caniculus (L.) (author's transl)

Electron microscopic, cytochemical and biochemical techniques were applied to study structural aspects and changes in nuclear components during the spermiogenesis of Scyliorhinus caniculus. Five major stages of nuclear differentiation were recognized and characterized by variations in the organization and chemical properties of chromatin. Stage I is analogous to a somatic nucleus with heterogeneous chromatin. At the second stage, the nuclear content is dispersed but the chromatin fibers are of the same diameter as those of the stage I. The nuclear elongation begins at stage III, the DNP fibers running preferentially parallel to the long axis of the nucleus. During these early modifications of chromatin structure appear two new basic nuclear proteins (S 1 and S 2) which migrate faster than histones but typical histones remain assosciated with these nuclei. In later elongation stage (stage IV), the chromatin fibers organize in a helical form and fuse side by side giving lamellar systems which have a reticular structure. At the end of this stage, the nuclear material has become uniformly compact. These late variations in chromatin organization are parallel to the association of chromatin with new basic nuclear proteins (S 3, S 4, Z 1, Z 2 and Z 3). The cytochemical and electrophoretical properties of one of these proteins (S 4) which appears at the end of spermiogenesis are similar to those of a protamine. In stage V, the chromatin is homogeneous and the nucleus assumes a helical configuration beginning at the posterior end. The deoxyribonucleoproteins of the mature sperm show some novel chemical characters, including the appearance of a stable nuclear acidophilia with the ALFERT and GESCHWIND method and extraction with 0.25 N HCl of one of the basic protein fractions newly appeared in late spermiogenesis (Z 3), two other fractions (Z 1 and Z 2) being extracted with a more drastic procedure. The other fractions described before are no more detectable.     

Cycle and duration of the seminiferous epithelium in puma (Puma concolor)

Puma or sussuarana (Puma concolor) is the second largest feline in the American continent and has an ample latitudinal distribution in very diverse habitats. In relation to its conservation status, the puma is considered an extinction-threatened species. The study of the testis morphology and the spermatogenic process in a species is fundamental for establishing the physiologic patterns that will make possible the selection of the protocols for assisted reproduction. A number of peculiarities associated with the reproductive biology of specific species such as the duration of spermatogenic process can be used to determine the frequency of sperm collection. Nine adult male pumas maintained in captivity were used to determine the relative frequency of stages in the seminiferous epithelium cycle. Three of them received intra-testicular injections of 0.1ml tritiated thymidine to determine the duration of the seminiferous epithelium cycle, and were subjected to biopsy 7 days later. The cycle of the seminiferous epithelium in puma was didactically described into eight stages by the tubular morphology method. The total duration of one seminiferous epithelium cycle in puma was calculated to be 9.89 days, and approximately 44.5 days are required for development of spermatozoon from spermatogonia. The duration of spermiogenesis, prophase and other events of meiosis were 14.08, 15.20 and 1.79 days, respectively. The relative frequency of the pre-meiotic, meiotic and post-meiotic phases were 3.98, 1.79 and 4.12 days, respectively.     

An assessment approach for application of spermatic data in phylogenetic analyses: within the genus Moenkhausia Eigenmann, 1903 (Characiformes: Characidae)

Spermiogenesis and sperm ultrastructure from 21 species of Moenkhausia and others related genera are described. To evaluate the phylogenetic signals, 18 unordered characters were utilized in implied weighting analysis through the program TNT 1.1. Four variations of spermiogenesis were found. In the earliest spermatids, the nucleus can be positioned lateral, eccentric, strongly eccentric or nearly medial in relation to the distal centriole. The nuclear rotation can be present or absent. These spermiogenesis processes are related or intermediate to Type I and Type III. Taking into account the degrees of nuclear rotation during the spermiogenesis and other characteristics, distinct forms of spermatozoa are observed among the species analyzed. The phylogenetic analysis yielded a single most parsimonious tree with fit value 2.70000 and the topology obtained founds Moenkhausia as non‐monophyletic. However, some hypothesis of relationships previously proposed viz the clade 20, which contains the type species Moenkhausia xinguensis, is recovered herein. This clade is supported by five synapomorphies, and it allows the supposition that these species constitute a monophyletic group. The whole topology is presented and discussed.