The characterization of the mitochondrial genome of Graptemys ouachitensis.

Graptemys ouachitensis (CAGLE, 1953) belongs to the Graptemys genus, the Emydidae family, and the Testudines order. This study involved sequencing the complete mitochondrial genome (mitogenome) of G. ouachitensis using next-generation sequencing, and analyzing the essential characteristics, and phylogenetic relationship. The results revealed that the G. ouachitensis mitogenome was 16,674 bp in length (A: 34.1%, C: 26.0%, G: 13.0%, T: 26.9%) and included 22 tRNAs, 13 protein-coding genes, two ribosomal RNA genes, and a non-coding control region (GenBank accession: NC071766). The genome composition of G. ouachitensis presented a slight A + T bias (61.0%) and exhibited a positive AT skew (0.118) and a negative GC skew (-0.333). A phylogenetic analysis based on the complete mitogenome indicated that the G. ouachitensis was more closely associated with Malaclemys terrapin than the other eight known Emydidae species. Thus, our findings present a novel mitogenome at the species level. This study introduces the first complete mitogenome of G. ouachitensis, providing valuable molecular information for phylogenetic and conservation genetics analyses of G. ouachitensis.

Auditory sensitivity and tympanic middle ear in a vocal and a non-vocal frog.

The tympanic middle ear is important for anuran hearing on land. However, many species have partly or entirely lost their tympanic apparatus. Previous studies have compared hearing sensitivities in species that possess and lack tympanic membranes capable of sound production and acoustic communication. However, little is known about how these hearing abilities are comparable to those of mutant species. Here, we compared the eardrum and middle ear anatomies of two sympatric sibling species from a noisy stream habitat, namely the "non-vocal" Hainan torrent frog (Amolops hainanensis) and the "vocal" little torrent frog (Amolops torrentis), the latter of which is capable of acoustic communication. Our results showed that the relative (to head size) eardrum diameter of A. hainanensis was smaller than that of A. torrentis, although the absolute size was not smaller. Unlike A. torrentis, the tympanic membrane area of A. hainanensis was not clearly differentiated from the surrounding skin. The middle ear, however, was well-developed in both species. We measured the auditory brainstem responses (ABRs) of A. hainanensis and compared the ABR thresholds and latencies to those previously obtained for A. torrentis. Our results suggested that these two species exhibited significant differences in hearing sensitivity. A. hainanensis (smaller relative eardrum, nonvocal) had higher ABR thresholds and longer initial response times than A. torrentis (larger relative eardrum, vocal) at lower frequencies. Neurophysiological responses from the brain were obtained for tone pips between 800 Hz and 7,000 Hz, with peak sensitivities found at 3,000 Hz (73 dB SPL) for A. hainanensis, and at 1,800 Hz (61 dB SPL) for A. torrentis. Our results suggest that the non-vocal A. hainanensis has lower hearing sensitivity than its vocal sister species (i.e., A. torrentis), which may be related to differences in tympanic or inner ear structure and morphology.

The Characterization of Mitochondrial Genome of Spotted Pond Turtle (Geoclemys hamiltonii).

The spotted pond turtle Geoclemys hamiltonii (Gray, 1830) is widely distributed in the Indus, Ganges, and Brahmaputra river basins. In this study, the complete mitochondrial genome (mitogenome) of G. hamiltonii was sequenced using the next-generation sequencing (NGS) and Sanger sequencing, and the essential characteristics, gene arrangement, and phylogenetic relationship were analyzed. The results showed that the G. hamiltonii mitogenome was 16,505 bp in length (A: 33.6%, C: 27.1%, G: 13.4%, T: 25.8%) and consisted of 22 tRNAs, 13 protein-coding genes, two ribosomal RNA genes, and a non-coding control region (GenBank accession ON243873). The genome composition of G. hamiltonii presented a slight A + T bias (59.4%), and showed a positive AT skew (0.131) and a negative GC skew (- 0.338). All tRNAs had the typical clover structure, except trnS1 (GCT). The gene order of the G. hamiltonii mitogenome was the same as other Geoemydidae mitogenomes. A phylogenetic analysis based on the complete mitogenome indicated that the G. hamiltonii grouped independently of other species in the family Geoemydidae, supporting the species' placement in the monotypic genus Geoclemys. Our results describe a novel genome at the species level. As the first complete mitogenome of G. hamiltonii, it provided valuable molecular information for phylogenetic and conservation genetics analyses of G. hamiltonii.

Single-cell transcriptomic landscape of peripheral blood cells provides insights into adaptation of red-eared sliders (Trachemys scripta elegans).

Red-eared sliders (Trachemys scripta elegans), as one of the 100 most threatening aliens, have stronger immunity than the native species in response to environmental stress. Blood cells are an important component of immunity in the body. However, the blood cell researches of turtle are still in the traditional blood cell classification and morphological structure observation. Furthermore, turtle granulocytes cannot be accurately identified using traditional methods. Single-cell RNA sequencing techniques have been successfully implemented to study cells based on the mRNA expression patterns of each cell. The present study profiled the transcriptomes of peripheral blood cells in red-eared sliders to construct a single-cell transcriptional landscape of the different cell types and explored environmental adaptation mechanism from the perspective of hematology. All 14 transcriptionally distinct clusters (platelets, erythrocytes1, erythrocytes2, CSF1R monocytes, POF1B monocytes, neutrophils, GATA2high basophils, GATA2low basophils, CD4 T cells, CD7 T cells, B cells, ACKR4 cells, serotriflin cells, and ficolin cells) were identified in the peripheral blood cells of the red-eared sliders. In particular, a subtype of erythrocytes (erythrocytes1) that expressed immune signals was identified. Peripheral blood cells were grouped into three lineages: platelet, erythroid/lymphoid, and myeloid cell lineages. Furthermore, based on differentiation trajectory and up-regulated gene expression, ACKR4 cells were newly identified as lymphocytes, and serotriflin and ficolin cells as granulocytes. The single-cell transcriptional atlas of the peripheral blood cells in red-eared sliders provided in the present study will offer a comprehensive transcriptome reference for the exploration of physiological and pathological hematology in this species.

Diversity of Underwater Vocalizations in Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

Sound communication is important for underwater species. The wild population of the Chinese soft-shelled turtle (Pelodiscus sinensis) is listed as vulnerable. However, its vocalization, which can serve as the basis for ecological and evolutionary research, has not been studied. Here, we performed underwater recordings of 23 Chinese soft-shelled turtles of different ages and sexes and identified 720 underwater calls. The turtle calls were manually divided into 10 call types according to visual and aural inspection properties. The similarity test indicated that the manual division was reliable. We described the acoustic properties of the calls and the statistical analysis showed that the peak frequency of calls was significantly different between adult females and males, and also between subadults and adults. Similar to other aquatic turtles that prefer to live in deep water, Chinese soft-shelled turtles have a high vocal diversity and many harmonic calls, indicating that this highly aquatic species developed a variety of vocalizations to enhance their underwater communication, which helped them adapt to the complex and dim underwater environment. Furthermore, the turtles showed a tendency for vocalization to become more diverse with age.

Chinese striped-neck turtles vocalize underwater and show differences in peak frequency among different age and sex groups.

Background: Turtle vocalizations play an important role throughout their lives by expressing individual information (position, emotion, or physiological status), reflecting mating preferences, and synchronizing incubation. The Chinese striped-neck turtle (Mauremys sinensis) is one of the most widely distributed freshwater turtles in China, whose wild population is critically endangered. However, its vocalization has not been studied, which can be the basis for behavioral and ecological studies. Methods: Five different sex-age groups of turtles were recorded underwater in a soundproof room. Cluster analysis and principal component analysis for classification of Chinese striped-neck turtle calls were unreasonable. The turtle calls were manually sought using visual and aural inspection of the recordings in Raven Pro 1.5 software and classified according to differences perceived through auditory inspection and the morphological characteristics of the spectrograms. The results of similarity analysis verified the reliability of manual classification. We compared the peak frequency of the calls among different age and sex groups. Results: We identified ten M. sinensis call types, displayed their spectra and waveforms, and described their auditory characteristics. Most calls produced by the turtles were low-frequency. Some high-frequency call types, that are common in other turtle species were also produced. Similar to other turtles, the Chinese striped-neck turtle generates harmonic vocalizations. Courtship behaviors were observed when one of the call types occurred in the mixed-sex group. Adult females produced more high-frequency call types, and subadult males had higher vocalizations than other groups. These results provide a basis for future research on the function of vocalizations, field monitoring, and conservation of this species.

Geographical variation of the acoustic signals in the spot-legged treefrog (Polypedates megacephalus) of Hainan Island.

Geographical divergence in acoustic signals is often affected by multiple selective pressures. Environmental factors are expected to shape signals by adjusting for greater propagation efficiency. Nevertheless, such habitat-driven hypotheses have not been well-examined with multi-signal and multi-habitat types included simultaneously. In this study, we evaluated call similarity of the spot-legged treefrog (Polypedates megacephalus), characterized by multiple call types, from 11 geographic populations across Hainan Island. We also examined multiple factors (i.e. body mass, temperature, altitude, and vegetation) among different acoustical geographic clusters, and tested whether the variation in acoustic length and frequency among different habitats was in accordance with the prediction derived from the acoustic adaptation hypothesis. We showed that P. megacephalus from Hainan Island included 3 acoustic geographic clusters: the northeastern flatlands, the southwest mountains, and the hills around the mountains. We also showed that environmental factors (i.e. altitude and vegetation) varied across these 3 acoustically distinct areas. Moreover, the duration of the different call types supported the hypothesis that they should have optimal transmission in their own habitats. Thus, our results reveal the roles of ecological selection in the geographical divergence of anuran acoustic signals.

Comparison of the Single-Cell Immune Landscape of Testudines from Different Habitats.

Testudines, also known as living fossils, have evolved diversely and comprise many species that occupy a variety of ecological niches. However, the immune adaptation of testudines to the different ecological niches remains poorly understood. This study compared the composition, function, and differentiation trajectories of peripheral immune cells in testudines (Chelonia mydas, Trachemys scripta elegans, Chelonoidis carbonaria, and Pelodiscus sinensis) from different habitats using the single-cell RNA sequencing (scRNA-seq) technique. The results showed that T. scripta elegans, which inhabits freshwater and brackish environments, had the most complex composition of peripheral immune cells, with 11 distinct immune cell subsets identified in total. The sea turtle C. mydas, had the simplest composition of peripheral immune cells, with only 5 distinct immune cell clusters. Surprisingly, neither basophils were found in C. mydas nor T cells in C. carbonaria. Basophil subsets in peripheral blood were identified for the first time; two basophil subtypes (GATA2-high-basophils and GATA2-low-basophils) were observed in the peripheral blood of T. scripta elegans. In addition, ACKR4 cells, CD4 T cells, CD7 T cells, serotriflin cells, and ficolin cells were specifically identified in the peripheral blood of T. scripta elegans. Furthermore, LY6G6C cells, SPC24 cells, and NKT cells were specifically observed in C. carbonaria. Moreover, there were differences in the functional status and developmental trajectory of peripheral immune cells among the testudine species. The identification of specific features of peripheral immune cells in testudines from different habitats may enable elucidation of the adaptation mechanism of testudines to various ecological niches.

Characterization and comparison of mitogenomes of three 'eyed' turtles Sacalia spp.

In this paper, we characterized the mitogenomes of three 'eyed' turtles, the Beal's Eyed turtle (Sacalia bealei), the Four Eye-spotted turtle (S. quadriocellata), and the Hainan Four Eye-spotted turtle which was a unique lineage in the four-eyed turtle and considered as an independent species S. insulensis recently. The full lengths of the S. bealei and S. quadriocellata mitogenomes are 16,564 bp, and 16,555 bp, respectively, while the length of partial mitochondrial genome of S. insulensis is 16,433 bp without tailed part of D-loop. All the genes exhibit the typical mitochondrial gene arrangement and transcribing directions of turtles. Phylogenetic analysis indicating that a deep divergence about 7.8% p distance was found between S. bealei and (S. quadriocellata + S. insulensis), and the divergence (2.8% in patristic distance) between S. quadriocellata and S. insulensis is comparable with other closely related species in turtles.

Phylogeography, speciation and demographic history: Contrasting evidence from mitochondrial and nuclear markers of the Odorrana graminea sensu lato (Anura, Ranidae) in China.

Understanding the process of speciation and the factors driving the geographical distribution patterns of species is of great interest in ecology and evolutionary biology. Herein, we investigated the phylogeographic patterns, speciation, demographic history and genetic structure of the widespread endemic Odorrana graminea sensu lato in Southern China and adjacent areas. A total of 439 specimens from 68 localities were sequenced and analyzed for both mitochondrial (12S and 16S rRNA) and nuclear markers (RAG-1 and ten microsatellite loci). Phylogenetic analyses of the concatenated mtDNA data revealed five major highly divergent lineages within the O. graminea sensu lato in Southern China, and these divergent lineages were highly concordant with five geographical regions. The nuclear data showed a discordant genetic structure compared to the mtDNA lineages (Clades A, B, and C) for O. graminea sensu stricto, with an admixed pattern in the RAG-1 data and two structure clusters in the microsatellite data. The species delimitation analyses, based on three methods, supported the species status of Odorrana zhaoi and Odorrana rotodora, and revealed the existence of putative cryptic species in the O. graminea sensu stricto. In addition, one statistically significant gene flow event was detected from Clade B to Clade C based on mtDNA and RAG-1 data, and the microsatellite data suggested gene flow within the O. graminea sensu stricto. Bayesian skyline plotting analyses and ecological niche modeling supported demographic and range expansions during the LGM for Clades A and C of the O. graminea sensu stricto. In addition, ecological niche models suggested the existence of ecological divergence among the three Clades (Clades A, B and C) of the O. graminea sensu stricto. The intense uplifting of the Qinghai-Tibetan Plateau, the Quaternary climate oscillations, and drainage changes may have driven the speciation, genetic structure and phylogeoraphic patterns of the O. graminea sensu lato.

Auditory brainstem responses in the red-eared slider Trachemys scripta elegans (Testudoformes: Emydidae) reveal sexually dimorphic hearing sensitivity.

Hearing sensitivity is of general interest from the perspective of understanding the functionality and evolution of vertebrate auditory systems. Sexual dimorphism of auditory systems has been reported in several species of vertebrates, but little is known about this phenomenon in turtles. Some morphological characteristics, such as middle ear and tympanic membrane that influence the hearing sensitivity of animals can result in hearing sexual dimorphism. To examine whether sexual dimorphism in hearing sensitivity occurs in turtles and to compare hearing characteristics with respect to the shape of the tympanic membrane, we measured the hearing sensitivity and tympanum diameter in both sexes of Trachemys scripta elegans. The results showed that, with the exception of 0.9 kHz, auditory brainstem response thresholds were significantly lower in females than in males for frequencies in the 0.2-1.1 kHz range, indicating that the hearing of females shows greater sensitivity. No significant differences were detected in the tympanum diameter of both sexes. These results showed that sexually dimorphic hearing sensitivity has evolved in turtles; however, this difference does not appear to be related to differences in the size of the tympanic membrane. The possible origin and function of the sexual differences in auditory characteristic are discussed.

Hierarchical Porous and Zinc-Ion-Crosslinked PIM-1 Nanocomposite as a CO2 Cycloaddition Catalyst with High Efficiency.

CO2 cycloaddition to epoxides is an effective and economical utilization method to alleviate the current excessive CO2 emission situation. The development of catalysts with both high catalytic efficiency and high recyclability is necessary but challenging. In this context, a heterogeneous catalyst was synthesized based on a zinc-ion-crosslinked polymer with intrinsic microporosity (PIM-1). The high microporosity of PIM-1 promoted a high Zn2+ loading rate. Additionally, the relatively stable ionic bond formed between Zn2+ and the PIM-1 framework through electrostatic interaction ensured high loading stability. In the process of CO2 cycloaddition with propylene epoxide, an optimized conversion of 90 % with a turnover frequency as high as 9533 h-1 could be achieved within 0.5 h at 100 °C and 2 MPa. After 15 cycles, the catalytic efficiency did not demonstrate a significant decline, and the catalyst was able to recover most of its activity after Zn2+ reloading. This work thereby provides a strategically designed CO2 conversion catalyst based on an ionic crosslinked polymer with intrinsic microporosity.

Complete mitochondrial genome of Goniurosaurus zhoui (Squamata: Sauria: Eublepharidae).

In this study, using Illumina sequencing data, we assembled the complete mitogenome of Goniurosaurus zhoui, which consists of 16,803 base pairs, comprising 13 protein-coding genes, 2 ribosomal RNA genes, 21 tRNA genes, and non-coding regions (D-loop), and has an overall A + T content of 60.39%. Using three complete 16S rRNA gene and 13 partial sequence data of the 16S rRNA gene, including that of G. zhoui, we reconstructed the phylogeny of the genus Goniurosaurus. The result of the Bayesian inference tree revealed four major, distinctly separated groups.

Atypical assortative mating based on body size in an explosive-breeding toad from a tropical island of southern China.

Mating patterns exhibit considerable intra- and interspecific variation. Sexual selection can lead to the occurrence of random and assortative mating in different populations of the same species. Thus, understanding variation in mating decisions is crucial to understanding variation in the direction of sexual selection. We investigated natural mating patterns in Black-spectacled toads (Duttaphrynus melanostictus), an explosive-breeding species that breeds throughout the year. We captured amplectant pairs (137) and non-amplectant males (212) during breeding seasons from November 2016 to April 2017 in tropical-island population of southern China. Our study found no significant difference in snout-vent length (SVL) between amplectant and non-amplectant males. Female and male SVL were positively correlated with each other. Small females were paired more frequently with small males, less frequently with large males, but had no preference for or against medium males. Medium females exhibited no preference. Large females showed no preference for large males, but were paired less frequently with small males. These data suggested that successful amplectant males had body sizes representative of the entire population. Both random and size-assortative mating were present simultaneously in the same population and within the same breeding season. Female choice was important in shaping the mating behavior of Black-spectacled toads, promoting genotype-frequency stabilization and body-size diversity in the population.

The complete mitochondrial genome of Feirana taihangnica (Anura: Dicroglossidae).

The complete mitochondrial genome sequence of the Feirana taihangnica (Anura: Dicroglossidae) was determined. It is a circular molecule of 17,412 bp in length, containing 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a control region. The tRNA(Thr) gene located in the LTPF tRNA gene cluster typically found in other anurans is absent from the F. taihangnica mtDNA and a tandem duplication of tRNA(Met) gene is observed. The A+T content of the overall base compositon of H-strand is 57.2% and the length of control region is 1972 bp with 61.8% A+T content.

Complete mitochondrial genome of the green odorous frog Odorrana margaretae (Anura: Ranidae).

The complete mitochondrial genome (mitogenome) of the green odorous frog Odorrana margaretae (Anura: Ranidae) has been studied. The 17,903 bp circular genome contains the typical complement of 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a control region. The AT content of the overall base compositon of H-strand is 56% and the length of control region is 2501 bp with 63.8% AT content. The arrangement of the protein-coding and ribosomal RNA genes was the same as that found in other anurans. The cluster of rearranged LTPF tRNA genes and the translocation of tRNA(His) gene into the D-loop region are observed.

Complete mitochondrial genome of the Ye's spiny-vented frog Yerana yei (Anura: Dicroglossidae).

Yerana yei (Anura: Dicroglossidae) is the only one species in the genus Yerana and the taxonomy of the Y. yei remains unresolved and controversial. The complete mitochondrial genome of Yerana yei (Anura: Dicroglossidae) was sequenced in the present study, and it is a circular molecule of 17,072 bp in length and contains the 38 genes typically found in other anurans: 13 protein-coding genes, 2 ribosomal RNA genes and 23 transfer RNA genes (including an extra copy of tRNA(Met)). The order and orientation of the genes is the same as that found in other Paini species. The A + T content of the overall base composition of H-strand is 58% (T, 29.4%; C, 27.3%; A, 28.6%; G, 14.7%) and the length of control region is 1580 bp with 67% A + T content.

Molecular phylogeny and diversification of the genus Odorrana (Amphibia, Anura, Ranidae) inferred from two mitochondrial genes.

A diversity of hypotheses have been proposed for phylogenetic relationships and taxonomy within the genus Odorrana, and great progress has been made over the past several decades. However, there is still some controversy concerning relationships among Odorrana species. Here, we used many paratypes and topotypes and utilized 1.81 kb of mitochondrial sequence data to generate a phylogeny for approximately 4/5 of Odorrana species, and Odorrana haplotypes form a strongly supported monophyletic group relative to the other genera sampled. The deepest phylogenetic divergences within Odorrana separate 3 lineages whose interrelationships are not recovered with strong support. These lineages include the ancestral lineage of O. chapaensis, the ancestral lineage of a strongly supported clade comprising many western species, and the ancestral lineage of a strongly supported clade comprising all other Odorrana sampled. Within the latter clade, the first phylogenetic split separates O. ishikawae from a well-supported clade comprising its other species. These divergences likely occurred in the middle Miocene, approximately 12-15 million years ago. Separation of the ancestral lineage of Odorrana from its closest relative, Babina in our study, likely occurred in the early Miocene or possibly late Oligocene. Rates of lineage accumulation remained high from the middle Miocene through the Pleistocene.