Molecular phylogenetic relationships of moles,shrew moles,and desmans from the new and old worlds

A Rich variety of anatomical and physiological specializations has enabled members of the family Talpidae (moles, shrew moles, and desmans) to exploit a diverse range of habitats: terrestrial, semi-aquatic, aquatic/fossorial, semi-fossorial, and fossorial. While numerous morphological and biochemical studies pertaining to the origin and radiation of the Talpidae have been completed, phylogenetic hypotheses remain controversial. To address this shortcoming we sequenced the mitochondrial DNA cytochrome b gene (1140bp) from 29 individuals spanning 12 talpid species. Phylogenetic trees incorporating 12 New and Old World genera (18 species; all 3 extant subfamilies) were then constructed using NJ, MP, ML, and NJ-ML (NJ with ML parameters) methods. Our results provide molecular support for a mononphyletic Talpidae, and suggest that the 12 genera are clustered into seven major clades; (1) Asiatic shrew-like moles (Uropsilus), (2) North American aquatic/fossorial moles (Condylura), (3) North American fossorial moles (Parascalops, Scalopus, and Scapanus), (4) North American semi-fossorial shrew moles (Neurotrichus), (5) Japanese semi-fossorial shrew moles (Dymecodon and Urotrichus), (6) European semi-aquatic desmans (Desmana), and (7) Eurasian fossorial moles (Euroscaptor, Mogera, and Talpa). None of these groupings comprised mole species from both continents. In fact, North American moles and shrew moles do not appear to have specific affinities with Asian moles and shrew moles, respectively. Although low bootstrap support was generally found for evolutionary nodes uniting the major talpid clades, all gene trees constructed identified fossorial North American and Eurasian mole lineages as nonmonophyletic groups, suggesting subterranean specializations arose independently at least twice during the evolution of the Talpidae. Additionally, our data set provides molecular support for a basal divergence and long independent history of Uropsilus from the main talpid line, and refutes the traditional taxonomic status and secondarily basal phylogenetic placement of the subfamily Desmaninae within the Talpidae.     

Evolution and biogeography of talpid moles from continental East Asia and the Japanese islands inferred from mitochondrial and nuclear gene sequences

We sequenced the cytochrome b gene from two little-studied mammal species from the highlands of Southwest China, the long-tailed mole Scaptonyx fusicaudus and the gracile shrew-like mole Uropsilus gracilis. This data was used to examine the phylogenetic relationships among 19 talpid species within the family Talpidae (Mammalia: Eulipotyphla). Cytochrome b gene trees supported a basal placement of shrew-like moles (Uropsilus) within the Talpidae, and suggested that fossorial specializations arose twice during talpid evolution. To assess the evolutionary relationships of moles endemic to this region, we additionally sequenced the 12S rRNA gene and the nuclear recombination-activating gene-1 from eight and ten East Asian taxa, respectively. Analyses of these single and concatenated data sets suggested that East Asian shrew moles diverged prior to the evolution of fossorial Eurasian moles. However, we were unable to determine whether semi-fossorial shrew moles are monophyletic. In contrast, fossorial Eurasian genera (Talpa, Mogera and Euroscaptor) were consistently found to form a monophyletic clade, with Mogera and Euroscaptor representing sister taxa. Furthermore, this fossorial clade grouped with the semi-aquatic Desmana, although with fairly low (35-62%) bootstrap support. Mogera imaizumii was found to be more closely related to M. wogura than to M. tokudae. This implies that the ancestors of these three species entered Japan from the Asian continent in this order via a series of migration events, suggesting that the Japanese Islands have played an important role in preserving mole lineages from ancient to recent times.     

Testes of XX ↔ XY chimeric mice develop from fetal ovotestes

The majority of XX ? XY chimeric mice develop into fertile males. The sexual differentiation of the gonads in these animals has been examined on days 12–14 postcoitum to determine if their development parallels that of normal testes. It was found that 50% of chimeric fetuses, the proportion predicted to be XX ? XY, had neither normal testes nor ovaries. Instead, ovotestes were present, with varying proportions of presumptive ovarian and testicular tissue. On day 12 the ovotestes were organized with testicular tissue in the central region and ovarian tissue at the craniad and/or caudad poles. In the more advanced fetuses there was evidence of regression of the ovarian portion, which would account for the testes found in adults. These results are discussed in light of current theories of sex determination and differentiation and what was previously known about gonads of sex mosaics.     

Testes of XX in equilibrium with XY chimeric mice develop from fetal ovotestes

The majority of XX in equilibrium with XY chimeric mice develop into fertile males. The sexual differentiation of the gonads in these animals has been examined on days 12-14 postocoitum to determine if their development parallels that of normal testes. It was found that 50% of chimeric fetuses, the proportion predicted to be XX in equilibrium with XY, had neither normal testes nor ovaries. Instead, ovotestes were present, with varying proportions of presumptive ovarian and testicular tissue. On day 12 the ovotestes were organized with testicular tissue in the central region and ovarian tissue at the craniad and/or caudad poles. In the more advanced fetuses there was evidence of regression of the ovarian portion, which would account for the testes found in adults. These results are discussed in light of current theories of sex determination and differentiation and what was previously known about gonads of sex mosaics.     

Females of four mole species of genus Talpa (Insectivora,mammalia) are true hermaphrodites with ovotestes

We studied the anatomical, histological, and genetic features of the sexual tract in four European mole species of the genus Talpa (Insectivora, mammalia): T. occidentalis, T. europaea, T. romana, and T. stankovici. All XY individuals had a normal male phenotype, whereas all XX individuals in all four species had features that identified them as intersexes. These individuals were nonetheless presumed to be functionally fertile females. Intersexuality was manifested mainly as gonadal hermaphroditism, with all females possessing bilateral ovotestes. The gonads were composed of a small portion of histologically normal ovarian tissue and a variably sized, generally large mass of disgenetic testicular tissue, accompanied by a small, rudimentary epididymis. The rest of the sexual tract was typically female, including oviducts, uterus, and vagina of normal appearance. Polymerase chain reaction (PCR) and Southern blotting analyses showed that the mammalian testis-determining gene SRY is present in males but not in females. Part of the conserved sequence of the mole SRY gene was cloned and sequenced after PCR amplification in two of the four mole species (T. occidentalis from Spain and T. romana from Italy). Sequences were identical in these two species and were very similar to those of the human and mouse SRY gene. Our findings constitute the first evidence of the existence of a genus-specific case of true hermaphroditism, probably due to a very ancient mutation that fixed in populations of the ancestral species from which contemporary moles evolved. The possible nature of this mutation is discussed with regard to the cytologic, histologic, and genetic features of the gonads in Talpa females. © 1996 Wiley-Liss, Inc.     

Mitochondrial phylogeny of hedgehogs and monophyly of Eulipotyphla

We sequenced the complete mitochondrial (mt) genomes of three insectivores: the long-eared hedgehog Hemiechinus auritus, the Japanese mole Mogera wogura, and the greater Japanese shrew-mole Urotrichus talpoides. These mtDNA data together with other previously sequenced mtDNAs were analyzed using a maximum likelihood method to infer their phylogenetic relationships among eutherians. Previous mitochondrial protein analyses used a simple model that did not consider site-heterogeneity, and Erinaceoidea (hedgehogs and moonrats) was placed at the basal eutherian position that is separated from Soricoidea (shrews) and Talpoidea (moles), suggesting the exclusion of the Erinaceoidea-Eulipotyphla tree. By including the new mtDNA sequences and introducing site-heterogeneity into the model, the Erinaceoidea-Eulipotyphla tree emerges as the best tree or as a tree with a log-likelihood score indistinguishable from that of the best tree. However, this conclusion depends on species sampling in Erinaceoidea, demonstrating the importance of both species sampling and use of an appropriate substitution model when inferring phylogenetic relationships.     

Humerus development in moles (Talpidae,Mammalia)

The humerus of fossorial moles has a highly derived anatomy, reflecting the ecological specialization of these animals for digging. It is short and broad, with enlarged muscle attachment sites and pronounced articulations compared to non‐fossorial sister taxa and other mammals. Both condyles are rotated in opposite directions, resulting in a torsion which is unique among eutherian mammals. The development of this exceptional bone was studied in embryonic stages of the fossorial Iberian mole (Talpa occidentalis) from mesenchymal condensation to incipient ossification based on histological serial sections using 3D reconstruction methods. For comparison, embryonic stages of the semi‐fossorial Japanese shrew mole (Urotrichus talpoides) as well as a sister taxon of moles, the terrestrial North American least shrew (Cryptotis parva), were studied. Results show that the humerus of Talpa already shows its derived anatomy with broadened muscle attachment sites and distinct articulations at early cartilaginous stages, when ossification has just started in the mid‐diaphyseal region. The torsion takes place simultaneously with the medial rotation of the forelimbs. The supracondylar foramen is closed in all studied Talpa embryos, but patent in Cryptotis and Urotrichus. This is an example of developmental penetrance, suggesting that variation of adult elements can be found at early stages as well.     

The reproductive biology of blackspot sea bream Pagellus bogaraveo in captivity. I. gonadal development, maturation and hermaphroditism

Gonadal development, maturation, spawning and hermaphroditism were investigated in captive blackspot sea bream, Pagellus bogaraveo Brünnich, 1768, during the second, third and fourth years of life. The gonads of 224 fish were examined macroscopically and histologically. Undifferentiated gonads were found in fish smaller than 22.0 cm. Adult fish showed four gonadal differentiations: ovotestes with functional testis and quiescent ovary (Mf), ovotestes with functional ovary and regressed testis (mF), ovotestes with both ovary and testis at a resting stage (mf), and ovaries with no male tissue (F). The overall incidence of gonochoric females F was 41%. Functional males Mf were more frequent in age classes 1+ and 2+, whereas functional females mF predominated in the 3+ age class, above 25.0 cm TL. Histological examination revealed testicular degeneration and atrophy in functional females mF. On the basis of both histological data as well as size and age frequency distribution, it is suggested that the reproductive strategy of P. bogaraveo in captivity is characterized by protandrous hermaphroditism, with a high incidence of female gonochorism. Spawning occurred in March–April, at a size of 28.0 cm and age 3 in males and at 29.5 cm and age 4 in females. The gonosomatic index (GSI) remained constantly low (≤ 0.05) throughout the second and third years of life. A significant increase in GSI was noted in both males and females at the accomplishment of the fourth year of life, coinciding with the spawning season. The results are compared with information available on wild P. bogaraveo and discussed with a view to a possible exploitation of this species in aquaculture, through a reliable control of reproduction.     

Xenotransplantation: a tool for reproductive biology and animal conservation?

The transplantation of reproductive organs, including ovaries and ovarian tissue, was pioneered over 100 years ago. In the 1960s, ovarian grafting was used as a tool to investigate ovarian function, but with the recent development of more effective cryopreservation protocols for ovarian tissue, germline preservation and propagation have now become realistic goals. This review describes progress in ovarian banking and ovarian tissue transplantation, with emphasis on how fresh and frozen ovarian tissue can be used in assisted reproduction for both humans and animals. This paper focuses most closely on the potential value of xenotransplantation, the transplantation of gonads from one species to another, to conserve rare and endangered species. Specific attention is drawn to the use of xenotransplantation as a strategy for generating viable gametes that can be used to produce live fertile offspring. Other upcoming xenogeneic technologies that may be of potential significance in animal conservation, such as transplantation of whole ovaries or isolated growing follicles, and even male germ cells, are discussed.     

Historical aspects of protandrous sex change in the anemonefish Amphiprion melanopus (Pomacentridae, Teleostei)

Gonadal structure and cellular composition were examined in juveniles, males and females of the protandric hermaphrodite, Amphiprion melanopus. Functional sex change was experimentally induced in the field and gonad structure was histologically examined both qualitatively and quantitatively at 10, 20, 30 and 45 days after its initiation. Juvenile gonads consist primarily of immature ovarian tissue. Functional male gonads are ovotestes with co-existing mature spermatogenic tissue and immature ovarian tissue, while females possess only ovarian tissue. The initiation of sex change is marked by a rapid maturation of spermatogenic tissue and proliferation of putative oogonia. Gonads were essentially female by 20 days into sex change, but evidence of mature female function (marked by the initiation of vitellogenesis) was not observed until 45 days. Considerable variation between individuals was seen in quantitative measures of gonadal change in the early stages of sex change, but not in later stages. Progress in sex change as indicated by histological indicators was, however, consistent within stages. Duet systems for gamete transport changed from the male to the female form after all male tissue had been replaced.     

Experimentally induced true hermaphroditism in genetically female fowl

Summary Two types of hermaphroditism were experimentally induced in genetically female fowls by grafting of embryonic testes in embryos. Of the 27 hermaphrodites observed during the 8 months after hatching, 20 possessed a right testis and a left ovary and 7 a right testis and a left ovotestis. The testes and ovotestes contained seminiferous tubules with a more or less developed germ cell complement, attaining in many cases the early spermatid stage. The interstitial tissue was poorly functional, as shown by the absence of male secondary sex characters. The ovary or ovarian part of the ovotestes possessed numerous small ovarian follicles. The female arrangement of the plumage and the absence of spurs demonstrated the secretion of oestrogens. A mechanism is proposed for explaining this partial masculinization of genetically female gonads, a phenomenon which occurs during the period of embryonic sex differentiation, and is responsible for this experimental true hermaphroditism.     

Reproductive Biology and Protandrous Hermaphroditism in Acanthopagrus latus

Detailed macroscopic and histological studies of the gonads of a full size and age range of Acanthopagrus latus from each season in Shark Bay, Western Australia, demonstrate that this species is a protandrous hermaphrodite in this large subtropical embayment. Although our scheme for the changes that occur in the ovotestes of A. latus during life is not consistent with some of the conclusions drawn for this species elsewhere, it is similar to that of Pollock (1985 J. Fish. Biol. 26: 301–311) for the congeneric Acanthopagrus australis. The ovotestes of males develop from gonads which contain substantial amounts of both testicular and ovarian tissue. The testicular component of the ovotestes of all males regresses markedly after spawning. During the next spawning season, the ovotestes either become gonads in which the testicular zone again predominates and contains spermatids and spermatozoa (functional males), or gonads in which the ovarian zone now predominates and contains mature oocytes (functional females). Once a fish has become a functional female, it remains a female throughout the rest of its life. In Shark Bay, A. latustypically spawns on a limited number of occasions during a short period in late winter and early spring and has determinate fecundity. The mean potential annual fecundity was ca. 2000000. The total length of 245mm, at which, during the spawning period, 50% of A. latus become identifiable as males, is very similar to the current minimum legal length (MLL) of 250mm, which corresponds to an age of ca. 2.5 years less than that at which 50% of males become females. Thus, although the spawning potential ratio suggests that the present fishing pressure is sustainable, the current MLL should be reviewed if recreational fishing pressure continues to increase.     

Normal Testis Determination in the Mouse Depends on Genetic Interaction of a Locus on Chromosome 17 and the Y Chromosome

We previously described a locus on chromosome (Chr) 17 of the mouse that is critical for normal testis development. This locus was designated "T-associated sex reversal" (Tas) because it segregated with the dominant brachyury allele hairpin tail (Thp) and caused gonads of C57BL/6J XY, Thp/+ individuals to develop as ovaries or ovotestes rather than as testes. To clarify the inheritance of Tas, we investigated the effects of T-Orleans (TOrl), another brachyury mutation, on gonad development. We found that gonads of C57BL/6J XY, Thp/+ and TOrl/+ mice develop ovarian tissue if the Y chromosome is derived from the AKR/J inbred strain, whereas normal testicular development occurs in the presence of a Y chromosome derived from the C57BL/6J inbred strain. From these observations we conclude that: (1) Tas is located in a region on Chr 17 common to the deletions associated with Thp, and TOrl, and (2) the Y-linked testis determining gene, Tdy, carried by the AKR/J inbred strain differs from that of the C57BL/6J inbred strain. We suggest that in mammals Tdy is not the sole testis determinant because autosomal loci must be genetically compatible with Tdy for normal testicular development.     

Gonadal Growth in Embryos of Sex reversed Mice

Gonadal volumes were measured in litters of mouse embryos, aged 15 and 16 days, which were segregating for the Sex reversed factor. The testes of XY embryos were much larger than the ovaries of female embryos and the testes of XX Sex reversed males were almost, but not quite, as large as those of XY males. One of the 13 Sex reversed embryos had ovotestes.
It is concluded that the Sex reversed factor causes an increase in the growth rate of XX gonadal rudiments and that this is a necessary prerequisite for testicular differentiation. It is further postulated that the failure of spermatogenesis seen in adult Sex reversed , XX males is a result of the suboptimal growth rate of the gonads, compared to that of normal testes seen in the embryos. The formation of ovotestes may result from a growth rate which is at a lower limit of that necessary for testicular differentiation.     

Lymphoid aggregates in gonads of embryos, hatchlings, and young of turtles with temperature-dependent sex determination

Cellular infiltrations forming lymphoid-like aggregates were previously observed in gonads of two turtle species exhibiting temperature-dependent sex determination (TSD): at hatching in Chelydra serpentina; at and after hatching in Emys orbicularis. We show here that such aggregates are also present in gonads of Testudo graeca by the end of embryonic development, suggesting that their occurrence is general in turtles. Since in C. serpentina, infiltrations were observed mainly in testes exhibiting remnants of the germinal epithelium, it was assumed that their occurrence was an expression of maleness leading to rejection of this epithelium. The generality of this hypothesis was tested in E. orbicularis by looking for lymphoid-like aggregates in three types of gonads (testes, ovotestes, and ovaries) and for the stages at which they occur. Gonads were from embryos, hatchlings, and young incubated at various temperatures. Ovotestes obtained by treatment with an aromatase inhibitor of eggs incubated at female-producing temperature were also examined. In these gonads, the differentiation of Sertoli cells in testicular cords/tubes was ascertained by expression of SOX9. Moreover, the cell composition of aggregates was determined on electron micrographs. Aggregates appear in ovaries and ovotestes by the end of embryonic development and are present in the majority of these gonads at hatching, and at least up to one year after hatching. They are composed mainly of lymphocytes and fibroblasts. Aggregates are not present in typical testes. Since they occur in most ovaries, they cannot be seen as an expression of maleness. Rather, lymphocytic infiltration and formation of lymphoid aggregates in turtle gonads can be seen as components of the immune system, and can be under the control of gonadal endogenous sex steroids.     

Female gonad of moles, genus Talpa (Insectivora, mammalia): ovary or ovotestis?

The female gonads of moles (genus Talpa) are composed of a cortex, functioning as an ovary, and a medulla, which is structurally similar to that of the testis. In the female reproductive apparatus there are masculine glandular annexes, such as a bilobate prostate, two Cowper glands, and a penis-like clitoris. All these features have recently led to the hypothesis of the presence of hermaphroditism due to sex-reversal in Talpa. The purpose of this study is to understand the functional significance of the structural organization of the female gonads in order to verify this hypothesis. Histological, histochemical and ultrastructural analyses have been carried out on several gonads of both sexes of two species: T. europaea and T. romana, including three fetuses. In both species, the cortical region of the female gonad shows a regular oogenetic activity. While the medulla is composed of interstitial cells that are partly organized in cord-like structures, no spermatogenetic activity has been ever observed inside of them. A histochemical analysis shows that in both sexes the interstitial cells secrete steroids, presumably estrogens as well as androgens. The presence of androgens in the female gonads would therefore explain the persistence of male glandular annexes in the female reproductive apparatus and both the sexual and behavioral monomorphism typical of the genus Talpa. Nonetheless, the female gonad of moles is a real ovary and a well-defined gonochorism exists. Therefore, there is no reason to assert the presence of hermaphroditism due to sex reversal.     

Life stages and reproductive components of the Marmorkrebs (marbled crayfish), the first parthenogenetic decapod crustacean

Recently, we briefly reported on the first case of parthenogenesis in the decapod Crustacea which was found in the Marmorkrebs or marbled crayfish, a cambarid species of unknown geographic origin and species identity. Curiously, this animal is known only from aquarium populations, where it explosively propagates. By means of light and electron microscopic techniques we have now investigated the reproductive components of this crayfish, using more than 100 specimens ranging from hatchling to repeatedly spawned adult. Additionally, we documented its principal life stages. Our results revealed that the external sexual characters and also the gonads of the marbled crayfish are purely female, making this fast-reproducing species a good model for investigating female reproductive features in crayfish. Testicular tissues, ovotestes, or male gonoducts, gonopores, or gonopods were never found, either in small juveniles or large adult specimens, confirming the parthenogenetic nature of this crayfish. Parthenogenesis may have arisen spontaneously or by interspecific hybridization since Wolbachia-like feminizing microorganisms were not found in the ovaries. The external sexual characters of the marbled crayfish are first recognized in Stage 4 juveniles and are structurally complete approximately 2 months after hatching in specimens of approximately 2 cm total length. In the same life stage the ovary is fully differentiated as well, although the oocytes are in previtellogenic and primary vitellogenic stages only. The architecture of the mature ovary and also the synchronous maturation of cohorts of primary vitellogenic oocytes by secondary vitellogenesis are in general agreement with data published on ovaries of bisexual crayfish. New results were obtained with respect to the muscular nature of the ovarian envelope and its extensive proliferation after the first spawning, the distribution of hemal sinuses in the ovarian envelope and in the interstitium around the oogenetic pouches, the high transport activity of the follicle cells, and the colonization of oogenetic pouches by previtellogenic oocytes that originate in the germaria. Investigation of the nuclei of oocytes in the germaria and oogenetic pouches revealed no signs of meiosis, as usually found in females of bisexual decapods, suggesting that parthenogenesis in the marbled crayfish might be an apomictic thelytoky. The detection of new rickettsial and coccidian infections in the ovary and further organs raises fears that the marbled crayfish might endanger native European species by transmission of pathogens once escaped into the wild.     

Abnormal sex-duct development in female moles: the role of anti-Müllerian hormone and testosterone

We have performed a morphological, hormonal and molecular study of the development of the sex ducts in the mole Talpa occidentalis. Females develop bilateral ovotestes with a functional ovarian portion and disgenic testicular tissue. The Müllerian ducts develop normally in females and their regression is very fast in males, suggesting a powerful action of the anti-Müllerian hormone in the mole. RT-PCR demonstrated that the gene governing this hormone begins to be expressed in males coinciding with testis differentiation, and expression continues until shortly after birth. Immunohistochemical studies showed that expression occurs in the Sertoli cells of testes. No expression was detected in females. Wolffian duct development was normal in males and degenerate in prenatal females, but developmental recovery after birth gave rise to the formation of rudimentary epididymides. This event coincides in time with increasing serum testosterone levels and Leydig cell differentiation in the female gonad, thus suggesting that testosterone produced by the ovotestes is responsible for masculinisation of female moles. During postnatal development, serum testosterone concentrations decreased in males but increased in females, thus approaching the levels that adult males and females have during the non-breeding season.     

Histology of the reproductive tract of hybrids between gonochoristic males and parthenogenetic females of Lepidodactylus lugubris in French Polynesia (Reptilia, Gekkonidae).

Parthenogenetic populations of the gecko Lepidodactylus lugubris are widespread throughout Polynesia. They often occur parapatrically, and occasionally syntopically, with the increasingly rare bisexual populations. In these instances, a small number of hybrid individuals occur and include both "female" and "male" external phenotypes, both with greatly reduced gonads. Histological examination demonstrates that these hybrids possess small ovotestes. The differentiation of the cortical tissue is identical in both "male" and "female" hybrids, but the medullary tissue is more developed in "males." The remainder of the genital tract in "females" resembles that of fertile females in the parthenogenetic and bisexual populations. By contrast, the "male" hybrids are markedly intersexual. In one of the two specimens autopsied, the hemipenes are more or less the same size as those of bisexual males, and the sexual segment of the kidney is hypertrophied and serous. In the other hybrid "male," the hemipenes have a structure similar to that seen in females, and the sexual segment of the kidney is poorly differentiated. In both hybrid "males," the ductus deferens is extremely narrow and further reduced in its middle portion; oviducts are present and resemble those of normal or hybrid females. Thus, embryonic-like gonads are associated with complete and normal female reproductive ducts in hybrid "females." Hybrid "males" also have embryonic-like gonads and feminized genital ducts but associated with secondary sexual characters that match those of sexually active or quiescent normal males.