Antifungal activity of the pygidial gland secretion of <Emphasis Type="Italic">Laemostenus punctatus</Emphasis> (Coleoptera: Carabidae) against cave-dwelling micromycetes

The antifungal potential of the pygidial gland secretion of the troglophilic ground beetle Laemostenus punctatus from a cave in Southeastern Serbia against cave-dwelling micromycetes, isolated from the same habitat, has been investigated. Eleven collected samples were analyzed and 32 isolates of cave-dwelling fungi were documented. A total of 14 fungal species were identified as members of the genera Aspergillus, Penicillium, Alternaria, Cladosporium, Rhizopus, Trichoderma, Arthrinium, Aureobasidium, Epicoccum, Talaromyces, and Fusarium. Five isolates were selected for testing the antifungal activity of the pygidial gland secretion: Talaromyces duclauxi, Aspergillus brunneouniseriatus, Penicillium sp., Rhizopus stolonifer, and Trichoderma viride. The microdilution method has been applied to detect minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs). The most sensitive isolate was Penicillium sp., while the other isolates demonstrated a high level of resistance to the tested agent. L. punctatus has developed a special mechanism of producing specific compounds that act synergistically within the secretion mixture, which are responsible for the antifungal action against pathogens from the cave. The results open opportunities for further research in the field of ground beetle defense against pathogens, which could have an important application in human medicine, in addition to the environmental impact, primarily.     

Evolution through cold and deep waters: the molecular phylogeny of the Lithodidae (Crustacea: Decapoda)

The objectives of this work are to use gene sequence data to assess the hypothesis that the Lithodinae arose from ancestors with uncalcified abdomens in shallow waters of the North-East Pacific, investigate the monophyly and interrelationships of genera within the Lithodinae and to estimate the scale and minimum number of biogeographic transitions from the shallow environment to the deep sea and vice versa. To do this, phylogenetic analysis from three mitochondrial and three nuclear markers was conducted using minimum evolution, maximum likelihood and Bayesian methods. The Lithodinae as defined to include North Pacific genus Cryptolithodes may be paraphyletic, with the Hapalogastrinae and Cryptolithodes as sister taxa. This implies that the soft-bodied abdomen of the Hapalogastrinae might not be plesiomorphic for the Lithodidae. Paralomis, Lopholithodes, Phyllolithodes, Lithodes and Neolithodes share a common ancestor, from which the North Pacific Hapalogastrinae did not descend. Lithodid ancestors are likely to have had a north Pacific, shallow water distribution and to have had planktotrophic larvae. North Pacific genus Paralithodes is paraphyletic; P. brevipes is the most basal member of the genus (as sampled) while P. camtschaticus and P. platypus are more closely related to the genera Lithodes and Neolithodes. Genera Lithodes, Neolithodes and Paralomis (as sampled) are monophyletic if Glyptolithodes is included within Paralomis. Lopholithodes is closely related to, but not included within, the Paralomis genus. Paralomis is divided into at least two major lineages: one containing South Atlantic, West African, and Indian Ocean species, and the other containing Pacific and South American species. Several species of Paralomis do not resolve consistently with any other groups sampled, implying a complex and possibly rapid global evolution early in the history of the genus. Relationships within the Lithodes genus vary between analytical methods, suggesting that conclusions may not be stable. Consistently, however, Indian Ocean and Pacific forms—L. murrayi, L. longispina and L. nintokuae form a group separated from Atlantic species such as L. santolla, L. confundens, L. maja and L. ferox.     

Climate change implications for the nest site selection process and subsequent hatching success of a green turtle population

Sandy beach habitat where sea turtles nest will be affected by multiple climate change impacts. Before these impacts occur, knowledge of how nest site selection and hatching success vary with beach microhabitats is needed to inform managers on how to protect suitable habitats and prepare for scientifically valid mitigation measures at beaches around the world. At a highly successful green turtle (Chelonia mydas) rookery at Akumal, Quintana Roo, Mexico, we measured microhabitat characteristics along the beach crawl (rejected sites) and related nest site conditions (selected sites) to subsequent hatching success rates for 64 nesting events. To our knowledge, this is the first study to report environmental data along the nesting crawl for a green turtle population and the first to use natural breaks in the data to describe their preferred habitat ranges. Our results indicate that turtles were likely using a combination of cues to find nest sites, mainly higher elevations and lower sand surface temperatures (Kruskal-Wallis test, H?=?19.84, p?<?0.001; H?=?10.78, p?<?0.001). Hatching success was significantly and negatively correlated to sand temperature at cloaca depth (Spearman’s ρ?=??0.27, p?=?0.04). Indeed, the preferred range for cloaca sand temperatures at the nest site (26.3–27.5 °C) had significantly higher hatching success rates compared to the highest temperature range (Tukey HSD?=?0.47, p?=?0.05). Sand temperatures at various depths were intercorrelated, and surface and cloaca depth sand temperatures were correlated to air temperature (ρ?=?0.70, p?=?0.00; ρ?=?0.26, p?=?0.04). Therefore, rising air temperatures could alter sand temperature cues for suitable nest sites, preferred nest site ranges, and produce uneven sex ratios or lethal incubating temperatures. Elevation cues and preferred ranges (1.4–2.5 m) may also be affected by sea level rise, risking inundation of nests.     

Computational molecular species delimitation and taxonomic revision of the gecko genus <Emphasis Type="Italic">Ebenavia</Emphasis> Boettger, 1878

Cryptic species have been detected in many groups of organisms and must be assumed to make up a significant portion of global biodiversity. We study geckos of the Ebenavia inunguis complex from Madagascar and surrounding islands and use species delimitation algorithms (GMYC, BOLD, BPP), COI barcode divergence, diagnostic codon indels in the nuclear marker PRLR, diagnostic categorical morphological characters, and significant differences in continuous morphological characters for its taxonomic revision. BPP yielded ≥?10 operational taxonomic units, whereas GMYC (≥?27) and BOLD (26) suggested substantial oversplitting. In consequnce, we resurrect Ebenavia boettgeri Boulenger 1885 and describe Ebenavia tuelinae sp. nov., Ebenavia safari sp. nov., and Ebenavia robusta sp. nov., increasing the number of recognised species in Ebenavia from two to six. Further lineages of Ebenavia retrieved by BPP may warrant species or subspecies status, but further taxonomic conclusions are postponed until more data become available. Finally, we present an identification key to the genus Ebenavia, provide an updated distribution map, and discuss the diagnostic values of computational species delimitation as well as morphological and molecular diagnostic characters.     

More than euglossines: the diverse pollinators and floral scents of Zygopetalinae orchids

Floral volatile organic compounds (VOCs) play important roles in plant-pollinator interactions. We investigated the reproductive ecology and floral VOCs of Zygopetalinae orchids to understand the relationship between floral scents and pollinators. We performed focal observations, phenological censuses and breeding system experiments in eight species in southeast Brazil. Floral scents were collected and analysed using SPME/GC-MS. We performed multivariate analyses to group species according to affinities of their VOCs and define compounds associated to each plant. Dichaea cogniauxiana was pollinated by weevils which use their developing ovules, while D. pendula was pollinated by the same weevils and perfume-collecting male euglossine bees. The other species were deceit-pollinated by bees. Zygopetalum crinitum was pollinated by carpenter bees, while W. warreana, Z. mackayi and Z. maxillare were bumblebee-pollinated. The latter was also pollinated by Centris confusa. Breeding system varied widely with no association to any pollinator group. Most VOCs are common to other floral scents. Zygopetalum crinitum presented an exclusive blend of VOCs, mainly composed of benzenoids. The scents of Pabstia jugosa, Promenaea xanthina and the Zygopetalum spp. were similar. The bumblebee-pollinated species have flowering periods partially overlapped, thus neither phenology nor pollinators constitute hybridization barriers among these species. Euglossines are not the only pollinators of Zygopetalinae. Different VOCs, size and lifespan of flowers are associated with distinct pollinators. A distinctive VOC bouquet may determine specialisation in carpenter bees or male euglossines within bee-pollinated flowers. Finally, visitation of deceit-pollinated flowers by perfume-collecting euglossines allows us to hypothesise how pollination by this group of bees had evolved.     

Spatial and Temporal Distribution of Norovirus and <Emphasis Type="Italic">E. coli</Emphasis> in Sydney Rock Oysters Following a Sewage Overflow into an Estuary

This paper reports a study of norovirus (NoV) GII distribution and persistence in Sydney rock oysters (SRO) (Saccostrea glomerata) located in an estuary after a pump station sewage overflow. SRO were strategically placed at six sites spanning the length of the estuary from the pump station to the sea. The spatial and temporal distribution of NoV, hepatitis A virus (HAV) and Escherichia coli (E. coli) in oysters was mapped after the contamination event. NoV GI and GII, HAV and E. coli were quantified for up to 48 days in oysters placed at six sites ranging from 0.05 to 8.20 km from the sewage overflow. NoV GII was detected up to 5.29 km downstream and persisted in oysters for 42 days at the site closest to the overflow. NoV GII concentrations decreased significantly over time; a reduction rate of 8.5% per day was observed in oysters (p < 0.001). NoV GII concentrations decreased significantly as a function of distance at a rate of 5.8% per km (p < 0.001) and the decline in E. coli concentration with distance was 20.1% per km (p < 0.001). HAV and NoV GI were not detected. A comparison of NoV GII reduction rates from oysters over time, as observed in this study and other published research, collectively suggest that GII reduction rates from oysters may be broadly similar, regardless of environmental conditions, oyster species and genotype.     

Differences in functional traits between invasive and native <Emphasis Type="Italic">Amaranthus</Emphasis> species under different forms of N deposition

Differences in functional traits between invasive and native plant species are believed to determine the invasion success of the former. Increasing amounts of anthropogenic nitrogen (N) are continually deposited into natural ecosystems, which may change the relative occurrence of the different N deposition forms (such as NH₄–N, NO₃–N, and CO(NH₂)₂–N) naturally deposited. Under high N deposition scenarios, some invasive species may grow faster, gaining advantage over native species. In a greenhouse experiment, we grew invasive and native Amaranthus species from seed both alone and in competition under simulated N enriched environments with different forms of N over 3 months. Then, we measured different leaf traits (i.e., plant height, leaf length, leaf width, leaf shape index, specific leaf area (SLA), and leaf chlorophyll and N concentrations). Results showed that the competition intensity between A. retroflexus and A. tricolor decreased under N deposition. This may be due to the large functional divergence between A. retroflexus and A. tricolor under simulated N deposition. Phenotypic plasticity of SLA and leaf chlorophyll concentration of A. retroflexus were significantly lower than in A. tricolor. The lower range of phenotypic plasticity of SLA and leaf chlorophyll concentration of A. retroflexus may indicate a fitness cost for plastic functional traits under adverse environments. The restricted phenotypic plasticity of SLA and leaf chlorophyll concentration of A. retroflexus may also stabilize leaf construction costs and the growth rate. Meanwhile, the two Amaranthus species possessed greater plasticity in leaf N concentration under NO₃–N fertilization, which enhanced their competitiveness.     

Testing the influence of gravity on flower symmetry in five <Emphasis Type="Italic">Saxifraga</Emphasis> species

Flower symmetry is considered a species-specific trait and is categorized in asymmetry, actinomorphic symmetry, bisymmetry and zygomorphic symmetry. Here we report on the intra-individual variation of flower symmetry in the genus Saxifraga and the influence of light, gravity and intrinsic factors on the development of flower symmetry. We tested five species—Saxifraga cuneifolia, Saxifraga imparilis, Saxifraga rotundifolia, Saxifraga stolonifera and Saxifraga umbrosa—concerning six flower parameters—angles between petals, petal length, petal pigmentation, angular position of carpels, movement of stamens and (only for S. imparilis and S. stolonifera) the length of the two lower elongated petals in regard to their position towards the stem. Specimens of all species were tested on a vertical clinostat as a gravity compensator, on a horizontal clinostat as a light incidence compensator and on a stationary control. The results show that the angle of incident light has no apparent impact on flower symmetry, whereas gravity affects the angular position of petals in S. cuneifolia and S. umbrosa and the petal colouration in S. rotundifolia. In S. cuneifolia and S. umbrosa, the absence of directional gravity resulted in the development of actinomorphic flowers, whereas the corresponding control flowers were zygomorphic. The development of flowers in S. rotundifolia was not altered by this treatment. The length of the two elongated petals in S. stolonifera and S. imparilis was not affected by gravity, but rather was determined by position of the flower within the inflorescence and resulted in asymmetrical flowers.     

The first Gondwanan borioteiioid lizard and the mid-Cretaceous dispersal event between North America and Africa

Borioteiioidea are an enigmatic group of Cretaceous lizards widely distributed in northern continents (Laurasia). Here, we describe the first borioteiioid lizard from Gondwana, represented by a new species of the polyglyphanodontine genus Bicuspidon, B. hogreli sp. nov., from the Cenomanian Kem Kem beds of Morocco. The discovery of Bicuspidon hogreli sp. nov., which is one of the oldest known member of Polyglyphanodontini, challenges previous assumptions on the center of origin and dispersal of the group. In addition, the known distribution of Bicuspidon (Cenomanian of Utah and Morocco, and Santonian–Maastrichtian of eastern Europe) suggests a complex palaeobiogeographical history for the genus. The existence of a terrestrial dispersal route persisting during the whole Early Cretaceous between North America and Africa is hypothesized to explain some similarities observed between the Cenomanian squamate assemblages of these two continents. Alternatively, dispersal between the two landmasses may have occurred by transatlantic rafting. During the Cenomanian–Santonian interval, Bicuspidon colonized the European archipelago probably from North Africa, like many “Eurogondwanan” taxa.     

Microanatomy and life history in <Emphasis Type="Italic">Palaeopleurosaurus</Emphasis> (Rhynchocephalia: Pleurosauridae) from the Early Jurassic of Germany

The tuatara (Sphenodon punctatus) from New Zealand is often—erroneously—identified as a ‘living fossil’, although it is the lone survivor of a large, successful radiation of Rhynchocephalia, sister taxon to squamates (lizards and snakes), that thrived through the Mesozoic and Cenozoic and experienced an intricate evolution of life histories and feeding habits. Within Rhynchocephalia, only Pleurosauridae are thought to be marine and piscivorous. Here, we present bone histological data of the Jurassic pleurosaurid Palaeopleurosaurus, showing osteosclerosis (i.e. bone mass increase) in its gastralia, and some osteosclerosis in its rib but no increase in bone mass in the femur, supporting a gradual skeletal specialization for an aquatic way of life. Similar to Sphenodon, the bone tissue deposited in Palaeopleurosaurus is lamellar zonal bone. The femoral growth pattern in Palaeopleurosaurus differs from that of terrestrial Sphenodon in a more irregular spacing of growth marks and deposition of non-annual (i.e. non-continuous) rest lines, indicating strong dependency on exogenous factors. The annual growth mark count in adult but not yet fully grown Palaeopleurosaurus is much lower when compared to adult individuals of Sphenodon, which could indicate a lower lifespan for Palaeopleurosaurus. Whereas the gastral ribs of Palaeopleurosaurus and Sphenodon are similar in composition, the ribs of Sphenodon differ profoundly in being separated into a proximal tubular rib part with a thick cortex, and an elliptical, flared ventral part characterised by extremely thin cortical bone. The latter argues against a previously inferred protective function of the ventral rib parts for the vulnerable viscera in Sphenodon.     

Extreme longevity in a deep-sea vestimentiferan tubeworm and its implications for the evolution of life history strategies

The deep sea is home to many species that have longer life spans than their shallow-water counterparts. This trend is primarily related to the decline in metabolic rates with temperature as depth increases. However, at bathyal depths, the cold-seep vestimentiferan tubeworm species Lamellibrachia luymesi and Seepiophila jonesi reach extremely old ages beyond what is predicted by the simple scaling of life span with body size and temperature. Here, we use individual-based models based on in situ growth rates to show that another species of cold-seep tubeworm found in the Gulf of Mexico, Escarpia laminata, also has an extraordinarily long life span, regularly achieving ages of 100–200 years with some individuals older than 300 years. The distribution of results from individual simulations as well as whole population simulations involving mortality and recruitment rates support these age estimates. The low 0.67% mortality rate measurements from collected populations of E. laminata are similar to mortality rates in L. luymesi and S. jonesi and play a role in evolution of the long life span of cold-seep tubeworms. These results support longevity theory, which states that in the absence of extrinsic mortality threats, natural selection will select for individuals that senesce slower and reproduce continually into their old age.     

Application of environmental DNA analysis to inform invasive fish eradication operations

Environmental DNA (eDNA) detection of non-native species has considerable potential to inform management decisions, including identifying the need for population control and/or eradication. An invasive species of European concern is the Asian cyprinid fish, topmouth gudgeon (Pseudorasbora parva). Here, eDNA analyses were applied at a commercial angling venue in southern England to inform operations aiming to eradicate P. parva, which had only ever been observed in one of the venue’s seven unconnected angling ponds. Eradication of P. parva was initially attempted by repeated depletion of the population using fish traps (crayfish traps fitted with 5 mm mesh netting) and the introduction of native predators over a 4-year period. The very low number of P. parva captured following these eradication efforts suggested a possible population crash. Conventional PCR analysis of water samples using species-specific primers was applied to all seven ponds to confirm that P. parva was present in only one pond, that the eradication attempt had indeed failed and that the species’ distribution in the pond appeared to be restricted to three bankside locations. The continued presence of P. parva at these locations was confirmed by subsequent trapping. Water samples from an adjacent, unconnected stream were also analysed using the eDNA methodology, but no DNA of P. parva was detected. The results suggest that further management action to eradicate P. parva be focused on the pond shown to contain the isolated P. parva population and thereby eliminate the risk of further dispersal. This study is the first to apply eDNA analysis to assess the efficacy of an eradication attempt and to provide evidence that the species was unlikely to be present in the other ponds, thus reducing the resources needed to control the species.     

Nocturnal herbivore-induced plant volatiles attract the generalist predatory earwig <Emphasis Type="Italic">Doru luteipes</Emphasis> Scudder

Numerous studies have demonstrated that entomophagous arthropods use herbivore-induced plant volatile (HIPV) blends to search for their prey or host. However, no study has yet focused on the response of nocturnal predators to volatile blends emitted by prey damaged plants. We investigated the olfactory behavioral responses of the night-active generalist predatory earwig Doru luteipes Scudder (Dermaptera: Forficulidae) to diurnal and nocturnal volatile blends emitted by maize plants (Zea mays) attacked by either a stem borer (Diatraea saccharalis) or a leaf-chewing caterpillar (Spodoptera frugiperda), both suitable lepidopteran prey. Additionally, we examined whether the earwig preferred odors emitted from short- or long-term damaged maize. We first determined the earwig diel foraging rhythm and confirmed that D. luteipes is a nocturnal predator. Olfactometer assays showed that during the day, although the earwigs were walking actively, they did not discriminate the volatiles of undamaged maize plants from those of herbivore damaged maize plants. In contrast, at night, earwigs preferred volatiles emitted by maize plants attacked by D. saccharalis or S. frugiperda over undamaged plants and short- over long-term damaged maize. Our GC-MS analysis revealed that short-term damaged nocturnal plant volatile blends were comprised mainly of fatty acid derivatives (i.e., green leaf volatiles), while the long-term damaged plant volatile blend contained mostly terpenoids. We also observed distinct volatile blend composition emitted by maize damaged by the different caterpillars. Our results showed that D. luteipes innately uses nocturnal herbivore-induced plant volatiles to search for prey. Moreover, the attraction of the earwig to short-term damaged plants is likely mediated by fatty acid derivatives.     

Inhibition of Murine Norovirus and Feline Calicivirus by Edible Herbal Extracts

Human noroviruses (HuNoVs) cause foodborne and waterborne viral gastroenteritis worldwide. Because HuNoV culture systems have not been developed thus far, no available medicines or vaccines preventing infection with HuNoVs exist. Some herbal extracts were considered as phytomedicines because of their bioactive components. In this study, the inhibitory effects of 29 edible herbal extracts against the norovirus surrogates murine norovirus (MNV) and feline calicivirus (FCV) were examined. FCV was significantly inhibited to 86.89 ± 2.01 and 48.71 ± 7.38% by 100 μg/mL of Camellia sinensis and Ficus carica, respectively. Similarly, ribavirin at a concentration of 100 μM significantly reduced the titer of FCV by 77.69 ± 10.40%. Pleuropterus multiflorus (20 μg/mL) showed antiviral activity of 53.33 ± 5.77, and 50.00 ± 16.67% inhibition was observed after treatment with 20 μg/mL of Alnus japonica. MNV was inhibited with ribavirin by 59.22 ± 16.28% at a concentration of 100 μM. Interestingly, MNV was significantly inhibited with 150 µg/mL Inonotus obliquus and 50 μg/mL Crataegus pinnatifida by 91.67 ± 5.05 and 57.66 ± 3.36%, respectively. Treatment with 20 µg/mL Coriandrum sativum slightly reduced MNV by 45.24 ± 4.12%. The seven herbal extracts of C. sinensis, F. carica, P. multiflorus, A. japonica, I. obliquus, C. pinnatifida, and C. sativum may have the potential to control noroviruses without cytotoxicity.     

Isotopic evidence for niche partitioning and the influence of anthropogenic disturbance on endemic and introduced rodents in central Madagascar

We applied a multi-isotope approach to examine aspects of niche partitioning, competition, and mobility for rodents in the Central Highlands of Madagascar. Specifically, we used carbon (δ¹³C), nitrogen (δ¹⁵N), and strontium (⁸⁷Sr/⁸⁶Sr) isotope ratios in bone to investigate diet and mobility for endemic tufted tail rats (Eliurus spp.), and introduced black rats (Rattus rattus) and house mice (Mus musculus) within and outside a fragment of montane humid forest in the Ambohitantely Special Reserve. There was a clear spatial segregation in trapping success for different species: Eliurus was only in the forest interior and edge, Mus only outside of the fragment in a marsh and park housing complex, and Rattus in all habitats except the housing complex. We find only moderate support for mobility of rodents among habitats. Mus may routinely move between the marsh and housing complex. However, regular movement between the forest edge and interior, or between the forest fragment and surrounding grassland is not supported. Taxa appear to target different foods: Rattus tends to feed at a higher trophic level than Eliurus, and Mus consumes some C₄ resources. To date, strontium isotopes have been underutilized in ecological research. Here, we show that they are highly complementary to carbon and nitrogen isotope data. Even in localities with relatively uniform underlying geology, it may be possible to distinguish individuals that regularly forage in different habitats.     

Isolation and Characterization of <Emphasis Type="Italic">Bacillus cereus</Emphasis> Bacteriophages from Foods and Soil

The aim of this study was to isolate and characterize Bacillus cereus bacteriophages of various origins. Twenty-seven bacteriophages against B. cereus were isolated from various Korean traditional fermented foods and soils. Plaque size, transmission electron microscopy, virulence profile, and in vitro lytic activity of bacteriophage isolates were examined. Transmission electron microscopy confirmed B. cereus bacteriophages belonging to the family Siphoviridae. Among B. cereus bacteriophages with broad host range, 18 isolates (66.7%) did not harbor any B. cereus virulence factors. Among them, bacteriophage strain CAU150036, CAU150038, CAU150058, CAU150064, CAU150065, and CAU150066 effectively inhibited B. cereus in vitro within 1 h. Therefore, they are considered potential candidates for controlling the contamination of B. cereus in food or other applications.     

Millennium-old farm breeding of Chinese softshell turtles (<Emphasis Type="Italic">Pelodiscus</Emphasis> spp.) results in massive erosion of biodiversity

Chinese softshell turtles (Pelodiscus spp.) are widely distributed, ranging from the Amur and Ussuri Rivers in the Russian Far East through the Korean Peninsula, Japan, and eastern, central, and southern China to southern Vietnam. In East and Southeast Asia, Chinese softshell turtles are traditionally exploited for food and have been farm-bred in China since the Spring and Autumn Period, more than 2400 years ago. Currently, the annual production of Pelodiscus amounts to 340,000 t in China alone. Using mitochondrial DNA (2428 bp) and five nuclear loci (3704 bp), we examined broad sampling of wild and farm-bred Pelodiscus to infer genetic and taxonomic differentiation. We discovered four previously unknown mitochondrial lineages, all from China. One lineage from Jiangxi is deeply divergent and sister to the mitochondrial lineage of Pelodiscus axenaria. The nuclear loci supported species status for P. axenaria and the new lineage from Jiangxi. Pelodiscus maackii and P. parviformis, both harboring distinct mitochondrial lineages, were not differentiated from P. sinensis in the studied nuclear markers. The same is true for two new mitochondrial lineages from Zhejiang, China, represented by only one individual each, and another new lineage from Anhui, Guangdong, Jiangxi and Zhejiang, China. However, Vietnamese turtles yielding a mitochondrial lineage clustering within P. sinensis were distinct in nuclear markers, suggesting that these populations could represent another unknown species with introgressed mitochondria. Its species status is also supported by the syntopic occurrence with P. sinensis in northern Vietnam and by morphology. In addition, we confirmed sympatry of P. axenaria and P. parviformis in Guangxi, China, and found evidence for sympatry of P. sinensis and the new putative species from Jiangxi, China. We also discovered evidence for hybridization in turtle farms and for the occurrence of alien lineages in the wild (Zhejiang, China), highlighting the risk of genetic pollution of native stock. In the face of the large-scale breeding of Pelodiscus, we claim that the long-term survival of distinct genetic lineages and species can only be assured when an upscale market segment for pure-bred softshell turtles is established, making the breeding of pure lineages lucrative for turtle farms. Our findings underline that the diversity of Pelodiscus is currently underestimated and threatened by anthropogenic admixture. We recommend mass screening of genetic and morphological variation of Chinese softshell turtles as a first step to understand and preserve their diversity.