Microbial Biogeography along the Gastrointestinal Tract Segments of Sympatric Subterranean Rodents (Eospalax baileyi and Eospalax cansus)

In this study, based on high-throughput sequencing technology, the biodiversity and the community structure of microbiota in different GIT segments (the stomach, small intestine, cecum and rectum) of plateau zokors and Gansu zokors were studied and compared. A source tracking analysis for the microbial communities of different GIT segments was carried out using the fast expectation–maximization microbial source tracking (FEAST) method. We found that, for both species, the microbial community richness and diversity of the small intestine were almost the lowest while those of the cecum were the highest among the four segments of the GIT. Beta diversity analyses revealed that the bacterial community structures of different GIT segments were significantly different. As for the comparison between species, the bacterial community compositions of the whole GIT, as well as for each segment, were all significantly different. Source tracking conducted on both zokors indicated that the soil has little effect on the bacterial community of the GIT. A fairly high percentage of rectum source for the bacterial community of the stomach indicated that both zokors may engage in coprophagy.

Identification and Functional Analysis of lncRNAs Responsive to Hypoxia in Eospalax fontanierii

Subterranean rodents could maintain their normal activities in hypoxic environments underground. Eospalax fontanierii, as one kind of subterranean rodent found in China can survive very low oxygen concentration in labs. It has been demonstrated that long non-coding RNAs (lncRNAs) have important roles in gene expression regulations at different levels and some lncRNAs were found as hypoxia-regulated lncRNAs in cancers. We predicted thousands of lncRNAs in the liver and heart tissues by analyzing RNA-Seq data in Eospalax fontanierii. Those lncRNAs often have shorter lengths, lower expression levels, and lower GC contents than mRNAs. Majors of lncRNAs have expression peaks in hypoxia conditions. We found 1128 DE-lncRNAs (differential expressed lncRNAs) responding to hypoxia. To search the miRNA regulation network for lncRNAs, we predicted 471 and 92 DE-lncRNAs acting as potential miRNA target and target mimics, respectively. We also predicted the functions of DE-lncRNAs based on the co-expression networks of lncRNA-mRNA. The DE-lncRNAs participated in the functions of biological regulation, signaling, development, oxoacid metabolic process, lipid metabolic/biosynthetic process, and catalytic activity. As the first study of lncRNAs in Eospalax fontanierii, our results show that lncRNAs are popular in transcriptome widely and can participate in multiple biological processes in hypoxia responses.

Evoked auditory potentials from African mole-rats and coruros reveal disparity in subterranean rodent hearing

Hearing in subterranean rodents exhibits numerous peculiarities, including low sensitivity and restriction to a narrow range of comparatively low frequencies. Past studies provided two conflicting hypotheses explaining how these derived traits evolved: structural degeneration and adaptive specialization. To further elucidate this issue, we recorded auditory brainstem responses from three species of social subterranean rodents that differ in the degree of specialization to the underground habitat: The naked mole-rat (Heterocephalus glaber) and the Mashona mole-rat (Fukomys darlingi) which represent the ancient lineage of African mole-rats (Bathyergidae) and the coruro (Spalacopus cyanus), a South American rodent (Octodontidae) which adopted a subterranean lifestyle in more recent geological time. Additionally, we measured call amplitudes of social vocalizations to study auditory vocal coupling. We found elevated auditory thresholds and severe low-frequency hearing range restrictions in the African mole-rats, with hearing in naked mole-rats tending to be more sensitive than in Mashona mole-rats. In contrast to that, hearing in coruros was similar to that of epigeic rodents, with its range extending into ultrasonic frequencies. However, as in the mole-rats, the coruros’ region of best hearing was located at low frequencies close to 1 kHz. We argue that the auditory sensitivity of African mole-rats, although remarkably poor, has been underestimated by recent studies, while data on coruros conform to previous results. Considering the available evidence, we propose to be open to both degenerative and adaptive interpretations of hearing physiology in subterranean mammals, as each may provide convincing explanations for specific auditory traits observed.

Telling the Seasons Underground: The Circadian Clock and Ambient Temperature Shape Light Exposure and Photoperiodism in a Subterranean Rodent

Living organisms anticipate the seasons by tracking the proportion of light and darkness hours within a day—photoperiod. The limits of photoperiod measurement can be investigated in the subterranean rodents tuco-tucos (Ctenomys aff. knighti), which inhabit dark underground tunnels. Their exposure to light is sporadic and, remarkably, results from their own behavior of surface emergence. Thus, we investigated the endogenous and exogenous regulation of this behavior and its consequences to photoperiod measurement. In the field, animals carrying biologgers displayed seasonal patterns of daily surface emergence, exogenously modulated by temperature. In the laboratory, experiments with constant lighting conditions revealed the endogenous regulation of seasonal activity by the circadian clock, which has a multi-oscillatory structure. Finally, mathematical modeling corroborated that tuco-tuco’s light exposure across the seasons is sufficient for photoperiod encoding. Together, our results elucidate the interrelationship between the circadian clock and temperature in shaping seasonal light exposure patterns that convey photoperiod information in an extreme photic environment.

Increased burrow oxygen levels trigger defensive burrow-sealing behavior by plateau zokors

Defensive behaviors are a response to immediate and potential threats in the environment, including abiotic and biotic threats. Subterranean rodents exhibit morphological and physiological adaptions for life underground, and they will seal with mounds and additional plugs when their burrow opened. However, little is known about the factors driving this defensive behavior. In this study, we selected a subterranean rodent, plateau zokor (Myospalax fontanieri), as a species to investigate (both in the laboratory and in the field) the possible factors responsible for burrow-sealing behavior. Our results showed that: (1) In the laboratory, the burrow-sealing frequency of plateau zokor in response to five factors were as follows: oxygen (52.63%) > light (34.58%) > temperature (20.24%) > gas flow (6.48%) > sound/control (0%). Except for light, the burrow-sealing frequency in response to other factors was significantly lower than that in response to oxygen (P < 0.05). (2) Burrow-sealing behavior in response to each treatment did not differ significantly between males and females in the laboratory experiment. (3) In the field, during the animal’s active periods in both the cold and warm season, the burrow-sealing frequency under the oxygen treatment was higher than that under the light and temperature treatments. Plateau zokors were found not to be sensitive to these treatments during their inactive periods during both the cold and warm season. (4) The latency to reseal the burrow showed no obvious differences between each treatment both in the laboratory and in the field. In conclusion, the main factor that influences the burrow-sealing behavior of plateau zokors is the variation in oxygen concentration, and this defensive behavior is related to their activity rhythm.

DNA metabarcoding Uncover the Diet of Subterranean Rodents in China

ObjectiveIn the past, the zokor, which lived in northern China, caused great harm to agriculture and forestry production due to its large and sophisticated diet. Since the rat lives underground for most of its life, researchers know little about its dietary habits. Further understanding of its diet in the field is of important meaning for developing green and sustainable control strategies for the rat.MethodsLongde County in Liupan Mountain area of Ningxia Hui Autonomous Region was selected as the interest area to capture zokor and investigate the species of habitat plants.We selected chloroplast trnL gene and eukaryotic internal transcription spacer 1 (ITS 1) primers to amplify DNA from the gastric contents of zokor,and then sequenced on Illumina Miseq PE300 platform.ResultsThe gastric contents of Eospalax smithii (n=16) and E.cansus(n=9) were analyzed by operational taxonomic units (OTU) clustering and amplicon sequence variants(ASVs).The OTU clustering method obtained 2,995 OTUs, and the ASV method obtained 4,657 ASVs. The ASV method was better than the OTU clustering method, and the ASV method was adopted in the subsequent analysis. The food list of 32 families, 80 genera and 154 species was obtained by ASV method after the error was removed. The food composition of zokor was evaluated by relative abundance(%RA) method and frequency of occurrence(%ROO) method. At the Family level, it was found that zokor mainly fed on Asteraceae, Poaceae, Rosaceae, Pinaceae, Brassicaceae, Apiaceae, etc. At the Genus level, they are mainlyEchinops, Littledalea,Artemisia,Picea, Cirsium, Elymus and so on. The diet alpha diversity of E.cansus was slightly higher than that of E.smithii (P > 0.05). The correlation coefficient between Sobs index of alpha diversity and body weight of zokor was −0.382 (P = 0.059). The diet beta diversity proved that most zokors (22/25) clustered together, with low heterogeneity. They fed positively on Calamagrostis, Cirsium, Echinops, Medicago, Sanguisorba and Taraxacum. We found that zokor mainly fed on the roots of perennial herbs(PH), which were rich in water, carbohydrate, fat and protein, which provided an important energy source for its survival.ConclusionHigh-throughput sequencing(HTS) based DNA metabarcoding technology has well revealed the diet of zokor, which are generalist.

Redefining the Distributional Boundaries and Phylogenetic Relationships for Ctenomids From Central Argentina

With about 68 recognized living species, subterranean rodents of the genus Ctenomys are found in a multiplicity of habitats, from the dunes of the Atlantic coast to the Andes Mountains, including environments ranging from humid steppes of Pampas to the dry deserts of Chaco region. However, this genus needs an exhaustive reevaluation of its systematic and phylogenetic relationships regarding the different groups that compose it. This knowledge is essential to propose biodiversity conservation strategies both at species level and at higher hierarchical levels. In order to clarify the taxonomy and the recent evolutionary history from populations of Ctenomys in the Pampas region, Argentina, phylogenetic relationships among them were evaluated using mitochondrial DNA sequences: gene encoding cytochrome b protein (1,140 bp) and the non-coding D-loop region (434 bp). To infer the divergence times inside the Ctenomys clade, a Bayesian calibrate tree using fossil remains data from different families within Caviomorpha was performed at first. Secondly, that calibration data was used as priors in a new Bayesian phylogenetic inference within the genus Ctenomys. This phylogenetic tree emphasized on species currently distributed on the Pampas region, more precisely considering both the talarum and mendocinus groups. Bayesian inferences (BI) were integrated with the results of a Maximum Likelihood approach (ML). Based on these results, the distributional limits of the mendocinus and talarum groups appear to be related to the physiognomy of the Pampas region soils. On the other hand, the validity of C. pundti complex as a differentiated species of C. talarum is debated. According to previous evidence from morphological and chromosomal studies, these results show a very low divergence between those species that originally were classified within the talarum group. Mitochondrial DNA sequences from populations associated with these putative species have not recovered as reciprocal monophyletic groups in the phylogenetic analyses. In conclusion, C. talarum and C. pundti complex might be considered as the same biological species, or lineages going through a recent or incipient differentiation process. The results obtained in this study have important implications for conservation policies and practices, since both species are currently categorized as Vulnerable and Endangered, respectively.

Description of the sound diversity of two species of tuco-tucos (Ctenomys torquatus and Ctenomys lami ) in natural environment

Sound signals can travel through long distances, becoming an important communication channel between animals that need to establish contact beyond the visual form. They can then be considered a relevant form of communication between species living in the underground environment. Ctenomys torquatus and Ctenomys lami are solitary subterranean rodents, thus demanding an improvement of the communicative channels, especially in territorial defense actions and meeting potential reproductive partners. This work was aimed to describe the variability of acoustic signals emitted by C. torquatus and C. lami by analyzing the physical-morphological characteristics of the signals. The study was carried out in two populations, one of each species and was selected 14 individuals of C. torquatus and 15 C. lami. The acoustic signals were recorded in a natural environment, obtaining the sounds straight from the animal tunnels. A total of 1,380 signals were captured and analyzed, 786 from C. torquatus and 594 from C. lami. It was possible to characterize 5 different types of signals, emitted by both species. Most of the analyzed sequences presented low frequency, and many of these calls exhibited characteristics of long-range signals. It was verified a sharing of sound signals in both species, as regarding the acoustic parameters as the morphology of the analyzed spectrograms. For the first time, it was possible to have access to sound data emitted by direct subterranean rodents from their tunnels in the natural environment.

Gene losses may contribute to subterranean adaptations in naked mole-rat and blind mole-rat

The naked mole-rats (Heterocephalus glaber, NMRs) and the blind mole-rats (Spalax galili, BMRs) are representative subterranean rodents that evolved many extraordinary traits, including hypoxia tolerance, longevity and cancer resistance. Although a batch of candidate loci responsible for these intriguing traits have been uncovered by genomic studies, many of them are limited to functional modifications of intact genes and little is known about the contributions of other genetic makeups. Here, to address this issue, we focused on gene losses (unitary pseudogenes) and systematically analyzed gene losses in NMRs and BMRs, as well as their respective terrestrial relatives, guinea pigs and rats, in a genome-wide scale. 167, 139, 341 and 112 pseudogenes were identified in NMRs, BMRs, guinea pigs and rats, respectively. Functional enrichment analysis identified 4 shared and 2 species-specific enriched functional groups (EFGs) in subterranean lineages. The pseudogenes in these EFGs might be associated with either regressive (e.g. visual system) or adaptive (e.g. altered DNA damage response) traits. In addition, several pseudogenes including TNNI3K and PDE5A, might be associated with their specific cardiac features observed in subterranean linages. Furthermore, we observed 20 convergent gene losses in NMRs and BMRs. Given that the functional investigations of these genes are generally scarce, we provided functional evidence that independent loss of TRIM17 in NMRs and BMRs might be beneficial for neuronal survival under hypoxia, supporting the positive role of eliminating TRIM17 function in hypoxia adaptation. We also demonstrated that pseudogenes, together with positively selected genes, reinforced subterranean adaptations cooperatively. Overall, our study provides new insights into the molecular underpinnings of subterranean adaptations and highlights the importance of gene losses in mammalian evolution.