Speciation across biomes: Rapid diversification with reproductive isolation in the Australian delicate mice.

Phylogeographic studies of continental clades, especially when combined with palaeoclimate modelling, provide powerful insight into how environment drives speciation across climatic contexts. Australia, a continent characterized by disparate modern biomes and dynamic climate change, provides diverse opportunity to reconstruct the impact of past and present environments on diversification. Here, we use genomic-scale data (1310 exons and whole mitogenomes from 111 samples) to investigate Pleistocene diversification, cryptic diversity, and secondary contact in the Australian delicate mice (Hydromyini: Pseudomys), a recent radiation spanning almost all Australian environments. Across northern Australia, we find no evidence for introgression between cryptic lineages within Pseudomys delicatulus sensu lato, with palaeoclimate models supporting contraction and expansion of suitable habitat since the last glacial maximum. Despite multiple contact zones, we also find little evidence of introgression at a continental scale, with the exception of a potential hybrid zone in the mesic biome. In the arid zone, combined insights from genetic data and palaeomodels support a recent expansion in the arid specialist P. hermannsburgensis and contraction in the semi-arid P. bolami. In the face of repeated secondary contact, differences in sperm morphology and chromosomal rearrangements are potential mechanisms that maintain species boundaries in these recently diverged species. Additionally, we describe the western delicate mouse as a new species and recommend taxonomic reinstatement of the eastern delicate mouse. Overall, we show that speciation in an evolutionarily young and widespread clade has been driven by environmental change, and potentially maintained by divergence in reproductive morphology and chromosome rearrangements.

Ovary of the southern hairy-nosed wombat (Lasiorhinus latifrons): its divergent structural organisation.

The organisation of the ovarian interstitial tissue in the southern hairy-nosed wombat Lasiorhinus latifrons was investigated. Unlike in most other marsupials, the outer cortical region of the ovary contains abundant luteinised interstitial tissue that largely occurs in discrete lobules, many of which contain a localised area of non-cellular, highly eosinophilic and periodic acid-Schiff-positive material. The findings suggest that the latter arises from the zona pellucida that surrounded the oocyte in growing follicles and that the luteinised interstitial tissue thus developed from transformed theca interna of degenerated atretic follicles. It is hypothesised that this tissue synthesises and secretes progestogens, which may result in the long, and variable, oestrous cycle length that has been found to occur in this species.

Interspecific diversity of testes mass and sperm morphology in the Philippine chrotomyine rodents: implications for differences in breeding systems across the species.

The high diversity of native Philippine murid rodents includes an old endemic group, the chrotomyines, which are the sister group of the Australasian hydromyines. Herein we detail their interspecific diversity of relative testes mass (RTM) and sperm morphology. We find that in chrotomyines, as in the Australasian hydromyines, testes mass relative to body mass differs by an order of magnitude across the species and ranges from a large RTM in Soricomys and Chrotomys species to a small RTM in Apomys. Sperm morphology is associated with these findings, with individuals in species of Soricomys and Chrotomys producing relatively larger spermatozoa with a prominent apical hook and long tail, whereas, by contrast, the Apomys species have a sperm head that either has a very short or no apical hook and a shorter tail. These findings indicate coevolution of RTM with sperm morphological traits across the species, with the marked interspecific differences in RTM suggesting differences in the intensity of intermale sperm competition and hence breeding system. Thus, we hypothesise that species of Soricomys and Chrotomys that produce more streamlined spermatozoa with longer tails have a polyandrous or promiscuous mating system, whereas the Apomys species, which produce smaller and less streamlined spermatozoa, may exhibit monogamy.

Sperm morphology of the Rattini - are the interspecific differences due to variation in intensity of intermale sperm competition?

It is widely accepted that in mammals a causal relationship exists between postcopulatory sexual selection and relative testes mass of the species concerned, but how much it determines sperm size and shape is debatable. Here we detailed for the largest murine rodent tribe, the Rattini, the interspecific differences in relative testes mass and sperm form. We found that residual testes mass correlates with sperm head apical hook length as well as its angle, together with tail length, and that within several lineages a few species have evolved highly divergent sperm morphology with a reduced or absent apical hook and shorter tail. Although most species have a relative testes mass of 1-4%, these derived sperm traits invariably co-occur in species with much smaller relative testes mass. We therefore suggest that high levels of intermale sperm competition maintain a sperm head with a long apical hook and long tail, whereas low levels of intermale sperm competition generally result in divergent sperm heads with a short or non-existent apical hook and shorter tail. We thus conclude that sexual selection is a major selective force in driving sperm head form and tail length in this large tribe of murine rodents.

The egg coat zona pellucida 3 glycoprotein - evolution of its putative sperm-binding region in Old World murine rodents (Rodentia: Muridae).

In eutherian mammals, before fertilisation can occur the spermatozoon has to bind to, and penetrate, the egg coat, the zona pellucida (ZP). In the laboratory mouse there is good evidence that the primary sperm-binding site is a protein region encoded by Exon 7 of the ZP3 gene and it has been proposed that binding is species specific and evolves by sexual selection. In the present study we investigate these hypotheses by comparing Exon 6 and 7 sequences of ZP3 in 28 species of murine rodents of eight different divisions from Asia, Africa and Australasia, in which a diverse array of sperm morphologies occurs. We found considerable nucleotide (and corresponding amino acid) sequence divergence in Exon 7, but not in Exon 6, across these species, with evidence for positive selection at five codon positions. This molecular divergence does not appear to be due to reinforcement to reduce hybridisation, nor does it correlate with divergence in sperm head morphology or tail length, thus it is unlikely to be driven by inter-male sperm competition. Other forms of post-copulatory sexual selection therefore appear to have resulted in the molecular divergence of this region of ZP3 in this highly speciose group of mammals.

Greater sperm complexity in the Australasian old endemic rodents (Tribe: Hydromyini) is associated with increased levels of inter-male sperm competition.

Spermatozoa exhibit considerable interspecies morphological variation across mammals, especially among murid rodents. In Australasia, most murids in the tribe Hydromyini have a spermatozoon with a highly complex head exhibiting an apical hook, characteristic of most murids, and two projections that extend from its upper concave surface, the ventral processes. In the present study we performed a phylogenetically controlled comparison of sperm morphology across 45 species of hydromyine rodents to test the hypothesis that the length and angle of both the apical hook and ventral processes, as well as the length of the sperm tail, increase with relative testes mass as a proxy for differences in levels of inter-male sperm competition. Although both sperm head protrusions exhibited considerable variation in their length and angle across species, only the angles increased significantly in relation to relative testes mass. Further, the length of the sperm flagellum was positively associated with relative testes mass. These results suggest that, in hydromyine rodents, the angle of the apical hook and ventral processes of the sperm head, as well as the sperm tail length, are likely to be sexually selected traits. The possible functional significance of these findings is briefly discussed.

Spermatozoa of the old endemic rodents of Australia - the possible functional significance of their ventral processes.

Spermatozoa of the plains mouse (Pseudomys australis), like those of most Australian old endemic rodents, contain, in addition to an apical hook, two further processes that extend from the upper concave surface of the head, the ventral processes. This study shows that these processes contain thiol-rich cytoskeletal proteins, which presumably help to maintain their rigidity during sperm transport, together with the overlying cell membrane having abundant intramembranous proteins. To determine the possible functional significance of these processes, an in vitro study of spermatozoon-zona binding was undertaken. The findings suggest that initial sperm binding occurs by way of the cell membrane over the acrosome of the apical hook and that, subsequently, the lateral surfaces of the ventral processes also become tightly bound to the zona matrix. These ventral processes may therefore have evolved to increase sperm adhesion to the outer zona surface and/or to enhance stabilisation of the spermatozoon at the time of zona binding and initial penetration of the egg coat.

Sperm competition intensity shapes divergence in both sperm morphology and reproductive genes across murine rodents.

It remains unclear how variation in the intensity of sperm competition shapes phenotypic and molecular evolution across clades. Mice and rats in the subfamily Murinae are a rapid radiation exhibiting incredible diversity in sperm morphology and production. We combined phenotypic and genomic data to perform phylogenetic comparisons of male reproductive traits and genes across 78 murine species. We identified several shifts towards smaller relative testes mass, presumably reflecting reduced sperm competition. Several sperm traits were associated with relative testes mass, suggesting that mating system evolution selects for convergent suites of traits related to sperm competitive ability. We predicted that sperm competition would also drive more rapid molecular divergence in species with large testes. Contrary to this, we found that many spermatogenesis genes evolved more rapidly in species with smaller relative testes mass due to relaxed purifying selection. While some reproductive genes evolved rapidly under recurrent positive selection, relaxed selection played a greater role in underlying rapid evolution in small testes species. Our work demonstrates that postcopulatory sexual selection can impose strong purifying selection shaping the evolution of male reproduction, and that broad patterns of molecular evolution may help identify genes that contribute to male fertility.

Molecular evolution of male reproduction across species with highly divergent sperm morphology in diverse murine rodents.

Sperm competition can drive rapid evolution of male reproductive traits, but it remains unclear how variation in sperm competition intensity shapes phenotypic and molecular diversity across clades. Old World mice and rats (subfamily Murinae) comprise a rapid radiation and exhibit incredible diversity in sperm morphology and production. We combined phenotype and sequence data to model the evolution of reproductive traits and genes across 78 murine species. We identified several shifts towards smaller relative testes mass, a trait reflective of reduced sperm competition. Several sperm traits were associated with relative testes mass, suggesting that mating system evolution likely selects for convergent traits related to sperm competitive ability. Molecular evolutionary rates of spermatogenesis proteins also correlated with relative testes mass, but in an unexpected direction. We predicted that sperm competition would result in rapid divergence among species with large relative testes mass, but instead found that many spermatogenesis genes evolve more rapidly in species with smaller relative testes mass due to relaxed purifying selection. While some reproductive genes evolved under positive selection, relaxed selection played a greater role underlying rapid evolution in small testes species. Our work demonstrates that sexual selection can impose strong purifying selection shaping the evolution of male reproduction.

TESTIS ABNORMALITIES IN A POPULATION OF SOUTH AUSTRALIAN KOALAS (PHASCOLARCTOS CINEREUS).

Testis asymmetry, in which the testes in an individual differ in size, has recently been reported in koalas (Phascolarctos cinereus) in the Mount Lofty Ranges population of South Australia. We describe the morphology and histology of both testes from affected individuals in this population (n=56) and the parameters of koalas with normal-sized testes based on age and breeding season (n=56). Morphologic measurements included testis weight, length, width, and volume; histologic parameters included seminiferous tubule diameter, seminiferous epithelial height, and seminiferous tubule (interstitial tissue ratio and presence or absence of spermatozoa). Of the 56 koalas with intraindividual variation in testes size, 47 were classified as asymmetric and nine as microtestes. For koalas with asymmetric testes, all morphologic parameters were significantly decreased in the smaller testes compared with normal-sized testes, but for the histologic parameters, only seminiferous tubule diameter was significantly less. Histopathologic examination of the asymmetric testes showed 38 with normal parenchyma histologically indistinguishable between intraindividual testes, four with degeneration and atrophy, and three with hypoplasia, whereas examination of microtestes showed degeneration and atrophy in seven, hypoplasia in one, and aplasia in one. No association of testis size difference with Chlamydia pecorum infection was found in a subset of animals. For the 56 koalas with normal-sized testes, morphologic parameters were found to increase with age, and juvenile and young adults were found to have smaller seminiferous tubule diameters than adults. No differences were found between testes of koalas in the breeding and nonbreeding season. Overall, these findings indicate that testis asymmetry in koalas from the Mount Lofty Ranges population is common but not associated with decreased function, except where testis malformations such as hypoplasia or aplasia occur or when parenchyma has been disrupted by acquired disease.

Temporal deposition and spatial distribution of cytoskeletal proteins in the sperm head of an Australian rodent.

The Australian murine rodent, the plains mouse (Pseudomys australis), possesses a highly complex sperm head, in which there are, in addition to an apical hook, two ventral processes that extend from its upper concave surface. The present study set out to determine the temporal deposition and distribution of the proteins within these structures during late spermiogenesis by light and electron microscopy using various antibodies to bull and laboratory rat sperm-head cytoskeletal proteins. The findings show that there are two phases of protein deposition. In the first phase, perinuclear theca proteins are deposited at the base of the ventral processes around the acrosomal extensions of the developing spermatids. In the second phase, as the ventral processes expand, actin and then perforatorial proteins are laid down during which time the processes become progressively more bilaterally flattened. These various proteins are moulded together to give rise to the two very large cytoskeletal structures that extend from the upper concave surface of the sperm head. They may be involved in binding the spermatozoon to the outer surface of the zona pellucida and/or in aiding the spermatozoon in zona penetration at the time of fertilisation.

Changes in distribution of basic nuclear proteins and chromatin organization during spermiogenesis in the greater bandicoot rat, Bandicota indica.

Male germ cells of the greater bandicoot rat, Bandicota indica, have recently been categorized into 12 spermiogenic steps based upon the morphological appearance of the acrosome and nucleus and the cell shape. In the present study, we have found that, in the Golgi and cap phases, round spermatid nuclei contain 10-nm to 30-nm chromatin fibers, and that the acrosomal granule forms a huge cap over the anterior pole of nucleus. In the acrosomal phase, many chromatin fibers are approximately 50 nm thick; these then thickened to 70-nm fibers and eventually became 90-nm chromatin cords that are tightly packed together into highly condensed chromatin, except where nuclear vacuoles occur. Immunocytochemistry and immunogold localization with anti-histones, anti-transition protein2, and anti-protamine antibodies suggest that histones remain throughout spermiogenesis, that transition proteins are present from step 7 spermatids and remain until the end of spermiogenesis, and that protamines appear at step 8. Spermatozoa from the cauda epididymidis have been analyzed by acid urea Triton X-100 polyacrylamide gel electrophoresis for basic nuclear proteins. The histones, H2A, H3, H2B, and H4, transitional protein2, and protamine are all present in sperm extracts. These findings suggest that, in these sperm of unusual morphology, both transition proteins and some histones are retained, a finding possibly related to the unusual nuclear form of sperm in this species.

Spermatozoa from a marsupial, the brushtail possum, contain beta1,4-galactosyltransferase.

Beta1,4-galactosyltransferase-I (GalTase-I) is one of the key molecules on the sperm surface of eutherian mammals that is likely to be involved in binding to the egg coat, the zona pellucida, to mediate sperm-egg interaction. In laboratory mice, the species for which most data are available, this protein functions as a receptor for the zona pellucida protein ZP3 of the oocyte and, upon binding, triggers the sperm acrosome reaction. In the present study, we investigated the presence and abundance of GalTase-I in epididymal sperm extracts of a marsupial, the brushtail possum, Trichosurus vulpecula. For this, spermatozoa were collected from cauda epididymides and the amount of beta1,4-galactosyltransferase activity in washed sperm extracts was compared with that of porcine spermatozoa. Overall beta1,4-galactosyltransferase enzyme activity was found to be more abundant in possum sperm extracts than those from porcine spermatozoa (P<0.05). Immunoblots with an antibody to mouse GalTase-I revealed that the molecular weight of possum spermatozoa GalTase-I was 66 kDa, which is similar to the molecular weight of GalTase-I in spermatozoa from eutherian mammals. The molecular weight of GalTase-I was the same in sperm extracts collected from the caput and cauda epididymides. These results demonstrate that GalTase-I is indeed present in possum spermatozoa and thus it may be a gamete receptor molecule on the sperm surface of marsupials as well as those of eutherian mammals.

Sperm morphology of the Eurasian beaver, Castor fiber: an example of a species of rodent with highly derived and pleiomorphic sperm populations.

The structural organization of the spermatozoon from the Eurasian beaver, Castor fiber (Family: Castoridae), was determined and compared to that of other sciuromorph rodents. The beaver spermatozoon has a head, which is variable in form but usually paddle-shaped, with a small nucleus and very large acrosome, and a tail that is relatively short compared to that of most other rodents. Transmission electron microscopy indicates that in most testicular spermatozoa the acrosome projects apically, although in a few it becomes partly flexed. During the final stages of maturation, however, the acrosome becomes highly folded so that the apical segment comes to lie alongside part of the acrosome that occurs lateral to the nucleus, with, in some cases, fusion taking place between the outer acrosomal membranes. The sperm nucleus is wedge-shaped, being broader basally and narrowing apically with an occasional large nuclear vacuole occurring. This spermatozoon structure is markedly different from that found in the other species of Geomyoidea, which is the sister group of the Castoridae. The findings thus emphasize the highly divergent nature of the beaver spermatozoon and demonstrate that, within the proposed Infraorder Castorimorpha, very large differences in sperm structure have evolved.

Evolution of the spermatozoon in muroid rodents.

In the rodent superfamily Muroidea, a model for the evolution of sperm form has been proposed in which it is suggested that a hook-shaped sperm head and long tail evolved from a more simple, nonhooked head and short tail in several different subfamilies. To test this model the shape of the sperm head, with particular emphasis on its apical region, and length of sperm tail were matched to a recent phylogeny based on the nucleotide sequence of several protein-coding nuclear genes from 3 families and 10 subfamilies of muroid rodents. Data from the two other myomorph superfamilies, the Dipodoidea and kangaroo rats in the Geomyoidea, were used for an outgroup comparison. In most species in all 10 muroid subfamilies, apart from in the Murinae, the sperm head has a long rostral hook largely composed of acrosomal material, although its length and cross-sectional shape vary across the various subfamilies. Nevertheless, in a few species of various lineages a very different sperm morphology occurs in which an apical hook is lacking. In the outgroups the three species of dipodid rodents have a sperm head that lacks a hook, whereas in the heteromyids an acrosome-containing apical hook is present. It is concluded that, as the hook-shaped sperm head and long sperm tail occur across the muroid subfamilies, as well as in the heteromyid rodents, it is likely to be the ancestral condition within each of the subfamilies with the various forms of nonhooked sperm heads, that are sometimes associated with short tails, being highly derived states. These findings thus argue against a repeated evolution in various muroid lineages of a complex, hook-shaped sperm head and long sperm tail from a more simple, nonhooked sperm head and short tail. An alternative proposal for the evolution of sperm form within the Muroidea is presented in the light of these data.

The spermatozoon of Eurasian murine rodents: Its morphological diversity and evolution.

The murine rodents are the most speciose subfamily of mammals. Here the morphology of the spermatozoon, as determined by scanning and transmission electron microscopy of representative species from four Eurasian clades, is described. Much interspecific variability in all components of the spermatozoon was found to occur, although most species have a bilaterally flattened sperm head with a single apical hook of variable length and orientation. Ultrastructural observations indicate that this apical hook invariably contains a nuclear projection as well as a large extension of the subacrosomal cytoskeleton, as a perforatorium rostrally, and a complex asymmetrical acrosomal extension. These spermatozoa also have relatively long tails that are attached to the lower concave surface of the sperm head. Uniquely, in species in the Apodemus clade, the apical hook is orientated caudally. In a few species a highly derived sperm head morphotype that does not contain an apical hook is present. These sperm heads vary in morphology from being globular in two species of Bandicota, to bilaterally flattened and paddle-shaped in Tokudaia and Micromys. In spermatozoa of the latter two genera the subacrosomal cytoskeleton, which is less extensive than in species with a hooked sperm head, forms an apical extension, but that is not the case in Bandicota. In all species where the sperm head lacks an apical hook the acrosome is more symmetrical. The sperm tail is much shorter in these species, with attachment to the head occurring on the ventral surface in Tokudaia and basal in Micromys and the two species of Bandicota. As the sperm head morphotype with a complex apical hook is present in all the major clades of murine rodents, it is likely to be a plesiomorphic character within each of these clades, with the nonhooked sperm heads, which vary greatly in structure between species of the different lineages, probably being independently derived. The ultrastructural organization of the sperm head of Bandicota, but not those of Micromys or Tokudaia, suggest divergence in some of the morphological events associated with sperm-egg interaction at the time of fertilization.

Morphological diversity and evolution of the spermatozoon in the mouse-related clade of rodents.

Most species in the three highly speciose families of the mouse-related clade of rodents, the Muridae, Cricetidae, and Nesomyidae (superfamily Muroidea), have a highly complex sperm head in which there is an apical hook but there are few data available for the other related families of these rodents. In the current study, using light and electron microscopies, we investigated the structure of the spermatozoon in representative species of four other families within the mouse-related clade, the Dipodidae, Spalacidae, Pedetidae, and Heteromyidae, that diverged at or near the base of the muroid lineage. Our results indicate that a diverse array of sperm head shapes and tail lengths occurs but none of the species in the families Spalacidae, Dipodidae, or Pedetidae has a sperm head with an apical hook. By contrast, a rostrally extending apical hook is present in spermatozoa of members of the Family Heteromyidae which also invariably have comparatively long sperm tails. These findings suggest that the hook-shaped sperm head in the murid, cricetid, and nesomyid rodents evolved after divergence of this lineage from its common ancestor with the other families of the mouse-related clade, and that separate, and independent, convergent evolution of a similar sperm head form, and long sperm tail, occurred in the Heteromyidae.

Plasma biochemistry and urinalysis variables of koalas (Phascolarctos cinereus) with and without oxalate nephrosis.

BACKGROUND: Oxalate nephrosis is a highly prevalent disease in the Mount Lofty Ranges koala population in South Australia, but associated clinicopathologic findings remain undescribed. OBJECTIVES: The aims of this study were to determine plasma biochemical and urinalysis variables, particularly for renal function and urinary crystal morphology and composition, in koalas with oxalate nephrosis. METHODS: Blood and urine samples from Mount Lofty Ranges koalas with oxalate nephrosis were compared with those unaffected by renal oxalate crystal deposition from Mount Lofty and Kangaroo Island, South Australia and Moggill, Queensland. Plasma and urine biochemistry variables were analyzed using a Cobas Bio analyzer, and urinary oxalate by high-performance liquid chromatography. Urinary crystal composition was determined by infrared spectroscopy and energy dispersive X-ray analysis. RESULTS: Azotemia (urea > 6.6 mmol/L, creatinine > 150 µmol/L) was found in 93% of koalas with oxalate nephrosis (n = 15). All azotemic animals had renal insufficiency (urine specific gravity [USG] < 1.035), and in 83%, USG was < 1.030. Koalas with oxalate nephrosis were hyperoxaluric compared with Queensland koalas (P < .01). Urinary crystals from koalas with oxalate nephrosis had atypical morphology and were composed of calcium oxalate. Mount Lofty Ranges koalas unaffected by renal oxalate crystal deposition had renal insufficiency (43%), although only 14% had USG < 1.030 (n = 7). Unaffected Mount Lofty Ranges and Kangaroo Island koalas were hyperoxaluric compared with Queensland koalas (P < .01). CONCLUSIONS: Koalas with oxalate nephrosis from the Mount Lofty Ranges had renal insufficiency, hyperoxaluria, and pathognomonic urinary crystals. The findings of this study will aid veterinary diagnosis of this disease.

Reproductive biology of an old endemic murid rodent of Australia, the Spinifex hopping mouse, Notomys alexis: adaptations for life in the arid zone.

The Spinifex hopping mouse (Notomys alexis Thomas, 1922) is an arid adapted Australo-Papuan old endemic rodent that undergoes boom and bust population cycles. In this communication, we review our findings on the timing of reproduction and the potential reproductive rate of this species. To investigate the first question, the reproductive condition of adult females, and occurrence of immatures, in a population on a cattle station in central Australia was determined and, for the second, data from a laboratory colony compared to those previously published on closely related species in other environments. The findings show that, at least in the population at the times that monitoring was performed, reproductive activity was only taking place in spring and early summer, whereas the laboratory study indicates that females have a similar gestation length and litter size to those of most close relatives occurring in other environments. Males have extremely small testes and store relatively few sperm. The findings suggest that Notomys alexis might show some seasonality of reproduction at least in this region of central Australia and that this species does not have a higher reproductive rate than that of related species in other, more predictable, environments.

The morphology of the squirrel spermatozoon: a highly complex male gamete with a massive acrosome.

This study describes the morphology of the spermatozoon from the cauda epididymidis of representative members of two squirrel subfamilies, the Sciurinae and Callosciurinae, as determined by fluorescent, scanning, and transmission electron microscopy. All species examined possess a massive apical segment of the sperm acrosome. It varied markedly in the extent of its caudal flexion but was always much larger, and more complex, than that of the spermatozoon of most other rodents so far documented, although somewhat similar to that of some hystricomorph species. Because this sperm form appears to be present within at least two of the three major living clades of Rodentia, it is possible that it is the ancestral condition within this mammalian order.