FGF-stimulated tendon cells embrace a chondrogenic fate with BMP7 in newt tissue culture.

Newts can regenerate functional elbow joints after amputation at the joint level. Previous studies have suggested the potential contribution of cells from residual tendon tissues to joint cartilage regeneration. A serum-free tissue culture system for tendons was established to explore cell dynamics during joint regeneration. Culturing isolated tendons in this system, stimulated by regeneration-related factors, such as fibroblast growth factor (FGF) and platelet-derived growth factor, led to robust cell migration and proliferation. Moreover, cells proliferating in an FGF-rich environment differentiated into Sox9-positive chondrocytes upon BMP7 introduction. These findings suggest that FGF-stimulated cells from tendons may aid in joint cartilage regeneration during functional elbow joint regeneration in newts.

Comparison of hypoxia- and hyperoxia-induced alteration of epigene expression pattern in lungs of Pleurodeles waltl and Mus musculus.

Epigenetics is an emerging field of research because of its involvement in susceptibility to diseases and aging. Hypoxia and hyperoxia are known to be involved widely in various pathophysiologies. Here, we compared the differential epigene expression pattern between Pleurodeles waltl and Mus musculus (commonly known as Iberian ribbed newt and mouse, respectively) exposed to hypoxia and hyperoxia. Adult healthy newts and mice were exposed to normobaric hypoxia (8% O2) and hyperoxia (80% O2) for 2 hours. We collected the lungs and analyzed the expression of hypoxia-inducible factor 1 alpha (Hif1α) and several key epigenes from DNA methyltransferase (DNMT) family, histone deacetylase (HDAC) family, and methyl-CpG binding domain (MBD) family. The exposure to hypoxia significantly increased the mRNA levels of DNA methyltransferase 3 alpha (Dnmt3α), methyl-CpG binding domain protein 2 (Mbd2), Mbd3, and histone deacetylase 2 (Hdac2) in lungs of newts, but decreased the mRNA levels of DNA methyltransferase 1 (Dnmt1) and Dnmt3α in lungs of mice. The exposure to hyperoxia did not significantly change the expression of any gene in either newts or mice. The differential epigene expression pattern in response to hypoxia between newts and mice may provide novel insights into the prevention and treatment of disorders developed due to hypoxia exposure.

Amphibian newts as experimental models for studying weight gain after castration.

Vertebrate animals often exhibit sexual dimorphism in body shape. In mammals, decreases in sex hormones caused by testicular castration can affect body shape and occasionally lead to pathologies such as obesity. Post-castration obesity can also be problematic for the health of companion animals, including non-mammals. In order to understand the mechanism of post-castration obesity in vertebrates other than mammals, experimental models are required. We examined whether the Iberian ribbed newt, which has recently become a popular experimental model for amphibian research, could serve as a model for analyzing changes in body shape after castration. In newts, new testes can be regenerated after removal of differentiated testes. We analyzed changes in body shape by removing the testes under conditions in which they could regenerate or conditions in which they could not regenerate. Removal of the testes reduced blood testosterone levels. The body weight and abdominal girth of the newts were increased compared with normal male newts. Transcriptome analysis of the liver showed that a set of genes related to lipid metabolism was continuously up-regulated in castrated newts. Our study suggests that changes in body shape after castration are common in vertebrates. Iberian ribbed newts are thus a suitable model for comparative studies of the long-term physiologic- and endocrine-level effects of castration.

Learning the native pond odor as one of the mechanisms of olfactory orientation in juvenile smooth newt Lissotriton vulgaris.

Olfaction is an important mechanism of orientation in amphibians toward the breeding site. It is known that anurans can memorize the odor of the native pond during larval development and prefer this odor prior to the beginning of dispersion. However, such a mechanism in urodeles has not been studied yet. We conducted experiments on recognition of the odor of a native water body in juveniles of the smooth newt Lissotriton vulgaris. One group of larvae were reared in pure water (control), the other group in water with morpholine (10-7 mol/L). A few days after metamorphosis, the newts were tested under paired-choice conditions in a T-maze. A total of 73 newts from the experimental group and 47 newts from the control group were tested. The results of the experiment show that the newts in the experimental group preferred the morpholine solution, whereas the individuals of the control group made the choice randomly. We conclude that newts can learn the odor of the environment in which they developed and use this memory for orientation in later stages of life.

Terrestrial amphibians respond to rapidly changing temperatures with individual plasticity of exploratory behaviour.

Terrestrial ectotherms react to acute changes in environmental temperatures by adjusting their behaviour. Evaluating the adaptive potential of these behavioural adjustments requires information on their repeatability and plasticity. We examined behavioural response (exploration) to acute temperature change in two amphibian taxa, alpine (Ichthyosaura alpestris) and smooth (Lissotriton vulgaris) newts. These responses were investigated at both population and individual levels under multiple thermal contexts (dimensions), represented by the direction and range of changing temperature and rearing thermal regimes. Population-level analyses showed species-specific, non-additive effects of direction and range of temperature change on acute thermal reaction norms for exploration, but explained only a low amount (7-23%) of total variation in exploration. In contrast, within- and among-individual variation in acute thermal reaction norm parameters explained 42-50% of total variation in the examined trait. Although immediate thermal responses varied among individuals (repeatability = 0.07 to 0.53), they were largely shaped by environmental contexts during repeated trials. We conclude that these amphibians respond to acute temperature change through individual plasticity of behavioural traits. A repeated-measures approach under multiple thermal contexts will be needed to identify the selective and plastic potential of behavioural responses used by juvenile newts and perhaps other ectotherm taxa to cope with rapidly changing environmental temperatures.

Fine-scale spatial genetic structure and dispersal among Italian smooth newt populations in a rural landscape.

Amphibians are particularly sensitive to habitat loss and fragmentation caused by the intensification and modernization of farming occurring in the second half of the twentieth century in the Mediterranean basin. However, artificial water bodies, associated with traditional husbandry, proved to be important surrogate for amphibian feeding and reproduction. Here, multilocus genotypes were used to investigate the spatial population structure of Lissotriton vulgaris meridionalis and the role of drinking troughs in supporting viable breeding populations within a rural landscape interested by traditional husbandry and agriculture. Our genetic analysis highlighted the conservation value and the potential stepping-stone function of artificial aquatic sites in the dispersal of the species and for the gene flow maintenance. Indeed, populations of drinking troughs show allelic richness and heterozygosity levels comparable to those from natural ponds and there is no great evidence of genetic bottlenecks. A complex system of artificial aquatic sites and few natural wetlands was identified sustaining a well-structured network of demes highly interconnected with themselves and natural aquatic sites. The conservation of the identified genetic clusters may be useful to prevent further population declines and future loss of genetic diversity within the study area characterized by scarce natural wetlands that frequently dried because of agricultural practices and strong seasonality. Site-specific protection measures are needed to contrast the progressive disappearance of drinking troughs observed in the last years in Italy because of the abandonment of traditional farming practices in favour of modern agriculture and intensive farming.

Putting infection on the map: Using heatmaps to characterise within- and between-host distributions of trematode metacercariae.

The location of parasites within individual hosts is often treated as a static trait, yet many parasite species can occur in multiple locations or organs within their hosts. Here, we apply distributional heat maps to study the within- and between-host infection patterns for four trematodes (Alaria marcianae, Cephalogonimus americanus, Echinostoma spp. and Ribeiroia ondatrae) within the amphibian hosts Pseudacris regilla and two species of Taricha. We developed heatmaps from 71 individual hosts from six locations in California, which illustrate stark differences among parasites both in their primary locations within amphibian hosts as well as their degree of location specificity. While metacercariae (i.e., cysts) of two parasites (C. americanus and A. marcianae) were relative generalists in habitat selection and often occurred throughout the host, two others (R. ondatrae and Echinostoma spp.) were highly localised to a specific organ or organ system. Comparing parasite distributions among these parasite taxa highlighted locations of overlap showing potential areas of interactions, such as the mandibular inner dermis region, chest and throat inner dermis and the tail reabsorption outer epidermis. Additionally, the within-host distribution of R. ondatrae differed between host species, with metacercariae aggregating in the anterior dermis areas of newts, compared with the posterior dermis area in frogs. The ability to measure fine-scale changes or alterations in parasite distributions has the potential to provide further insight about ecological questions concerning habitat preference, resource selection, host pathology and disease control.

OCCURRENCE OF PATHOGENIC CHYTRID FUNGI BATRACHOCHYTRIUM SALAMANDRIVORANS AND BATRACHOCHYTRIUM DENDROBATIDIS IN THE HONG KONG NEWT (PARAMESOTRITON HONGKONGENSIS) AND OTHER WILD AND IMPORTED AMPHIBIANS IN A SUBTROPICAL ASIAN REGION.

One of the major threats for the massive loss in global amphibian diversity is chytridiomycosis, caused by chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Following its discovery in 2013, Bsal has emerged as a severe threat to the global survival of urodelans. In 2018, a study reported a high prevalence of Bsal (65.6%) in the Hong Kong newts (Paramesotriton hongkongensis, Near Threatened) of a southern China population adjacent to Hong Kong (HK). Uncertainty regarding the Bsal infection status of P. hongkongensis inhabiting HK raised deep concern over the risk of introducing Bsal from that population. We screened the skin swabs from wild individuals of P. hongkongensis, 15 sympatric amphibian species, and 16 imported amphibian species in HK for chytrids. We found that both Bsal and Bd occur in low prevalences in P. hongkongensis (Bsal 1.7%, 5/293; Bd 0.34%, 1/293), Hong Kong cascade frog, Amolops hongkongensis, family Ranidae (Bsal only, 5.26%, 1/19), and Asian common toad, Duttaphrynus melanostictus, family Bufonidae (Bsal only, 5.88%, 1/17), populations of HK, with infected individuals being asymptomatic, suggesting a potential role of these species as reservoirs of Bsal. Conversely, Bd, but not Bsal, was present on 13.2% (9/68) of imported amphibians, indicating a high chytrid introduction risk posed by international amphibian trade. Long-term surveillance of the presence of Bd and Bsal in wild and captive amphibians would be advisable, and we recommend that import and export of nonnative chytrid carriers should be prevented, especially to those regions with amphibian populations naïve to Bd and Bsal.

Demographic and ecological niche dynamics of the Vietnam warty newt, Paramesotriton deloustali: Historical climate influences.

Quaternary climatic cycles strongly affected the genetic diversification and ranges of organisms, shaping current genetic structures and distribution patterns. Urodeles provide ideal examples for exploring these dynamics over time and across space. In this study, we integrated a phylogeographic approach and ensemble species distribution modeling (eSDM) to infer the historical demography and distribution patterns of the Vietnam warty newt, Paramesotriton deloustali. Mitochondrial data revealed two groups, West and East, which diverged approximately 1.92 million years ago (Mya). Diversification was likely driven by change in the climate during early stages of the Pleistocene, with increasing monsoon and drought intensities. Biogeographic analysis indicated that the newt's current distribution formed as a result of vicariance events. In addition, the two groups occupy distinct ecological niches. Demographic reconstruction showed signs of expansion in the effective population sizes of the two major groups beginning around 0.11 and 0.15 Mya, respectively. However, eSDM showed fluctuating predicted distributions during the last interglacial, last glacial maximum, mid-Holocene, and present. Mountain systems in northern Vietnam are likely to have served as climatic refuges and to have played a crucial role in safeguarding species from the effects of climate change.

A synoptic review of the Amphibians of Iran: bibliography, taxonomy, synonymy, distribution, conservation status, and identification key to the eggs, larvae, and adults.

This study provides an illustrated account, a comprehensive update of the systematics, and a bibliography of the 15 species of anurans in five families, eight genera; and of the six species of urodeles in two families, four genera in Iran. Bufonidae, with eight species, is the most diverse family; Salamandridae has five species and Ranidae has four species. This study also presents updated identification keys for the eggs, larvae, and metamorphosed amphibians of Iran. We designated specimen NMW 19855.1 as neotype of Pelophylax persicus (Schneider, 1799) comb. nov.. Along with distribution maps obtained from all the reliable localities and museum specimens known at this time, the modelled habitat of species, and for the first time, the National Red List of amphibians based on the IUCN red list categories and criteria. Based on our evaluation we propose to categorize Bufo eichwaldi, Paradactylodon persicus, Neurergus derjugini, and N. kaiseri as Vulnerable at National Red List, and to move Bufotes (Calliopersa) luristanicus, B. (C.) surdus, Firouzophrynus olivaceus, and Rana pseudodalmatina from the category of Least Concern (LC) to Near Threatened (NT). The National Red List of amphibians that we propose has significant implications for endangered species management and conservation. Forty-one percent of amphibian species in Iran are endemic to the country, and more than forty percent of the Iranian amphibians are at risk of extinction. Zagros Mountain forest and Hyrcaniain forests have more than 80% (i.e. 18 species) of the diversity of Iranian amphibians. A considerable amount of scientific literature published on Iranian amphibians in Persian language is not easily accessible to researchers outside Iran. This monograph attempts to remedy the situation and provides broader access to international herpetology. We recognize that taxonomy is always in a state of flux, and the names and synonymies used here reflect our current view.

Regionalization and morphological integration in the vertebral column of Eurasian small-bodied newts (Salamandridae: Lissotriton).

Serially homologous structures may have complex patterns of regionalization and morphological integration, influenced by developmental Hox gene expression and functional constraints. The vertebral column, consisting of a number of repeated, developmentally constrained, and highly integrated units-vertebrae-is such a complex serially homologous structure. Functional diversification increases regionalization and modularity of the vertebral column, particularly in mammals. For salamanders, three concepts of regionalization of the vertebral column have been proposed, recognizing one, two, or three presacral regions. Using three-dimensional geometric morphometrics on vertebra models acquired with microcomputerized tomography scanning, we explored the covariation of vertebrae in four closely related taxa of small-bodied newts in the genus Lissotriton. The data were analyzed by segmented linear regression to explore patterns of vertebral regionalization and by a two-block partial least squares method to test for morphological integration. All taxa show a morphological shift posterior to the fifth trunk vertebra, which corresponds to the two-region concept. However, morphological integration is found to be strongest in the mid-trunk. Taken jointly, these results indicate a highly integrated presacral vertebral column with a subtle two-region differentiation. The results are discussed in relation to specific functional requirements, developmental and phylogenetic constraints, and specific requirements posed by a biphasic life cycle and different locomotor modes (swimming vs. walking). Further research should be conducted on different ontogenetic stages and closely related but ecologically differentiated species.

An experimental assessment of detection dog ability to locate great crested newts (Triturus cristatus) at distance and through soil.

Detection dogs are increasingly used to locate cryptic wildlife species, but their use for amphibians is still rather underexplored. In the present paper we focus on the great crested newt (Triturus cristatus), a European species which is experiencing high conservation concerns across its range, and assess the ability of a trained detection dog to locate individuals during their terrestrial phase. More specifically, we used a series of experiments to document whether a range of distances between target newts and the detection dog (odour channelled through pipes 68 mm in diameter) affects the localisation, and to assess the ability and efficiency of target newt detection in simulated subterranean refugia through 200 mm of two common soil types (clay and sandy soil, both with and without air vents to mimic mammal burrows, a common refuge used by T. cristatus). The detection dog accurately located all individual T. cristatus across the entire range of tested distances (0.25 m- 2.0 m). The substrate trials revealed that the detection dog could locate individuals also through soil. Contrary to existing studies with detection dogs in human forensic contexts, however, detection was generally slower for T. cristatus under sandy soil compared to clay soil, particularly when a vent was absent. Our study provides a general baseline for the use of detection dogs in locating T. cristatus and similar amphibian species during their terrestrial phase.

Widespread introgression of MHC genes in Iberian Podarcis lizards.

Major histocompatibility complex (MHC) genes are crucial for the adaptive immune response of jawed vertebrates. Their variation, reaching extreme levels, is driven mainly by an arms race between hosts and pathogens. One hypothesised mechanism contributing to MHC polymorphism is adaptive introgression, the exchange of genetic variants between hybridising species favoured by selection, yet its effect on MHC variation is poorly understood. Detection of adaptive MHC introgression, though challenging, may be facilitated by the analysis of species complexes forming multiple hybrid zones. Here, we investigated MHC introgression in six hybrid zones formed by seven species of Podarcis lizards inhabiting the Iberian Peninsula. To differentiate adaptive introgression from neutral introgression, we compared the patterns of gene exchange in MHC and genome-wide markers. We found elevated sharing of MHC alleles in the proximity of contact beyond the areas of detectable genome-wide admixture in most hybrid zones and, in half of them, asymmetric MHC exchange. In general, the elevated MHC allele sharing between species pairs with abutting ranges compared to geographically isolated species pairs also supports the prevalence of introgression. Collectively, our results demonstrate widespread MHC introgression in the Iberian Podarcis complex and suggest its adaptiveness. Contrary to previous results from Triturus newts, we did not observe differences in the rate of introgression between MHC classes. Our work adds support to the emerging view of adaptive introgression as a key mechanism shaping MHC diversity. It also raises questions about the effect of elevated MHC variation and factors leading to the asymmetry of adaptive introgression.

Step-by-step protocol for alternative injury models in newt cardiac regeneration.

Although the heart is one of the most important organs for animal survival, its regenerative capacity varies among animal species. Notably, adult mammals cannot regenerate their hearts after damage such as acute myocardial infarction. In contrast, some vertebrate animals can regenerate the heart throughout their lives. Cross-species comparative studies are important to understand the full picture of cardiac regeneration in vertebrates. Among the animal species able to regenerate the heart, some urodele amphibians, such as newts, possess a remarkable capacity for this process. Standardized methods of inducing cardiac regeneration in the newt are needed as a platform for studies comparing newts and other animal models. The procedures presented here describe amputation and cryo-injury techniques for the induction of cardiac regeneration in Pleurodeles waltl, an emerging model newt species. Both procedures consist of simplified steps that require no special equipment. We additionally show some examples of the regenerative process obtained using these procedures. This protocol has been developed for P. waltl. However, these methods are also expected to be applicable to other newt and salamander species, facilitating comparative research with other model animals.

Coccidiosis in Wild Eastern Newts (Notophthalmus viridescens): An Unexpected Finding in Consecutive Mortality Events in Tennessee, USA.

Wildlife diseases are a major threat for species conservation, and there is a growing need to implement more comprehensive disease response programs to better identify these diseases of concern. During March 2017, we observed moribund and dead eastern newts, Notophthalmus viridescens, in a single pond in middle Tennessee. All moribund individuals were emaciated. We euthanized and processed all individuals immediately on-site and later performed histopathology and quantitative PCR for ranavirus, the protist Perkinsea, and chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans. One newt was positive for ranavirus. Histopathology showed no evidence of ranavirosis but did reveal overwhelming coccidiosis. Overlapping partial sequences of coccidian 18S subunit DNA showed a 96.4% match with Eimeria steinhausi, suggesting that lesions were due to a previously undescribed Eimeria sp. In 2019, two more moribund newts were encountered at the same pond. Histopathology revealed the same suspicious parasitic organisms, and one individual was positive for B. dendrobatidis. Further research on how seasonal and other environmental parameters may influence coccidia-associated morbidity and mortality is warranted. These events highlight the importance of histopathologic evaluation of mortality events and provide guidance for investigation of future outbreaks.

Histological changes of the skin during postembryonic development of the crested newt Triturus ivanbureschi (Urodela, Salamandridae).

BACKGROUND: Amphibian skin has been studied for many decades, especially the metamorphic changes in the skin of frogs. Less attention has been paid to salamander skin. Here, we describe changes in the skin structure during postembryonic development in a salamandrid species, the Balkan crested newt Triturus ivanbureschi. METHOD: Using traditional histological techniques we examined the skin in the trunk region of three premetamorphic larval stages (hatchling, mid larval and late larval) and two postmetamorphic stages (juvenile, just after metamorphosis, and adult). RESULTS: In larval stages, skin consists only of the epidermis, which gradually develops from the single epithelial cell layer in hatchlings, to a stratified epidermis with gland nests and characteristic Leydig cells at the late larval stage. During metamorphosis, Leydig cells disappear, and the dermal layer develops. In postmetamorphic stages, skin is differentiated on stratified epidermis and the dermis with well-developed glands. Three types of glands were observed in the skin of the postmetamorphic stages: mucous, granular and mixed. Gland composition appears to be stage- and sex-specific, with juveniles and adult female being more similar to each other. In juveniles and adult female, there are a similar proportion of glands in both dorsal and ventral skin, whereas in adult male granular glands dominated the dorsal skin, while mixed glands dominated the ventral skin. CONCLUSION: Our results provide a baseline for future comparative research of skin anatomy in salamanders.

Structural and functional analysis of the newt lymphatic system.

Regeneration competent vertebrates such as newts and salamanders possess a weakened adaptive immune system characterized by multiple connections between the lymphatic system and the blood vascular system called lymphatic hearts. The role of lymphatic vasculature and these lymphaticovenous connections in regeneration is unknown. We used in-vivo near-infrared lymphangiography, ultra-high frequency ultrasonography, micro-CT lymphangiography, and histological serial section 3-dimentional computer reconstruction to evaluate the lymphatic territories of Cynops pyrrhogaster. We used our model and supermicrosurgery to show that lymphatic hearts are not essential for lymphatic circulation and limb regeneration. Instead, newts possess a novel intraosseous network of lymphatics inside the bone expressing VEGFR-3, LYVE-1 and CD-31. However, we were unable to show Prox-1 expression by these vessels. We demonstrate that adult newt bone marrow functions as both a lymphatic drainage organ and fat reservoir. This study reveals the fundamental anatomical differences between the immune system of urodeles and mammals and provides a model for investigating lymphatics and regeneration.

Senescent cells enhance newt limb regeneration by promoting muscle dedifferentiation.

Salamanders are able to regenerate their entire limbs throughout lifespan, through a process that involves significant modulation of cellular plasticity. Limb regeneration is accompanied by the endogenous induction of cellular senescence, a state of irreversible cell cycle arrest associated with profound non-cell-autonomous consequences. While traditionally associated with detrimental physiological effects, here, we show that senescent cells can enhance newt limb regeneration. Through a lineage tracing approach, we demonstrate that exogenously derived senescent cells promote dedifferentiation of mature muscle tissue to generate regenerative progenitors. In a paradigm of newt myotube dedifferentiation, we uncover that senescent cells promote myotube cell cycle re-entry and reversal of muscle identity via secreted factors. Transcriptomic profiling and loss of function approaches identify the FGF-ERK signalling axis as a critical mediator of senescence-induced muscle dedifferentiation. While chronic senescence constrains muscle regeneration in physiological mammalian contexts, we thus highlight a beneficial role for cellular senescence as an important modulator of dedifferentiation, a key mechanism for regeneration of complex structures.

Characterizing the lens regeneration process in Pleurodeles waltl.

BACKGROUND: Aging and regeneration are heavily linked processes. While it is generally accepted that regenerative capacity declines with age, some vertebrates, such as newts, can bypass the deleterious effects of aging and successfully regenerate a lens throughout their lifetime. RESULTS: Here, we used Spectral-Domain Optical Coherence Tomography (SD-OCT) to monitor the lens regeneration process of larvae, juvenile, and adult newts. While all three life stages were able to regenerate a lens through transdifferentiation of the dorsal iris pigment epithelial cells (iPECs), an age-related change in the kinetics of the regeneration process was observed. Consistent with these findings, iPECs from older animals exhibited a delay in cell cycle re-entry. Furthermore, it was observed that clearance of the extracellular matrix (ECM) was delayed in older organisms. CONCLUSIONS: Collectively, our results suggest that although lens regeneration capacity does not decline throughout the lifespan of newts, the intrinsic and extrinsic cellular changes associated with aging alter the kinetics of this process. By understanding how these changes affect lens regeneration in newts, we can gain important insights for restoring the age-related regeneration decline observed in most vertebrates.

Identification and characterization of sperm motility-initiating substance-2 gene in internally fertilizing Cynops species.

Sperm motility-initiating substance (SMIS) is an oviductal protein critical for internal fertilization in urodeles. It contributes to the establishment of various reproductive modes in amphibians and is thus a unique research model for the gene evolution of gamete-recognizing ligands that have diversified among animal species. In this study, a paralogous SMIS gene, smis2, was identified via the RNA sequencing of the oviduct of the newt, Cynops pyrrhogaster. The base sequence of the smis2 gene was homologous (˃90%) to that of the original smis gene (smis1), and deduced amino acid sequences of both genes conserved six cysteine residues essential for the cysteine knot motif. Furthermore, smis2 complementary DNA was identified in the oviduct of Cynops ensicauda, and the base substitution patterns also suggested that the smis gene was duplicated in the Salamandridae. Nonsynonymous/synonymous substitution ratios of smis1 and smis2 genes were 0.79 and 2.6, respectively, suggesting that smis2 gene evolution was independently driven by positive selection. Amino acid substitutions were concentrated in the cysteine knot motif of SMIS2. The smis2 gene was expressed in some organs in addition to the oviduct; in contrast, SMIS1 was only expressed in the oviduct. The SMIS2 protein was suggested to be produced and secreted at least in the oviduct and redundantly act in sperm. These results suggest that smis1 plays the original role in the oviduct, whereas smis2 may undergo neofunctionalization, which rarely occurs in gene evolution.