Ecology

Analysis of the roof failure characteristics of coal seamBefore mining, fracturing was conducted on a portion of gritstone in the lower section of the Naoro Formation and then entered the mining stage. Figure 9 shows the influence law of coal roof rupture under different periodic pressures. With mining of the #2 coal seam working face, the direct roof of the coal seam partially broke and collapsed, forming gangue in the goaf. There is a clear separation between the direct and basic roof. When the working face advanced to 228.2 mm, the old roof ruptured, and the working face started to enter the periodic pressure-bearing stage. As the working face advanced to 592.9 mm, the roof exhibited the fourth periodic pressure. The overlying layer roof in the excavation area was affected by the upper bearing arch pressure, leading to the collapsed rock to not completely contact the upper roof. With the increasing distance of coal seam mining, the roof developed significant subsidence, and the influence range of the bedrock boundary caused by the mining was still in the isolation layer fracturing zone. The bedrock influence boundary angle reached 73.57°, and the rock fracture angle was 56.95°. When the working face advanced to 726.5 mm, the fifth periodic pressure on the roof occurred. The bedrock layer in the upper right of the workings was near the right boundary of the first isolated coal seam rupture. Then, coal mining was suspended, and a second isolated seam fracturing process was conducted. The bedrock influence boundary angle reached 73.57°, and the rock rupture angle was 56.95°.Figure 9Influence law of coal roof rupture during different periodic pressure.Full size imageWhen the processing was advanced to 798.4 mm, the bedrock layer in the upper right of the processed area became close to the right boundary of the second isolated seam fracturing. After the third isolated layer fracturing process, the rock impact boundary angle reached 75.33°, and the rock fracture angle was 50.39°. Proceeding to 1031.6 mm, eighth periodic pressure was generated on the roof. The falling gangue in the mined-out area was in contact with the roof, with the bedrock impact boundary angle reaching 74.77° and the rock fracture angle reaching 57.06°. Thereafter, the bedrock layer of the roof gradually entered the full-scale mining stage. As the working face continues to advance, the bedrock impact boundary caused by coal seam mining should be in isolated coal seam fractures. When the bedrock layer at the working face is close to the right boundary of the isolation layer fracturing, the next isolation layer fracturing should be performed.Analysis of roof stress evolution lawFigure 10 illustrates the change law of the roof support pressure when mining of the working face, in which the roof support pressure curve is the stress change minus the initial value of the sensor before mining. After the excavation of the working face, the surrounding rock will exhibit stress redistribution. The increase in tangential stress in front of the working face or on both sides is called the support pressure. The peak value of the support pressure generally occurs on the front of the working face. As the working face advanced to 228.2 mm, the direct roof gradually broke and collapsed with mining. Due to the redistribution of surrounding rock stress, the stress fluctuation at the open cut was clear. In front of the working face, the overlying rock stress was redistributed due to mining, and the vertical pressure peak area appeared, with a stress increment of 0.03 MPa. When the working face advanced to 360.8 mm, the first cycle pressure on the roof occurred. The falling gangue in the mine-out area gradually approached its upper strata, and the peak support pressure increments reached 0.05 MPa. During the advancement of the working face to 592.9 mm, the direct roof continued to collapse. The gangue at the cuttings was gradually compacted with the roof, and the stresses gradually restored to stability. Coal seam mining led to the decompression of the floor, and the vertical stress maximum reduction at the working face was 0.045 MPa. The peak vertical pressure in front of the working face shifted to the right as mining progressed. When degradation reached 726.5 mm, the fifth periodic pressure on the roof occurred. Figure 10b shows that the fracture of the isolation layer had no apparent effect on the change in roof stress. Within 560 mm from the open excavation, the mine-out area gangue gradually compacted with the roof. Vertical pressure changes between the fourth and fifth periodic pressures are slight and practically nonsignificant.Figure 10Vertical pressure variation law with coal mining. (a) First pressure and First periodic pressure difference. (b) Fourth and First periodic pressure difference. (c) Eighth and Ninth periodic pressure difference. (d) Eleventh and Twelfth periodic pressure difference. (e) Variation laws of vertical pressure with mining.Full size imageWhen the mining reached 1031.6 mm, the directly caving gangue completely filled the goaf and was compacted with the roof. The upper roof of the caving rock was supported again, and the compaction range of the mining area extended to 821 mm. As the working face advanced to 1338.9 mm, the peak vertical pressure appeared at 1400 mm, with a maximum increment of 0.375 MPa. The compaction range of the mining area extends to 1200 mm. Then, the fractured isolation layer can be grouted. The subsequent working face advances until the end of mining, and the rock movement above the mine-out zone will exhibit a periodic “falling-filling-cutting-compaction” process. Fracture grouting of the flexible isolation layer has no significant effect on the vertical stress changes, and the stress unloading area and the peak vertical pressure will continue to change with mining. Nevertheless, consideration needs to be given to the adequacy of the gangue falling from the roof for isolation layer grouting.Roof displacement and development pattern of water-conducting fracture zoneFigure 11 shows the development law of the roof water-conducting fissures in the roof of the coal seam during different pressure periods, where the illustration shows the von Mises equivalent strain. Figure 12 shows the development trend of the water-conducting fracture zone height. From the whole observation, although the isolation layer is treated by fracturing before back mining, it has less influence on the displacement and deformation of the overlying rock layer because it is restricted by the surrounding rock of the model. When the working face was mined to 228.2 mm, the upper roof of the mining face collapsed, and the first periodic pressure occurred on the roof. The roof displacement reached the Yan’an Group mudstone layer, and the roof collapse height was only 104.3 mm. As the mining advanced, the roof fractures in the mining-out area continued to develop upwards. When the working face was mined to 360.8 mm, the first cycle pressure on the roof occurred, and the roof collapse height extended upwards to the siltstone of the Yan’an Formation, with a collapse range of 117.6 mm. At this point, only a small displacement change occurred around the direct roof, and the flexible isolation layer was basically not affected by any impact.Figure 11Development regularity of roof water-conducting fissures during different period pressure.Full size imageFigure 12Development height curve of water-conducting fracture zone.Full size imageFrom the second cycle pressure onwards, the development trend accelerated significantly, and the collapsed height rose rapidly to 210.9 mm. When the working face advanced to 537.1 mm, the third cycle pressure occurred on the roof. The collapsed Yan’an Formation mudstone layer was further pressurized by its upper layers and collapsed to a height of 344.7 mm. The roof displacement had spread to the coarse sandstone of the Naoro Formation, but the height of the water-conducting fracture zone had not reached the bottom of the isolation layer. When the workings reached 592.9 mm, the roof collapsed again, showing the fourth periodic pressure. The water-conducting fissure zone continues to develop upwards to 355.3 mm, which passes through the fissure isolation layer and reaches the gritstone at the top of the isolation layer. The fractured isolation layer is in an “activated” state.When the working face reached 1031.6 mm, fallen gangue completely filled the mining-out area and compacted with the roof, and eighth periodic pressure occurred on the roof. The height of the water-conducting fracture zone developed to 496.8 mm, which was lower than the height of the water-conducting fracture zone of 565.8 mm at the seventh periodic pressure. After that, the old roof collapsed as a cantilevered beam. The development height of the water-conducting fracture zone was allegedly less than 565.8 mm. Afterwards, the roof fracturing direction was consistent with the direction of working face advancement, from left to right. Displacement and fracture of the overlying rock layer were mainly caused by the overall downwards sliding of the upper rock seam due to the collapse of the bottom rock seam. At different heights of the coal seam roof, the degree of displacement damage decreased with increasing height.When the working face reached 1178.7 mm, the roof covering the open cut stabilized. The fractured isolation layers in the 1st ~ 13th groups were grouted, and then the coal was mined only after the slurry had completely solidified and reached a certain strength. The eleventh periodic pressure occurred on the roof, with a water-conducting fracture height of 367.6 mm at this time. When the working face was advanced to 1471.9 mm and 1645.2 mm, the roof had twelfth and fourteenth periodic pressures, and the heights of the water-conducting fracture zone were 332.0 mm and 416.0 mm, respectively. Then, the 14th ~ 15th and 16th ~ 17th group isolation layers of the upper coal seam were grouted while fracturing the right isolation layer. However, the disruption of displacement towards the extent of the development had a relatively small impact, mainly on the roof rock layer above the mining face. Table 2 indicates the development height of the water-conducting fracture zone and the fracture and grouting sequence of the isolated layer.Table 2 Development pattern of water-conducting fracture zone and fracture and grouting sequence of isolated layer.Full size tableDuring the mining process, damage to the water-conducting fissure zone was always a major factor in the displacement of the roof slab. Nonetheless, after fracturing and grouting measures, the effects of the damage were significantly reduced such that the damage to the roof rock was contained within the flexible isolation layer. After grouting, the enhanced strength of the isolation layer ensured that mining was carried out normally. During the mining period, four grouting reforms were made, and the isolation layer was fractured six times, with the maximum development height of the water-conducting fracture zone located at the seventh periodic pressure, reaching 565.8 mm.Analysis of water flow evolution law of overburden roofTo analyse the seepage law of the overburden roof, seven water flow monitoring lines were arranged from the top of the flexible isolation layer to the direct roof of the coal seam. The No. 1 water flow monitoring line was placed in the position of the third group of the isolation layer, which is initially located outside the deformation range of bedrock disturbed by mining and outside the stop line. The flow line was mainly used to monitor the influence of the rock disturbance boundary above the open cut on isolated seam fracturing and grouting. No 2–3 water flow monitoring lines were placed at the isolation layer positions of Group 12 and Group 14, which were initially located near the maximum height of the water-conducting fracture zone and were mainly used to monitor the change laws of the water-conducting fracture zone with mining impact. Monitoring Lines 4–6 were placed in isolation layers No. 17, No. 22 and No. 26 to study the impact of water flow changes with mining disturbance and the advanced influence scope. Water flow monitoring line No. 7 was placed in the thirtieth group of isolated layers, which was originally outside the cut-off line. As shown in Fig. 13, white arrows are water flow vectors in mL/min. Fracturing the 1–18 isolation layers before mining, the water tank hot water was injected into the flexible isolation layer such that the iodized salt in the flexible isolation layer was completely dissolved, and the infrared monitor showed the yellow area in the image. At this point, the water flow monitoring Lines 1–3 and 5–7 show yellow status, indicating that after the fracturing of the isolation layer, the aquifer water flows downwards along the fracture. The lower part of monitoring Line 4 was compacted at the top of the coal seam, indicating that the cracks between the roof and the aquifer had not been communicated. Therefore, the water flow rate was 0 mL/min until the sixth periodic pressure. Mining was then undertaken on the working face. The No. 1 monitoring line was therefore less affected by mining due to its layout outside the stop line, and there was no significant change in water flow before the first grouting.Figure 13Water flow evolution of the overburden roof with coal mining.Full size imageAs shown in Fig. 13, when the working face progressed to second periodic pressure, with the collapse of the coal seam, the stress of the surrounding rock was redistributed, the height of the water flowing fractured zone of the roof increased, and the water flow of the No. 2 monitoring line increased from the initial 9.1 mL/min to 14.0 mL/min. As the working face was advanced above the No. 2 monitoring line, the fifth periodic of pressure were generated in the roof. The development height of the roof water flowing fractured zone reached 504.4 mm. The roof was separated and collapsed, the cracks in the monitoring line communicated with each other, and the rock stress was released. The water flow in the No. 2 monitoring line increased significantly. Monitoring line No. 3 was affected by advanced mining, resulting in the coal seam roof’s increased rock fissures, the water flow path and resistance were reduced, and the water flow reached 48.3 mL/min. At the same time, the influence range of working face bedrock was close to the boundary of the first fracturing of the flexible isolation layer, and Groups 20–22 of isolation lays had been fractured.When mining started at the sixth periodic pressure, the roof water-conducting fracture zone gradually reached the maximum height and penetrated the fractured isolation layer, and the fracture of the roof rock increased. Lines No. 2 and No. 3 reached 44.4 mL/min and 85.6 mL/min, respectively. In fact, the encounter may indicate that the confined water of the gritstone aquifer was released, and the water flow of the working face increased. Then, the working face progressed, and the collapsed gangue above the mining-out area was compacted into the bedrock roof. The stress in the goaf did not change significantly, and the cracks in the strata decreased. The No. 2 and No. 3 water flows of the monitoring line gradually dropped. During this period, the change law of monitoring Lines 4–7 was similar to that of No. 2 and No. 3. During coal seam mining, the roof underwent a process of fracture, collapse, compaction and full mining, and the water flow monitoring line also went through a process of rising and then falling.When the working face was advanced to the eleventh periodic pressure, the grouting transformation of isolation layers 1–12 was conducted. The slurry was injected into the flexible isolation layer by hand pressure pump along the grouting pipe. After the slurry solidified, the colour of the No. 1 and No. 2 monitoring lines gradually became shallow, and the water flow gradually decreased under infrared observation. As the extraction of the coal seam progressed and the flexible insulation layer was broken and grouted, the colour of observation Lines 1–4 turned black in the infrared observation until the fourth grouting of insulation layer 18–19, and the water flow rate all showed 0 mL/min. However, the lower strata of the flexible isolation layer were not yet stabilized, so monitoring Lines 5–7 did not undergo any grouting transformation and still had a large water flow until the end of mining. Flow metre and infrared observations show that the destruction and grouting of the flexible isolation layer had a noticeable effect on the seepage characteristics of the overburden. In particular, after the grouting of the isolation layer, the slurry filled and solidified rapidly, the water flow decreased rapidly, and the water plugging effect of flexible isolation layer grouting was remarkable.Discussion and analysisDuring coal seam mining, the fracturing of the flexible isolation layer should be based on the premining overtopping influence range; that is, when the boundary line of bedrock influence extends to the range of the flexible isolation layer reached by the fracturing area of the flexible isolation layer, the next fracturing should continue. The average boundary angle range of the bedrock was 76.7°, and the field angle should not be less than 73.57°. The grouting of the flexible isolation layer considers the full mining degree of the coal seam. When there is no significant change in stress in the mined area, grouting of the flexible isolation layer at the top of the goaf is conducted. According to the simulation experiment in this paper, the full mining distance of the working face is 1338.9 mm, and the actual distance on site is 187.446 m. It is calculated that the distance between the fracture of the flexible isolation layer should be no less than 854.8 mm away from the working face, and the actual distance on site is 119.672 m. After the working face enters full mining, the shortest distance between the fracturing grouting range of the flexible isolation layer and the working face is not less than 242.6 mm, and the actual distance on site is 33.964 m.As seen from the previous analysis, with the advancement of the working face, the bedrock influence boundary angle of the coal seam does not change significantly, which only plays a guiding role in the fracturing sequence of the flexible isolation layer. The fracturing of the flexible isolation layer had an clear influence on the seepage of water-rich bedrock at the bottom of the Zhiluo Formation. The water-flowing fractured zone formed in the process of coal seam mining promoted the release of fractured water in the water-rich bedrock at the bottom of the Zhiluo Formation. The higher the height of the water-flowing fractured zone is, the greater the seepage of the water-rich bedrock. Coal seam mining had little effect on the seepage characteristics of the water-rich bedrock layer at the bottom of the Zhiluo Formation in the range of not disturbed by mining and advanced influence.In accordance with the stress sensor data, when the working face passed a certain distance, the bottom plate of the extraction area was compacted by the falling gangue, and the sensor pressure data did not change with the mining face. At this time, the grouting of the fracturing area of the flexible isolation layer corresponding to the above goaf was not affected by the mining face. For example, the stress in the goaf of 1200 mm had no clear change. Therefore, the first grouting was conducted in the fracturing area. After the solidification of the grouting slurry, the water flow of monitoring lines No. 1 and No. 2 decreased significantly. This minimized the impact on the original geological environment and at the same time reduced the goaf water drainage of the working face. The sealing effect of the isolation layer has an important influence on promoting water-retaining coal mining.The experimental application of the flexible isolation layer has realized its feasibility from the physical simulation test method in this paper. The realization of a flexible isolation layer requires premining fracturing and postmining isolation grouting. At present, premining fracturing can be achieved by directional drilling technology. There are also examples of roof separation grouting for postmining flexible isolation layer grouting28,29. Therefore, there is no technical bottleneck in field applications. Moreover, there is still a certain distance from the specific engineering application. According to the results of this study, it is predicted that the implementation of a flexible isolation layer will have great significance for water conservation coal mining in western China, which can reduce soil erosion and protect surface ecology. More

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18 January 2022

Portugal leads with Europe’s largest marine reserve

Filipe Alves

 ORCID: http://orcid.org/0000-0003-3752-2745

0
,

João G. Monteiro

 ORCID: http://orcid.org/0000-0002-3401-6495

1
,

Paulo Oliveira

2
&

João Canning-Clode

 ORCID: http://orcid.org/0000-0003-2143-6535

3

Filipe Alves

MARE Marine and Environmental Sciences Centre, Madeira, Portugal.

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João G. Monteiro

MARE Marine and Environmental Sciences Centre, Madeira, Portugal.

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Paulo Oliveira

Institute of Forests and Nature Conservation (IFCN, IP-RAM), Funchal, Madeira, Portugal.

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João Canning-Clode

Smithsonian Environmental Research Center, Maryland, USA.

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Marine conservation is central to the United Nations’ Sustainable Development Goals 13 (climate action) and 14 (life below water). Portugal has now created the largest marine reserve with full protection in Europe and the North Atlantic, an achievement that other nations could follow.

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Nature 601, 318 (2022)
doi: https://doi.org/10.1038/d41586-022-00093-8

Competing Interests
The authors declare no competing interests.

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Data and softwareThis analysis used two main data sources: (1) annual (through 2020) summaries of landings by species and by region provided by the Atlantic Coastal Cooperative Statistics Program (ACCSP), and (2) vessel-tracking data provided by Global Fishing Watch. The ACCSP is a cooperative state-federal program of U.S. states and the District of Columbia; it was established in 1995 to be the principal source of fisheries-dependent information on the Atlantic Coast of the United States. For the ACCSP data, I obtained annual landings by species for the North Atlantic region, Mid Atlantic region, and South Atlantic region (excluding landings from the Gulf of Mexico). The weekly cumulative landings data was obtained from the NOAA Fisheries Greater Atlantic Quota Monitoring website. Global Fishing Watch is an organization that provides access to information on commercial fishing activities, in particular information on the identity and location of fishing vessels34. Many large vessels use a system known as the Automatic Identification System (AIS) to avoid collisions at sea, broadcast their location to port authorities and other vessels, and to view other vessels in their vicinity. Vessels fitted with AIS transceivers can be observed by AIS base stations and by satellites fitted with AIS receivers. The US Coast Guard requires all vessels larger than 65 feet to have an AIS receiver onboard. Global Fishing Watch obtains AIS data for fishing vessels and enables users with Internet access to monitor fishing activity globally, and to view individual vessel tracks. They also partner with academic researchers to provide more fine-scale data.To obtain the vessel-tracking data for the relevant fisheries, I reviewed NOAA databases of squid and mackerel permits (2019 version; vessels with squid permits are automatically issued a Butterfish permit), and the Atlantic tuna permits (2020 version) and matched each permitted vessel to its unique Maritime Mobile Service Identity (MMSI) number, which is associated with Global Fishing Watch tracking information. I was able to identify 84% (187/224) of squid/butterfish permitted vessels (I focused on the SMB1A (Tier 1) permit category associated with the vast majority ( approx. 99%) of squid catch35), 100% of Tier 1 and Tier 2 mackerel-permitted vessels (56/56 vessels), and 74% of active tuna longline vessels (100/135 vessels). “Active” is defined as having reported successfully setting pelagic longline gear at least once between 2006 and 201236. This translates to a total of 17.55 million observations on fishing vessel locations for all three fisheries. I drop any observations that are missing either a latitude or longitude entry. For the squid and mackerel fisheries, I drop any observations with unusual longitudes ((ge 0^{circ }) and ( More

Experimental animalsGymnocypris przewalskii used in this study were obtained from the Rescue Center for Naked Carp of Lake Qinghai in Xining. Only healthy fish without visible body damage were used. Wet mass and body length were recorded before the fish were sampled. All fish were collected under permits issued by local and national authorities, and experimental procedures were in accordance with national animal care regulations. Experimental waters were prepared daily, and water qualities were measured before each experiment. The water temperature and salinity were measured using an YSI6600 multiprobe sensor (YSI Incorporated, Ohio, USA), and the carbonate alkalinity was determined by titration39. All fish (average body weight: 33.21 ± 2.74 g; average body length:14.81 ± 0.35 cm) were held in an indoor RAS system at a density of approximately 6.5 kg m−3. The holding and experimental water were filtered tap water (Canature/AC/KDF150-1–300) (salinity 0.16, pH 7.56, carbonate alkalinity 2.7 mmol L−1, temperature 17.1 ± 0.61 °C). Fish were fed daily with commercial feed. Fish husbandry and experimental procedures were approved by the Second Scientific Research Ethics Committee of East China Sea Fisheries Research Institute.Experimental designFish were placed on a 14:10-h light:dark (5:00–19:00 with light intensity of 600–1000 lx; 19:00–5:00 with light intensity of 0 lx) photoperiod aquaculture system. To examine the effect of rhythm on osmoregulation and acid–base balance, this study measured four endpoints: drinking rate, self-feeding intake, mRNA expression and the single cell expression level of osmoregulation and acid–base regulation relevant proteins. Fish held in filtered tap water were transferred directly to saline-alkaline lake water with salinities of 15 (L15, salinity 14.83, pH 8.65, carbonate alkalinity 30.54 mmol L−1) and 17 (L17, salinity 16.80, pH 9.02, carbonate alkalinity 34.61 mmol L−1), which was prepared by adding the same ratio of NaCl, MgCl2.6H2O, Na2SO4, CaCl2, KCl, NaHCO3 and Na2CO3 as in Qinghai Lake. The experimental period was 4–5 days.Drinking rate during high saline-alkaline transferFish were placed on a 14:10-h light:dark (5:00–19:00 with light intensity of 600–1000 lx;19:00–5:00 with light intensity of 0 lx) photoperiod aquaculture system. In this experiment, feeding was stopped 48 h prior to the experiments. Fresh water fish were transferred directly to L15 and L17 PEG-4000 free water for up to 4 days. For drinking rate analysis, new tanks were prepared which contained 50L saline-alkaline lake water with salinity 15 or 17 and with PEG-4000 (final concentration was 1.00 g L−1) during the day (10:00–16:00) and night (4:00–22:00) on the fourth day respectively. Nine fish per treatment were individually transferred from PEG-4000 free water to each tank which contained 1.00 g L−1 PEG-4000 at 10:00 or 4:00. Water samples were collected at 15 min after the fish were transferred to each treatment group for the determination of PEG-4000 concentration. The fish were terminally anesthetized with MS-222 (0.40 g L−1) after 6 h. The intestines were then quickly dissected out from nine individual fish per treatment group and the intestinal fluid were collected and stored at 4 °C. All fish were weighed before sampling.Self-feeding intake during high saline-alkaline transferFish were placed on a 14:10-h light:dark (5:00–19:00 with light intensity of 600–1000 lx;19:00–5:00 with light intensity of 0 lx) photoperiod aquaculture system. In this experiment, fish were kept in freshwater (FW) or acclimated to L15 for more than 15 days before the experiment started. Six RAS glass tanks (95 cm × 60 cm × 60 cm), which belong to two circulatory systems (3 tanks for FW and 3 for L15), were used for self-feeding experiment. Each tank had 15 individuals.Before the experiment, fish were trained by a custom-made self-feeding system (Fig. S2). Trained fish triggered the self-feeder when they want to feed. In the self-feeding system, the photoelectric sensor converts the change of optical signal into the change of electrical signal, and the feeder release feed by recognizing level fluctuation.During the formal experiment, we collected feed data at 5:00 and 19:00, which were the time points of the light and dark transition. Feed intakes of naked carp were calculated by weighing the feed quantities at two time points. The experiment lasted 5 days.mRNA expression of osmoregulation and acid–base regulation relevant proteins during high saline-alkaline transferFish were placed on a 14:10-h light:dark (5:00–19:00 with light intensity of 600–1000 lx;19:00–5:00 with light intensity of 0 lx) photoperiod aquaculture system. In this experiment, feeding was stopped 48 h prior to the experiments. Fresh water fish were transferred directly to L17 for up to 4 days. There were 24 fish per tank in triplicate. At the fourth day, six fish per tank were individually removed and terminally anesthetized with MS-222 (0.40 g L−1) at 4:00, 10:00, 16:00 and 22:00, respectively. The mid-intestine was quickly dissected out from six individual fish at each time point. Mid-intestine tissues for mRNA expression analyses were immediately snap-frozen in liquid N2, and stored at − 80 °C until analysis.Single cell positive rate of osmoregulation and acid–base regulation relevant proteinsFish were placed on a 14:10-h light:dark (5:00–19:00 with light intensity of 600–1000 lx;19:00–5:00 with light intensity of 0 lx) photoperiod aquaculture system. In this experiment, feeding was stopped 48 h prior to the experiments. To analyze the single cell positive rate of acid–base relevant proteins, a separate experiment was conducted. Fresh water fish were transferred directly to L17 for up to 4 days. There were 3 tanks (6 fish per tank) in this experimental group. At the fourth day, three fish per tank were individually removed and terminally anesthetized with MS-222 (0.40 g L−1) at 16:00 and 22:00, respectively. The mid-intestine was quickly dissected out from nine individual fish and immediately prepared for single-cell suspensions.Analytical techniquesDrinking rate analysisThe measurement of drinking rate was performed according to the study of Buxton et al.40. After weighing the collected intestinal fluid, it was centrifuged at 13,000g for 1 min, and 50 μL of the supernatant was taken, added dropwise to 350 μL of 72% pre-cooled (4 °C) acetone, and vortexed to mix. Samples were then centrifuged at 2000g for 10 min at 4 °C, the supernatant filtered with 0.45 μm filter paper, followed by addition of 100 μL of filtrate to 175 μL 25 mg L−1 gum arabic and vortexed to mix. Finally, 200 μL of TCA-CaCl2 (trichloroacetic acid-calcium chloride, 30% and 5% by mass) was added to the mixture and the reaction allowed to proceed at room temperature for 20 min. An Epoch microplate (Bio Tek) spectrophotometry unit was used to measure the absorbance at 650 nm. The remaining solution was weighed again after drying at 60 °C for 48 h, and the volume of intestinal fluid was determined (quantity of collected intestinal fluid-mass after drying). The same method as above was used to process the standard solution. Solute concentrations for standard curve were prepared as 0.00 g L−1, 0.10 g L−1, 0.20 g L−1, 0.40 g L−1, 0.60 g/L−1, 0.80 g L–1, 1 g L−1, and 2 g L−1 PEG-4000. The PEG-4000 concentration of intestinal fluid was calculated based on the standard curve. Drinking rate (μLg-1h-1) = 1000 × (CI × VI)/(CW × W × t), where CI is the concentration of PEG-4000 in the intestinal fluid (gL-1), VI is the volume of intestinal fluid (mL), CW is the concentration of PEG-4000 in experimental water (gL-1),W is the body weight of the fish (g), t is the duration of the experiment (h).Molecular biologyThe known sequences of the NKA-α gene of naked carp were compared with the corresponding genes of other species in GenBank, and highly conserved regions were selected for primer design (Table 1). The reference gene EF1α was used according to Yao et al.3. Previously published primers were used for SLC26A6 and SLC4A4 genes6. After extracting total RNA with Trizol (Invitrogen), the integrity of RNA was detected by 1% agarose gel electrophoresis, and the concentration and purity of total RNA were determined by a Bio Tek Epoch microplate spectrophotometer. The Rever Tra Ace-α (TOYOBO) kit was used to reverse transcribe mRNA to cDNA. Fluorescence quantitative PCR analysis was performed using a QuantStudio™ Real-Time PCR (Thermo life) with the SYBR Premix Ex TaqIII (TaKaRa) kit: total reaction volume of 10 μL, including 5 μL SYBR Premix Ex Taq, 2 μL upstream primers, 2 μL downstream Primers, and 1 μL cDNA template. The amplification procedure was as follows: 95 °C 30 s, 1 cycle; 95 °C 5 s, 60 °C 20 s, 40 cycles. Three replicates were included for each sample, with EF1α as the internal reference gene. The relative expression of each gene was calculated using the 2−ΔΔCt method41. Melting curve analysis was performed following each reaction to confirm that there was only a single product and no primer-dimer artifacts. In addition, representative samples were electrophoresed to verify that only a single product was present. Negative control reactions were performed for representative samples using RNA that had not been reverse transcribed to control for the possible presence of genomic DNA contamination. No-template control reactions were also performed to verify the absence of contaminating DNA or primer-dimer amplification in the reactions.Table 1 Nucleotide sequences of the primers used for amplification.Full size tableSingle cell staining analysisThe naked carp mid-intestine was isolated and transferred to HBSS on ice. The mid-intestine was washed by HBSS (Corning, 21-022-CV) and transferred to pre-warmed digestion medium containing 0.2 mg·mL−1 Collagenase I (Gibco, 17100-017), 0.06 mg mL−1 Collagenase II (Gibco, 17101-015) and 0.2 mg mL−1 Collagenase IV (Gibco, 17104-019), which was shaken vigorously for 30 s and further incubated at 37 °C for about 30 min in incubator with gentle shaking every 5 min to release cells. Cells were then collected by centrifuging at 300 × g for 5 min, and resuspended in D-PBS (BBI, E607009-0500). Then taken an appropriate amount of single cell suspension and dropped it on poly-L-lysine-coated slides where the experimental area was drawn with a hydrophobic marker to allow the single cells to settle freely. When the cell sedimentation density was moderate, aspirated the excess cell suspension, slides were fixed with 4% paraformaldehyde fix solution (BBI, E672002-0500) for 10 min, and blocked with 3% BSA (Sigma, B2064) for 1 h, three washes in D-PBS. Subsequently, slides were incubated in NKA-α or SLC26A6 (antibody dilution ratio was 1:100) for overnight at 4 °C. The NKA-α antibody was a commercial polyclonal rabbit Na+/K+-ATPase α antibody (Santa Cruz Biotechnology, sc-28800). The SLC26A6 antibody was a commercial polyclonal rabbit SLC26A6 antibody (Abcam, ab-172684). After the incubation, three washes in D-PBS. The secondary antibodies consisted of Alexa flour 568 goat anti-rabbit IgG (Thermo Fisher Scientific, A11036) (antibody dilution ratio was 1:400). Slides were incubated in room temperature for 1 h, followed by three washes in D-PBS. Finally, incubate with Hochest for 30 min. Cells were then photographed with a fluorescence microscope. For every fish, positive protein expression was counted using at least three pictures. Image J was used to analyze the fluorescence intensity and record the positivity rate.Statistical analysisThe data was expressed as mean ± standard error (SE). Two-way ANOVA and One-way ANOVA with LSD multiple comparison were employed to compare drinking rate, food intake and relative gene expression among different treatments and time courses respectively. Differences in single cell positive rate between 16:00 and 22:00 in L17 were evaluated by chi-square test. Assumptions for all parametric models (normality and equal residuals) were assessed via diagnostic plots. Means were considered significantly different when P  More

Phylogenetic relations and systematics of AllactaginaeIntergeneric relationsOur data produced a robust phylogeny for Allactaginae above species level and thereby firmly proved that Allactaga s.l. (as recognised by Holden and Musser17) is paraphyletic to both Pygeretmus and Allactodipus. Both of the latter taxa are morphologically distinct from Allactaga by a number of unique apomorphies: a unique molar pattern and glans penis morphology in Allactodipus as well as high-crowned terraced molars, reduction of the premolar, and particular glans penis morphology in Pygeretmus. At the same time, the morphology of all other five-toed jerboas is relatively monotonous with variation only in terms of body size, relative molar crown height, size of auditory bullae, m1 morphotype frequency, and the rate of M3 reduction1,45. Such level of differences never allowed recognition of more than one genus.Thus, allactagines represent a case when descendant lineages with derived morphology are nested within a group with overall conserved morphology. This can be compared to paraphyly of white-toothed shrews Crocidura relative to Diplomesodon46, rorquals (Balaenoptera) relative to humpback whales (Megaptera)47, or tits (Parus s.l.) relative to morphologically aberrant ground tit (Pseudopodoces humilis)48. In such cases, the taxonomy should be changed in accordance with the monophyly principle, which is achieved by combining genera (as done in whales) or splitting the genus in question into new taxa (as done in tits). Unfortunately, any decision in this context is arbitrary as it is based on subjective weighting of morphological differences. For Allactaginae, the splitting approach was implemented18, which resulted in the elevation of Scarturus and Orientallactaga to the generic rank2, despite the fact that a synapomorphy-based morphological diagnosis of Scarturus can hardly be formulated.As an alternative to the morphology-based approach, temporal banding—a method which uses node age as a measure of rank49—was suggested as a standardised method for taxonomic ranking. In the present study, the age of divergence of major Allactaginae lineages was dated to the Pliocene. However, in other groups of Myodonta, Pliocene divergences were found both among genera (as in voles50 or hamsters51) and among congeneric species (as in Sicista52). Thus, the ambiguity remains unresolved; we see no better option than to retain the generic classification established by Michaux & Shenbrot2 (Table S10). However, it should be noted that the inferred age of divergence between S. tetradactylus + S. hotsoni and the VECE clades (3.9–4.1 Mya) is comparable or even larger than the divergence time of Allactodipus from Allactaga. If the temporal criterion (sensu Avise, Johns49) is accepted, one should consider elevating the VECE clade at least to subgeneric rank, with Scarturus proper including only two species. The diagnosis of the new taxon should be polythetic (medium to small jerboas with five-toes, bullae not enlarged, glans penis with longitudinal fold, molar low-to medium crowned, M3 not reduced). Although the name Paralactaga is traditionally used as a subgeneric for the S. euphraticus group and therefore may have been applied to the whole VECE clade, we believe that this is incorrect. The type species of Paralactaga—P. anderssoni Young, 1927—was described from the Late Miocene of China, which is inconsistent with the estimated time of origin of the VECE clade. Apparently all similarities between S. euphraticus group and Paralactaga proper are because of plesiomorphy. Therefore, we suggest that Paralactaga should be attributed to fossil taxa only.Species groups within ScarturusIn the present study, we analysed in detail the phylogenetic reconstructions and divergence times estimations for the species and species groups of the genus Scarturus. Our study is the first to examine the phylogenetic position of the enigmatic taxon described from Afghanistan and which is currently termed Scarturus williamsi caprimulga. The mitochondrial data provided clear evidence that this taxon is not closely related to any member of the S. euphraticus species group including S. williamsi. Instead, it belongs to a separate divergent lineage of Scarturus, which should be considered a separate species, Scarturus caprimulga. It also includes the jerboa from Kopet Dag provisionally classified by Hamidi et al25 as Paralactaga cf. williamsi. The mitochondrial difference between specimens from Afghanistan and those from Kopet Dag suggested a potential subspecies rank of the latter form, which is provisionally referred to as S. aff. caprimulga. More research on the distribution and genetic structure of this species is needed for further clarification. Our study has added more representative genetic data on the poorly known S. vinogradovi and confirmed it as a separate divergent branch within Scarturus s.l. and likely a distant sister group of S. caprimulga.Previous phylogenetic reconstructions of the S. euphraticus species group based on mtDNA data recovered a divergent branch within S. euphraticus53, which was subsequently classified as S. aulacotis2. With further addition of comprehensive nuclear data, the full species rank of this taxon is now completely supported. The relationships among the three species in the S. euphraticus group correspond to a hard trichotomy dated to the late Early Pleistocene.Nuclear data strongly support deep structuring within the S. elater species group, as previously demonstrated using mtDNA19,22,54, and confirmed the species status of S. indicus and S. heptneri. The divergence between S. elater and S. indicus estimated based on the nuclear loci was dated to approximately 1.5 Mya, which was slightly older than the 1.26 Mya inferred from mtDNA by Bannikova et al.22. Both S. indicus and S. elater included allopatric lineages that have separated 600–800 kya (i.e. dzungariae and strandi within elater, and aralychensis within indicus). Their formal taxonomic rank appears controversial: the level of divergence apparently conforms to species rank, whereas genetic data indicates potential gene flow between them. Thus, the mtDNA haplotypes of Scarturus specimens from the Zaisan depression (S. e. zaisanicus) form a subclade within S. elater s.str., whereas nuclear data suggest that S. e. zaisanicus is relatively close to S. e. dzungariae. This pattern suggests that the Zaisan population, while being a derivative of the Dzungar form, experienced mtDNA capture as a result of a past hybridisation event with S. elater. Gene flow between S. strandi and S. elater proper was indicated by the occurrence of elater mtDNA haplotypes in certain populations of strandi from north-western Kyzylkum22. All these taxa require additional research to produce a more accurate evaluation of gene flow intensity. Nevertheless, we suggest that dzungariae, strandi, and aralychensis should be considered semispecies or species in statu nascendi. Taxonomically, we regard them as parts of elater and indicus superspecies and refer to them as S. (elater) dzungariae, S. (elater) strandi, and S. (indicus) aralychensis, respectively.Phylogenetic relations within OrientallactagaWithin Orientallactaga, O. bullata and O. balikunica were supported as sister taxa based on nuclear data, which is consistent with their common morphology (enlarged bullae). However, mtDNA suggested that O. bullata is a sister taxon to O. sibirica, and the reason for this discrepancy is unclear, with ancient mtDNA introgression being the most obvious explanation. The crown age of Orientallactaga was dated to the early Early Pleistocene (Gelasian). Neither O. bullata nor O. balikunica show substantial intraspecific variation.In contrast, O. sibirica consists of several genetic lineages, which partly correspond to recognised subspecies. The mtDNA data tentatively supported subdivision of O. sibirica into western and eastern groups separated by the Tianshan–Altay zoogeographic boundary. The structure of variation in the eastern portion of the range (Mongolia, China) is well-studied23; however, the genetic data on the western portion are still fragmentary. Available mtDNA data provisionally support recognition of western subspecies such as O. s. ognevi (north-eastern to central Kazakhstan), O. s. dementjevi (Issyk-Kul region), and O. s. altorum (central Tianshan). The latter two forms are distributed in high-altitude areas of Tianshan, thus indicating that, in contrast to most other jerboa species, mountain areas might serve as foci of diversification in O. sibirica.The westernmost part of the range (western Kazakhstan, Qyzylkum) was assumed to be inhabited by a single O. s. suschkini subspecies after morphological revision1. However, three divergent mtDNA lineages were recovered based on the preliminary analysis of mtDNA data retrieved from museum specimens from the area, which suggests that the diversity of western populations is likely underestimated and in need of further examination.The crown age of O. sibirica was estimated at 500–600 kya, which was substantially younger than 2.2–3.2 Mya as inferred by Cheng et al.23; this discrepancy, however, can be explained by mtDNA saturation effects and usage of inaccurate secondary calibrations in their study.Variation within Allactaga and PygeretmusConsidering the phylogenetic position of Pygeretmus, our data firmly corroborated its separate phylogenetic position and rejected any affinity with Orientallactaga bullata as reconstructed by Wu et al.55. The latter result should be attributed to identification error. In our study, all three species of Pygeretmus were analysed to confirm phylogenetic proximity of P. shitkovi and P. platyurus relative to P. pumilio. Thus, the subgeneric status of Alactagulus containing the latter species was not contradicted; however, the split age between Pygeretmus s.str. and Alactagulus is relatively young, dated as Pliocene/Pleistocene boundary, indicating that morphological and life history traits of the former (e.g. slower locomotion) have evolved rather recently.A further taxon demonstrating a complex structure is Allactaga major. Our mtDNA data indicated that A. major consisted of several genetic lineages partly corresponding to morphological subspecies (A. m. spiculum, A. m. djetysuensis). A high level of divergence was observed between specimens from the northern Caucasus and Kazakhstan. A specimen of morphologically distinct A. m. spiculum (north-eastern Kazakhstan, western Siberia) was placed as a sister species to all other A. major with a divergence level compatible with species status.Several other species included unexpected genetic lineages that were apparently divergent at subspecies level (e.g. a southern Uzbekistan lineage of A. severtzovi and an Ili lineage of P. shitkovi). However, the resolving power of the employed set of 15 nuclear genes is insufficient for clarifying relationships within species. Therefore, these cases should be studied using larger samples and further nuclear loci.Divergence time estimates within AllactaginaeOur estimated divergence times were generally more recent than those produced by most previous studies. The root node of crown Allactaginae was dated to 7.7 (5.4–9.9) Mya by Wu et al.55, 8.1 (4.2–12.7) Mya by Zhang et al.56, or 8.87 (8.3–9.85) Mya by Pisano et al.4. The results by Wu et al.55 may be affected by a node density effect as their re-analysis with reduced taxon sampling of Allactaginae and Dipodinae produced younger dating at 5.8 (3.1–8.6) Mya. The latter two studies used only one to four nuclear loci and calibrated their analysis using non-Dipodidae calibration points. In both cases, the Early Miocene age of Sicista primus was used to calibrate crown Sicista, which lacks proper justification and may result in upward bias, as argued by Rusin et al.57.The earliest Allactaginae appeared in the Early Miocene and, in the Middle Miocene, the members of the primitive genus Protalactaga Young, 1927 became a common element of the Asian fauna3. During the Late Miocene, the diversity of allactagines persisted, and new genera emerged including Paralactaga Young, 1927 which is morphologically similar to Allactaga and is often considered its subgenus3,45. However, as can be derived from our results, all but one of the Middle and Late Miocene lineages went extinct without leaving any recent descendants, and all current diversity is a product of the Pliocene–Pleistocene evolution. This diversification pattern is unlike that observed in a different jerboa subfamily, Dipodinae, which includes lineages that had diverged in the Middle and early Late Miocene (Paradipus and Dipus, respectively)4,58.As estimated here, the onset of radiation among crown Allactaginae occurred in the latest Messinian and thus was nearly coincident with the Messinian crisis. However, it remains unclear how (or whether at all) climatic perturbations at the Miocene /Pliocene boundary affected the evolution of Allactaginae. The results of the diversification analysis suggested that, throughout the Pliocene and Pleistocene, the rate and mode of speciation in five-toed jerboas remained constant, indicating high tolerance of this group towards the climatic changes of this period.The minimum age of split observed between sympatric species was approximately 1 Mya as demonstrated by heptneri versus elater s.str. (and strandi). This was the estimate for the minimum time necessary for formation of effective reproductive barriers in allactagines (post- or pre-zygotic). Other phylogenetically close sympatric species pairs were S. elater/S. indicus (1.5 My), O. bullata/O. balikunica (1.5 My), and A. major/A. severtzovi (2.0 My).Geography of speciationOf 17 analysed episodes of speciation in Pliocene–Pleistocene, the patterns of range fragmentation in 10 episodes matched well to the classical vicariance scenario and those of six episodes matched to the founder-event speciation scenario; in one episode, both scenarios were equally probable. As the location of arising isolation barriers within the ancestor range seemed incidental, only in three cases the ancestors’ range was subdivided into two parts which were more or less equal in size: first, into East and West Central Asia; second, into Turan and Iran; third, into Anatolia with trans-Caucasus and northern Zagros and Levant with northern Mesopotamia and southern Zagros. In all other cases, the ancestors’ range was subdivided into the main part and relatively small peripheral isolates. As can be expected from the modern patterns of species diversity of Allactaginae, the discovered speciation events were unequally distributed: one episode in North Africa, one in the eastern part of Central Asia, three in the Middle East, four in the Iranian highland, four in Turan, and five in Kazakhstan. In most cases, range fragmentation coincided with extreme climate conditions within the analysed time periods: warmest and wettest (decrease of the area of arid lands: nodes 2–3, 5, 10, 12, and 14–15) or coldest and driest (closing narrow mountain passages due to mountain glaciation: nodes 4, 6–9, 13, and 16–18). In one case (node 11), fragmentation of the range coincided with moderate climate conditions.Successful modelling of fragmentation of geographic ranges as a base of speciation events seemed to agree with the hypothesis of Peterson et al.15, which states that ecological niches evolve little at or around the time of speciation events, whereas niche differences accumulate later. This hypothesis was supported by Peterson’s analysis59 of data published between 1999 and 2008 which demonstrated that niche conservatism was found in more than 70% of comparisons within species and between sister species, but in less than 50% of comparisons among closely-related (but not sister) species and across monophyletic lineages of species. Moreover, analysis of habitat niche evolution of arvicoline rodents16 demonstrated that closely related species with allopatric or parapatric distribution demonstrated small niche differences, whereas they were larger in species with sympatric distribution. This is a clear indication that interspecific competition forces natural selection to increase niche differences resulting in species co-occurrence. It was demonstrated that niche divergence/conservatism can be differently expressed between different niche/resource axes60. In voles, which have a highly specialised folivorous diet, habitat segregation seems to be the only type of niche differentiation. Closely related Allactaginae species are similar in diet and typically occur in allopatric or parapatric distribution patterns1, which may indicate their niche conservatism. The only exception to a pattern where species with similar diets show widely overlapping geographic distributions are Scatrurus elater and S. heptneri (these two species are similar in both, macro- and micro-habitat niches, and it is unclear which mechanisms allow them to co-occur22). Distantly related sympatric species typically show similarities regarding macro-habitat niches but marked differences in terms of micro-habitat niches (Allactaga major and Orientallactaga sibirica; O. sibirica and O. bullata; O. sibirica and O. balikunica; Pygerethmus pumilio and P. platyurus; P. pumilio and P. shitkovi; personal observations) and diet (Allactaga and Allactodipus; Allactaga and Scarturus; Allactaga and Pygeretmus; Orientallactaga and Pygeretmus; Scarturus and Pygeretmus1,61). Thus, macro-habitat niche conservatism may be expected even in sympatric species. More

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