Ecology

Model equationsOur host-parasite mathematical model involves the following host population components: ‘susceptible’ hosts denoted by (S), and hosts infected by k distinct types of parasites ((k=1,2,…,n)), the corresponding population numbers of infected hosts are denoted by (I_{i_1,i_2,…,i_k}), where each index (i_j) can take a value from 1, …, n (to avoid repeated counting of the same infection configuration, we require throughout the paper that (i_1 More

Data collectionStudy sitesWe selected four study sites in the Upper Gunnison Basin in Colorado, USA. As we intended for these sites to capture a large amount of variation in environmental conditions, the primary criteria we used for site selection was elevation. Mirroring patterns of mountain weather observed worldwide, elevation is correlated with a multitude of environmental factors in the Rocky Mountains. Predominant trends include a negative correlation between altitude and temperature, a positive correlation between altitude and precipitation34, and transitions in plant community composition (from ‘mountain shrub’ to ‘montane’ to ‘subalpine’ to ‘alpine’) with increasing altitude35.Our sites span a large elevation gradient of approximately 1000 m that captures significant variation in environmental conditions. These sites should not be interpreted as a direct ‘elevation for climate substitution’, especially considering the significant changes in weather at each site that occur over the course of our observation periods, which ran from June to July. The lowest site, ‘cement creek’ (CC; approximately 38.82156° N, 106.86893° W), falls within a sage brush meadow on the boundary between the mountain shrub and montane vegetation zones at 2440 m elevation. The second lowest site, ‘bus turnaround’ (BT; approximately 38.97130° N, 106.99595° W), is situated in an open subalpine meadow at the base of Gothic Mountain at ~ 2940 m. The second highest site, ‘gothic mountain’ (GM; approximately 38.97969° N, 107.01937° W), is also in the subalpine zone on a steep hillside within a clearing in an evergreen forest on the lower slopes of Gothic Mountain at 3220 m. The highest site, ‘high meadow’ (HM; approximately 38.96779° N, 107.02184° W), is on an exposed meadow at the upper fringes of the subalpine zone on the shoulder of Gothic Mountain at 3,410 m.We began our observations at each site by mapping the flax population (see “Population mapping” sub-section below). This occurred on 6/15/2020 for CC, 6/17/2020 for BT, 6/16/2020 for GM, and 6/18/2020 for HM. We concluded all observations at CC on 7/27/2020, at BT on 7/29/2020, at GM on 7/28/2020, and at HM on 7/10/2020 (aside from spore trap collection which concluded on 7/21/2020).Weather monitoringWe deployed environmental sensors to record longitudinal weather data at each of our sites. We recorded temperature, humidity, rainfall, and wind speed and direction (at ~ 1 m above ground) every five minutes (see Supplementary Text 1 for logger specifications). We used temperature and humidity data to calculate absolute humidity (Supplementary Text 2). Due to logistical constraints, rainfall and wind speed/direction loggers were deployed later than temperature and humidity loggers.We extracted weather metrics for a given observation period from this data as follows: We calculated the mean, maximum, and minimum temperature as the mean, maximum, or minimum of temperature readings. Likewise, we calculated mean absolute humidity as the mean of absolute humidity records (calculated from temperature and relative humidity every five minutes). Daily mean rainfall was calculated as of mean rainfall records multiplied by the number of 5 min increments in a day (i.e. 288).Future climate dataTo enable forecasting of the effects of climate change on within and between host transmission processes, we compiled projected future weather data. We extracted downscaled climate and hydrological data generated using the Localized Constructed Analogs (LOCA) method and the CESM1(CAM5) model from the “Downscaled CMIP3 and CMIP5 Climate and Hydrology Projections” archive36,37,38,39,40. Specifically, we extracted values for daily mean temperature, relative humidity, and rainfall from the CESM1(CAM5) hydrology model, and values for daily maximum and minimum temperature from the CESM1(CAM5) climate model for both the RCP4.5 and RCP8.5 emissions scenarios41. Temporally, this data spanned June through July in 2020, 2030, 2040, 2045, 2050, 2060, and 2070. Spatially, this data represents projections for the area spanning (38.9375, 39.0) latitude and (− 107.0, − 106.9375) longitude. This area is the finest grained spatial extent including the BT site for which projections were available.Population mappingIn order to uncover drivers of population level epidemiological dynamics, it is necessary to characterize the ‘landscape of susceptibility’ in the host population. For spatially structured infectious disease systems such as flax rust, this involves documenting not only the infection status of each individual and any likely covariates of contact rate or susceptibility (e.g. size), but also the spatial arrangement of individuals. To accomplish this, we established 10 by 20 m transects at each of our field sites. We recorded the coordinates of each transect corner and the compass bearings associated with the transect axes (so that wind direction could be translated to the transect coordinate system) using a Garmin GPS unit (part #: 010-01735-10). At the beginning of the field season, we mapped the location, height, and infection status (healthy or diseased) of each plant within each transect. Plants with heights less than five centimeters were denoted as seedlings. While other metrics of size such as stem count could be used to form a more complete picture of plant size, we chose to use height alone as a proxy for size as it was the only measure that we could feasibly record for the many hundreds of plants within each transect. At the CC site, we were unable to map all healthy plants in certain areas of the transect due to time constraints, but we did record data for all diseased plants in these regions.Epidemiological surveysAfter documenting the initial epidemiological conditions, we tracked the spatiotemporal spread of flax rust within each transect. Approximately once per week, we visually inspected each flax individual in the transect for signs of disease, recording the location of any newly infected plants so they could be matched to a previously uninfected plant. We also recorded the height of all newly infected plants. We conducted these epidemiological surveys at least seven times at each site. Plants identified as infected in the initial population mapping and in these subsequent epidemiological surveys were marked with flags to ensure that they would not be repeatedly recorded as newly infected. Although we did not map the entirety of the CC transect, we covered the entire 10 by 20 m area in all epidemiological surveys.Diseased focal plantsWithin each transect, we designated a subset of diseased plants as ‘focal diseased plants’ and used them to make detailed measurements of within-host disease spread processes. These processes included changes in infection intensity, which we measured at the plant scale, as well as pustule growth, which we measured at the sub-plant scale using changes in pustule area and as a proxy.All initially infected plants were designated as diseased focal plants, and we continued to give this designation to newly diseased plants (except in some cases, diseased plants with height of approximately 5 cm or less, as these plants were too delicate to be manipulated) until we obtained at least 25 infected focal diseased plants per site. This occurred on 7/20/2020 at CC, on 7/1/2020 at BT, and on 7/9/2020 at GM and HM. We inserted metal tags into the ground at the base of diseased focal plants so they could be quickly and reliably identified. To facilitate longitudinal measurements of disease spread at sub-plant scales, we marked up to three infected stems on each focal diseased plant with pieces of colored flagging tape tied around the base of the stem. These stems were chosen haphazardly. On each of these stems (when possible), we marked the tip of one infected leaf with black ink. We preferentially marked leaves with one or a few pustules. Approximately once every three days, we recorded detailed measurements of plant height and within-host disease spread processes.To measure pustule growth, we photographed each marked leaf with a millimeter ruler in frame using a Cannon EOS Rebel T7i DSLR camera fitted with a Canon EF-S 35 mm f/2.8 Macro IS STM lens and a Cannon MR-14EX II Macro Ring Lite. While achieving consistent photographic angles and lighting conditions is impossible in a field setting, we attempted to keep the leaf and the scale in the same plane while maintaining a perpendicular shooting angle. We also adjusted camera settings so that pustule boundaries could be clearly distinguished in images. We used ImageJ software42 to measure the maximum and minimum diameters of each photographed pustule, and calculated pustule area assuming pustules to be elliptical. To enable the size of individual pustules to be tracked across observations, each measured pustule in each image was labeled so that it could be re-identified in subsequent images based on its location on the leaf and position relative to other pustules. We omitted data for any pustule that could not be confidently identified or whose borders became indistinct from other pustules. We marked a new leaf when we were unable to find the previously marked leaf, when the leaf was accidentally removed during data collection, or when the condition of the leaf deteriorated significantly. We suspect that either the presence of ink or our handling of the plant contributed significantly to the rate at which leaf condition deteriorated, but nevertheless we were able to observe the growth of many pustules over many weeks.To measure infection intensity, we first counted the number of infected stems. Next, for each marked stem, we recorded the length of infected tissue as the length of the stem segment spanning the lowest (relative to the ground) pustule to the highest pustule. Finally, we counted the number of pustules present on a haphazardly selected leaf in the middle of the region of infected tissue on each marked stem. We did not use the same leaf at each observation as the middle of the region of infected tissue changed due to disease spread. Using these measurements, we calculated an infection intensity metric as the product of the number of infected stems, the average length of infected tissue, and the average number of pustules present on a leaf in the middle of the region of infected tissue.Healthy focal plantsAs we wished to investigate patterns of growth in both healthy and infected plants, we designated certain uninfected plants as healthy focal plants, and measured their height whenever we measured the height of focal diseased plants. To control for potential effects of stem marking on the growth of diseased focal plants, we made these same markings on healthy focal plants. Healthy focal plants were selected haphazardly, and we added new healthy focal plants over time to keep the number of diseased and healthy focal plants approximately equal. At some sites, the number of healthy focal plants fell towards the end of the observation period as many healthy focal plants became infected. When this occurred, we stopped recording data for the plant, and in some cases designated it as a diseased focal plant.Spore depositionTo measure the distribution of spore deposition from an infected plant, and how it relates to the infection intensity of the source plant and wind patterns, we deployed spore traps in arrays around a subset of focal diseased plants. We chose focal disease plants that were as removed as possible from other diseased plants to minimize the number of spores originating from other plants that would be caught in the spore traps. These spore traps consisted of a ~ 2 cm2 section of Scotch Permanent Clear Mounting Tape (part #: MT76272-5) affixed to plastic backing with double sided tape. These spore traps were secured into the ground with ~ 5 cm nails. We deployed these traps at distances of either {~ 5 cm} (traps at ~ 5 cm were placed as close to the plant as possible), {~ 5 and 25 cm}, {~ 5, 25, and 50 cm}, or {~ 5, 25, 50, and 100 cm} in each of the four directions corresponding to the axes of the transect in which they were located. The traps were left in the field for approximately one week, and then collected. The sections of mounting tape were transferred to microscope slides and sealed in place using clear packing tape. We then used a light microscope to count the number of spores present in a known area. We counted spores in an area of 0.8 cm2 for most spore traps that contained few or no spores, but we counted a minimum area of 0.1 cm2 for all spore traps, including those with greater than 10,000 spores per cm2. Melampsora lini spores were identified visually using a slide prepared with spores as a reference for spore size, shape, and color. The spores matched descriptions in the literature43. We did not observe any rust fungi other than M. lini in or around our transects, so we can be reasonably certain that our counts represent only M. lini spores from infected focal diseased plants.Statistical analysesPlant growth and within-host disease spreadTo infer how different weather factors affect plant growth, pustule growth, and changes in infection intensity, we fit generalized additive models18. We used longitudinal observations of plant height in healthy and diseased focal plants to fit the model of plant growth, and longitudinal observations of within-host disease spread at various scales in focal diseased plants to fit all other models. Because the observation periods associated with our measurements of these processes varied, we formatted the response variables in change-per-day units: For our analyses of plant growth, pustule growth, and infection intensity progression we used change in plant height per day, change in pustule area per day, and change in infection intensity per day respectively as the response variables.To infer the effects of weather variables, we included smooth terms for these factors as predictors in each model. For each observation of change in plant height, pustule area, or infection intensity, we determined the start and end timepoints of the observation period to extract the corresponding weather data. For observations of plant height, we used 12:00:00 on the day of the observation as the start/end timepoints of the observation period. For observations of pustule area, we used photograph timestamps as the start and end timepoints. For observations of infection intensity, we used the average of the timestamps of photographs taken of pustules on that plant’s leaves as the start and/or end timepoints if such photographs were taken, because all data for an individual diseased focal plant was generally collected at once. If such photographs were not taken, we used the mean timestamp of all photographs taken at that plant’s site on the observation date as the start and/or end timepoint. To focus our analysis on fine-grained relationships between weather and within-host disease spread, we discarded data corresponding to observation windows of eight or more days. According to this criterion, 13 of 609 data points were discarded in the analysis of plant growth, 216 of 3535 data points were discarded in the analysis of pustule growth, and 25 of 338 data points were discarded in the analysis of infection intensity progression. Using the start and end timepoints of the observation period as bounds, we extracted the following variables from the weather data: temperature (mean, maximum, and minimum), absolute humidity, and mean daily rainfall.To account for the nested nature of our data, we included smooth terms for study site and plant identity as random effects (accomplished by setting bs = ”re” in the s() function in mgcv) in each model. In the case of the analyses of pustule growth, plant identity refers to the plant on which the pustules were observed. We also included a term in each model that accounted for the value of the previous observation to capture any allometric effects. In the model of change in plant height per day, this term was a full tensor smooth product of last observed height and infection intensity. In the model of change in pustule area per day, this term was a smooth of last observed pustule area. In the model of change in infection intensity per day, this term was a full tensor smooth product of the base 10 logarithm of the last observed infection intensity and last observed maximum plant height.We fit these models using the gam() function in the mgcv R package and the restricted maximum likelihood parameter estimation method18,44. We used gaussian response distributions with identity link functions. The dimensionality of all basis functions was set to the default value determined by mgcv. The one exception to this involved the marginal basis for log10 infection intensity (a part of the tensor term) in the model of change in infection intensity per day. The dimensionality of this marginal basis was increased to 15 after model diagnostics indicated that the default basis dimension was insufficient. To implement variable selection so that insignificant predictor terms would be effectively removed from the models, we used the double penalty approach of Mara and Wood45 (via the ‘select = TRUE’ option in the mgcv gam() function).The coefficients of the fit models can be used to infer the effects of weather factors on plant growth and within host disease spread. To translate these inferences into predictions about how climate change might affect plant growth and within-host disease spread, we simulated trajectories of plant growth, pustule area, and infection intensity under various climate conditions. For each class of simulation, we started by defining a hypothetical starting state. For simulations of plant growth, this was a 10 cm tall uninfected plant (the plant was assumed to remain uninfected for the entire simulation). For simulations of pustule growth, this was a pustule with area 0.1 cm2. For simulations of infection intensity, this was a 25 cm tall plant with infection intensity 1.0. Next, we simulated 100 trajectories from the model posteriors using observed weather data for each site and a step size of seven days. In these simulations, we restricted plant height, pustule area, and infection intensity to remain greater or equal to 5 cm, 0 mm2, and 0.1, respectively. We included the random effect of site (but not plant identity) when making these predictions. After confirming that these simulation results qualitatively recapitulated observed patterns, we simulated sets of 100 trajectories for future climate data sets. We again excluded the random effect of plant identity when making these projections. We used the same set of 100 random number seeds for each set of 100 trajectory simulations.While performing these simulations, we extracted weather variables as follows: When extracting weather variables from observed data sets, we defined the observation period as 00:00:00 on the first day in a step to 00:00:00 on the last day in a step (e.g. midnight on July 1st to midnight on July 8th). We extracted weather metrics as described in the “Weather data” subsection of the “Data collection” section above. Applying these same methods to the projected weather data is not possible, as these data sets only give only daily mean values. Instead, we calculated mean temperature as the mean of daily mean temperature values, maximum temperature as the maximum of daily maximum temperature values, and minimum temperature as the minimum of daily minimum temperature values. Similarly, we used daily mean temperature and relative humidity to calculate daily mean absolute humidity, and calculated mean absolute humidity as the mean of these values. We applied this same procedure to calculate mean rainfall.Spore dispersalTo infer how spore deposition is related to source plant infection intensity, wind speed, and wind direction, we fit a tilted gaussian plume model22 (TGPM) to our spore deposition data. The equation specifying the TGPM is as follows:$$begin{aligned} & dleft(I,H,s,X,Yright)=frac{I k {W}_{s}}{2 pi s {sigma }^{2} }{e}^{frac{{-Y}^{2}}{2{sigma }^{2}}-frac{{(H-frac{{W}_{s }X}{s})}^{2}}{2{sigma }^{2}}} \ & for quad X >0 0 \ & for quad Xle 0.end{aligned}$$This equation describes the concentration of spores deposited at a given point ((d)) as a function of wind speed ((s)), the infection intensity of the source plant ((I)), a constant relating (I) to the source concentration of spores ((k)), and the coordinates of the point (X,Y), relative to the source. The coordinate system has the source at the origin, the X-axis parallel to the wind direction (with wind flowing in a positive direction), the Y-axis perpendicular to the X-axis on the plane defined by the ground (assumed to be flat), and the vertical Z-axis orthogonal to the X and Y axes. The shape of the three dimensional spore plum emanating from the source is defined by (s), along with constants specifying the falling velocity of spores (({W}_{s})), the height of the source ((H)), and the standard deviation of spore dispersion (({sigma }^{2})) in the horizontal and vertical directions. Following Levine and Okubu22, we calculate ({sigma }^{2}) as a function of a diffusion coefficient, (A), assuming that the variance in spore distribution increases linearly with time. We also assume that spores diffuse along the Y and Z axes at equal rates.$${sigma }^{2}=frac{2AX}{s}.$$We fit the parameters (k, {W}_{s},) and (sigma ) by minimizing the sum of squared differences between model predictions and the spore deposition data we collected from the spore traps arranged around diseased focal plants. We assumed that all spores deposited on a spore trap originated from the associated diseased focal plant, and that spore deposition occurred between noon on the day of deployment and ceased at noon two days post-deployment as the spore traps lost their adhesive properties and became saturated with dust. When predicting spore deposition using the TGPM, we set (I) equal to the infection intensity of the source diseased focal plant on the day of spore trap deployment and (H) equal to half of the maximum height of the source diseased focal plant at the day of spore trap deployment. As wind speed and direction were recorded every 5 min, we calculated total spore deposition as the sum of predicted deposition across five minute time windows. In each individual estimate, we set (s) equal to wind speed and calculated (X) and (Y) from wind direction and the location of the spore trap relative to the focal diseased plant (see Supplementary Text 3).TransmissionUsing the fitted TGPM we next sought to connect patterns of spore dispersal to transmission. The first step in this process was to align data on the spatiotemporal spread of disease with the locations of all healthy and infected plants (including non-focal plants) within each transect so that the relative locations of these could be determined (see Supplementary Text 4). Next, we built a dataset (‘corrected plant height data’) describing the height of all plants on the dates of epidemiological surveys. We preferentially used actual measurements from the initial population survey, epidemiological surveys, or focal plant measurements where they existed. For the majority of plants, height was not recorded on the date of a given epidemiological survey. In these cases, we fore- or hind-casted height from a previous or future record of plant height (whichever was closer) using the fitted generalized additive model of plant growth and observed weather data spanning the period between the date of interest and the closest record. We included the random effect of study site when making predictions, but not the random effect of plant identity as we wished to forecast heights of both focal and non-focal plants and only focal plant data was used to fit the model of plant growth.After completing these steps, we next built a ‘transmission data set’ connecting weather conditions, predicted spore deposition, plant height, and infection occurrence across the time windows between each epidemiological survey. We included separate entries for each healthy plant (focal and non-focal) during each time window. We limited the scope of this data to the periods for each transect in which all infected plants (that were not seedlings) were tracked as diseased focal plants. Using noon on the date of one epidemiological survey as the beginning of the time window, and noon on the date of the next epidemiological survey as the end of the time window, we extracted the same weather variables as described in “Plant growth and within-host disease spread”. Plant height values were extracted from the ‘corrected plant height’ data set. We determined that a plant became infected if a plant with identical coordinates (after data alignment) was recorded as newly infected in the epidemiological survey that took place at the end of the observation period. Total spore deposition for each healthy plant was calculated as the sum of predicted spore deposition originating from each of the plants in the same transect that was infected at the beginning of the time window. To predict the spore deposition experienced by a healthy plant that originated from an individual diseased plant, we used the TGMP(.) We extracted the height of the diseased plant at the beginning of the time window and set (H) to half of this value. Likewise, we extracted the infection intensity of the diseased plant at the beginning of the time window, and set I equal to this value. For diseased seedlings that were not tracked as diseased focal plants, we set I = 0.1, as most were observed to be lightly infected. There were 14 instances of missing infection intensity data for a diseased focal plant on a certain date. In these cases, we fore- or hind-casted infection intensity for that plant using the generalized additive model of infection intensity progression fit in “Plant growth and within-host disease spread” (while including the random effect for study site, but not for plant identity), the closest observation of infection intensity for that plant, and the weather data spanning the time period from the missing observation to the closest measurement. We set the values of (k, {W}_{s},) and (sigma ) to those fit in the “spore deposition” section above. As wind speed and direction were recorded every five minutes, we summed spore deposition over 5 min windows that matched the resolution of the wind speed and direction data. For each of these windows, we predicted spore deposition after setting the values of X and Y based on the relative location of the healthy and diseased plants and the direction of the wind (see Supplementary Information).Using this data set, we modeled infection outcomes in a generalized additive model framework. We assumed a binomial response distribution, coding the outcome of an infection as 1 and the outcome of no infection as 0. To account for variation in the duration of observation period, we used a complementary log–log link function, and included the log time as an offset in the model46. We included smooth terms for all weather variables as predictors, along with smooth terms study site and plant identity as random effects (accomplished by setting bs = ”re” in the s() function in mgcv). To infer the connection between spore deposition and infection, we also included a full tensor smooth product between the base 10 logarithm of mean total spore deposition per day and maximum plant height. When fitting the tensor, we fixed the smoothing parameter for the marginal smooth for log10 total spore deposition to (1times {10}^{10}). This effectively sets the shape of this marginal smooth to be linear, preventing any overfitting involving non-monotonic effects of spore deposition. Height was included as a factor in the tensor for two reasons. First, we calculated spore deposition for a point. In reality, the spore deposition experienced by a plant depends on the size of the ‘target’ that it presents. As such, larger plants are presumably challenged by a greater number of spores than a smaller plant for the same level of predicted spore deposition. Secondly, plant size could be correlated with quantitative resistance, because leaf age can influence pustule development20. To achieve computational feasibility, we fit this model using the bam() function in the mgcv R package and the fast restricted maximum likelihood parameter estimation method (“fREML”). We again implemented variable selection so that insignificant predictor terms would be effectively removed from the models via the double penalty approach.To infer the effects of climate change and to illustrate the relationships between spore deposition, plant height, and infection risk, we predicted odds of infection using the estimated parameters of the fitted GAM (Fig. 5; Supplementary Fig. S14) and various weather data sets. In all of these projections, we included the random effect of site but excluded the random effect of plant identity.Epidemiological modelModel descriptionTo predict how the population level epidemiological dynamics of flax-rust might be affected in various climate change scenarios, we constructed a spatio-temporal epidemic model. This model tracks the infection status, infection intensity, and height of plants over time as infection spreads within the modeled population. There are three components required to simulate the model: (1) initial conditions describing the locations and starting states of all plants, (2) weather data, and (3) models of spore dispersal, transmission, infection intensity progression, and plant growth.The initial conditions for individual simulations of the epidemic model were in all cases constructed to represent either the BT or GM study populations on the date of an early epidemiological survey. For BT, this date was chosen as the first which had complete weather data (spanning 00:000:00 to 24:00:00), which was 6/24/2020. For GM, this date was set to 7/2/2020. Weather data was available for earlier dates, but a sharp increase in prevalence occurred during the prior week, perhaps due to prior missed observations of new infections. The model consistently underestimated observed prevalence when initialized prior to this increase in prevalence. We did not use the HM study population to initialize the model as no epidemiological surveys occurred on a date with complete weather data. Likewise, we do not use the CC study population to initialize the model because not all regions of the transect were mapped (see “Population mapping” sub-section in “Data collection” above).The construction of initial conditions to represent a given site proceeded as follows. The locations of all plants were taken from the ‘corrected plant height’ data set. Plants observed to be infected on the date of the first epidemiological survey were initialized as infected, and all other plants were initialized as uninfected. Following the first epidemiological survey, all infected plants (except for several that were approximately 5 cm or less in height) were designated diseased focal plants and their infection intensity was recorded. We used these measurements as the initial infection intensities of infected plants. We fore- or hind-casted infection intensities for any missing records using the method described in the transmission sub-section of the statistical analyses section above. Plant heights were taken from the ‘corrected plant height’ data set.The weather data component of the model is used to predict spore dispersal, transmission, infection intensity progression, and plant growth in conjunction with the fitted statistical models of these processes. As such, it needs to describe mean weather conditions over the period spanning a simulation step. Additionally, because the TGPM is calibrated to make predictions over 5 min intervals, the weather data must also describe average wind speed and direction over five minute windows.We used the fitted statistical model relating weather to the processes spore dispersal (TGPM), transmission, infection intensity progression, and plant growth to simulate these processes in the epidemiological model. These models contain random effects for both site and plant identity. The random effect of plant identity was excluded when making predictions about infection intensity progression and plant growth as random effects were only fit for focal plants. We included random effects of plant identity when making predictions using the transmission model because random effects were fit for all plants in the modeled populations.Using these components, the model simulates changes in infection status, infection intensity and plant height in time steps of 7 days. The simulation for each time step proceeds as follows:

(1)

First, the model predicts the number of spores deposited at the location of each healthy plant over the course of the entire time step. As all infected plants are assumed to be sources of spore deposition, the model predicts total spore deposition as the sum of spore deposition originating from each infected plant. The spore deposition originating from a single infected plant is calculated as the sum of predictions over 5 min windows spanning the time step. Individual predictions were generated using the TGPM with I is set to the infection intensity of the source plant at the beginning of the time step, H set to 0.5 times the height of the source plant at the beginning of the time step, s set to average wind speed, and X and Y set according to locations of the source and target plants and wind direction (see Supplementary Information). The parameters (k, {W}_{s},) and (sigma ) were set to the values fit to spore trap data.

(2)

Next, for each healthy plant, the model infers the probability of that plant becoming infected from predicted spore deposition at that plant’s location and mean weather metrics using the transmission model. Following this, a random number in [0,1] is drawn, and if it is equal or lower to the probability of infection that plant becomes infected. All newly infected plants are set to have an infection intensity of 1.0.

(3)

After simulating the transmission process, the model next simulates changes in infection intensity. For each plant, we simulated a prediction of the change in infection intensity per day by drawing randomly from the posterior of the infection intensity progression model. We used the infection intensity and height of that plant at the beginning of the time step along with mean weather metrics spanning the time step as the model inputs. We multiplied the predicted change in infection intensity per day by the length of the time step and added it to the plant’s infection intensity at the beginning of the time step to calculate the new infection intensity of the plant.

(4)

Finally, we used a similar process to simulate change in plant height. For each plant, we simulated a prediction of the change in height per day by drawing randomly from the posterior of the plant growth model. Again, we used the height of that plant at the beginning of the time step along with mean weather metrics spanning the time step as model inputs. To calculate the new height of the plant, we multiplied the predicted change in height per day by the length of the time step and added this number to the plant’s height at the beginning of the time step.

There are two sources of stochasticity within the epidemiological model. The first of these sources pertains to the probability of infection. Although the predicted probability of infection for a certain combination of predictor values is always the same, we compare this probability to a random number to determine if infection occurs. The second source of stochasticity pertains to predicted changes in infection intensity and plant height. We predict rates of infection intensity change and plant growth by drawing randomly from the posterior distributions of the corresponding fitted models. These sources of stochasticity allow us to understand the variation in epidemiological model predictions via repeated simulation.SimulationTo validate the epidemiological model, we simulated 10 epidemiological trajectories for the BT and GM sites and compared the results to our observations of flax-rust spread to ensure a qualitative match. For each of these sites, we initialized the model using data corresponding to the site as described above and used observed weather (from the same site) data to run the simulation. When extracting weather variables from observed weather data sets, we defined the observation period as 00:00:00 on the first day in a step to 00:00:00 on the last day in a step (e.g. noon on July 1st to noon on July 8th). We included the random effects of site and plant identity when making predictions from the transmission model, and the random effects of site when making predictions from the infection intensity and plant growth models.To infer the effects of climate change of flax-rust epidemiology, we again initialized the model using data corresponding to the for BT and GM sites, and then simulated 10 epidemiological trajectories for each set of weather data (RCP4.5 and 8.5 emissions scenarios and the years 2020, 2030, 2040, 2045, 2050, 2060 and 2070). We extracted weather metrics as described in the “Weather data” subsection of the “Data collection” section above. We used observed records of wind speed and direction to simulate spore deposition using the TGPM as the projected data sets lack these measures. As before we included the random effects of site and plant identity when making predictions from the transmission model, and the random effects of site when making predictions from the infection intensity and plant growth models. For each set of 10 model simulations corresponding to a unique site and weather data set combination, we used the same 10 random number seeds. More

The top-brand nonglutinous rice variety ‘Koshihikari’, which has a high palatability, is extremely susceptible to blast. Therefore, farmers apply fungicides over four times during the rice production season. As Koshihikari is sold by the Niigata brand, it has been traditionally viewed as having a high eating quality in Japan, and because of this, both farmers and consumers have requested that the multiline variety KO-BL be tested to determine if it is equivalent to Koshihikari before its introduction. Trials comparing Koshihikari and KO-BL were carried out in 2003 and 2004 in 594 and 622 fields covering 236 and 315 ha, respectively. These trials evaluated plant homogeneity, eating quality, and blast suppression using fewer fungicidal sprays. Following favorable results, in 2005, all Koshihikari were converted to KO-BL multiline variety covering an area of 94,000 ha. In addition, seed use and cultivation were restricted to the Niigata area to distinguish KO-BL from Koshihikari grown in other prefectures.Seed production and mixture processes are managed with precision by each prefectural official member (Fig. 1a). Original isogenic lines (ILs) were separately produced from the original stock in the original strain fields by the Niigata prefectural government. Using a precise mixture machine, the mixture of four ILs was then blended by weight in 2000 kg volumes, all multiplied by ten (giving a total volume of 20 t). Original production fields and commercial fields all used blended seeds that had been authorized by seed production farmers and commercial farmers in the 2003 and 2004 trials. Thus, it takes two years for seed production at the original strain field followed by the original production field for the preparation of commercial fields; thus, the seed mixture composition needs to be determined at least two years before introduction. Susceptible and resistant (effective) ILs were mixed at a ratio of 3:7 from 2005 to 2019 (Fig. 1b, Supplementary Table S1). Susceptible ILs, possessing Pia and Pii genes, were always mixed at a ratio of 1:2, but the composition of resistant ILs, containing Pita-2, Piz, Pib, Piz-t, and Pit genes, was changed every two to three years to avoid the breakdown of resistance6. These changes were determined by annually monitoring blast race distributions.Figure 1Representative seed production flow from original stock to commercial field and history of Koshihikari BL composition from 2005 to 2019 in Niigata Prefecture. (a) S1–S2, susceptible KO-BL; R1-R2, resistant KO-BL. Seeds obtained from original stock field at the Niigata Agricultural Research Institute. Seeds obtained from the original strain field and the original production field at both designated farmers’ fields. Commercial field (general farmers field) used for KO-BL production. Each field requires a year for seed production. (b) Pia and Pii, susceptible; Pita-2, Piz, Pib, Piz-t, and Pit, resistant. The proportion of susceptible KO-BLs and resistant KO-BLs was consistently 3:7 across years.Full size imageIn Niigata Prefecture, the predominant 5 blast races distributed from 1994 to 2004 were 001.0 (virulent to Koshihikari [Pik-s]), 003.0 (virulent to Pik-s and Pia), 005.0 (virulent to Pik-s and Pii), 007.0 (virulent to Pik-s, Pia, and Pii), and 037.1 (virulent to Pik-s, Pia, Pii, and Pik) (Fig. 2a, Supplementary Table S2). Because all the 5 races were virulent to Koshihikari, which had been widely cultivated in Niigata area during the years, there were no drastic race changes. In addition, genetic variations in blast resistance indicated that Koshihikari also harbored the Pish gene, and that the Pia, Pii, and Pik genes were also dominant in the Hokuriku region, including Niigata Prefecture21. Virulent blast races against the resistance genes Pish, Pia, Pii, Pi3, Pi5(t), Pik, Pik-s, and Pi19(t) were dominantly distributed in Niigata Prefecture22. These reports confirmed that Koshihikari had been susceptible to dominant blast races before KO-BL introduction.Figure 2Blast race change during the 1994–2019 period in Niigata Prefecture and the worst-case simulation of blast race dynamics in KO-BL during the 2005–2019 (years 1–15) period. Races and virulences are shown in Table 1. (a) A red line indicates the year (2005) when KO-BL was introduced. Races 007.0 and 037.1 became dominant after the introduction. (b) Actual races and their rates in 2004 and annual KO-BL compositions from 2005 to 2019 were set in the simulation. Parameters set in the simulation were as follows: maximum lesion number in a year, 10,000,000; weather condition, 10 (favorable); virulent mutation rate, 10–5; overwintering probability, 0.01; number of simulated years, 15; and number of simulation trials, 1000. The 1000 trial results for the lesion number increase in each race were averaged in each year and transformed into rates to show race dynamics. All simulation results are shown in Supplementary Table 6 in Supplementary information 2. The races 007.0 and 037.1 were also dominant until year 15 (correspond to 2019). Both actual and simulated race dynamics showed no outbreaks of the resistant composition of KO-BL.Full size imageIn the 2005 release year of KO-BL, the predominant blast races, 001.0 (virulent to Pik-s) and 003.0 (virulent to Pik-s and Pia), drastically decreased in distribution from 41.8% to 22.3% and 27.6% to 17.3%, respectively (Fig. 2a, Supplementary Table S2). Interestingly, races 001.0 and 003.0 rapidly decreased by 5.4% and 1.3% in 2006, respectively, even though especially Pia, which can be infected by the race 003.0, was used in the KO-BL composition. Because all ILs in the composition of KO-BL were resistant to race 001.0, and race 003.0 was only virulent to Pia, which made up 10% of the annual KO-BL composition (Table 1). In contrast, races 007.0 (virulent to Pik-s, Pia, and Pii) and 037.1 (virulent to Pik-s, Pia, Pii, and Pik) dominated from 2005 to 2019. The higher rate of race 007.0 detection was affected by 30% of the ILs composing the annual KO-BL were susceptible. The second highest rate of race 037.1 detection was affected by a number of factors: the high susceptibility of a minor cultivar that had Pii and Pik, the mosaic configuration of fields typical in Niigata, and the air-borne spread of race 037.1. To maintain consensus on KO-BL cultivation based on total blast suppression in Niigata, rarely detected races virulent to resistant ILs in commercial fields are strictly supervised by the prefectural government to avoid unnecessary confusion in Niigata residents.Table 1 Susceptible or resistant reaction of Koshihikari and KO-BL against blast races.Full size tableIn 2008, to mathematically support KO-BL composition changes, we developed a simulation software to estimate long-term blast race dynamics in multilines using a plant‒pathogen coevolution system23. The model calculated the persistence of resistant ILs to determine the optimal timing of changes to multiline variety compositions. To simulate race dynamics in KO-BL, we set five currently investigated races, 001.0 (virulent to Pik-s), 003.0 (virulent to Pik-s and Pia), 005.0 (virulent to Pik-s and Pii), 007.0 (virulent to Pik-s, Pia, and Pii), and 037.1 (virulent to Pik-s, Pia, Pii, and Pik), and their rates in 2004, as well as five emerging races, 043.0 (virulent to Pik-s, Pia, and Piz), 303.0 (virulent to Pik-s, Pia, and Pita-2), 003.2 (virulent to Pik-s, Pia, and Pib), 403.0 (virulent to Pik-s, Pia, and Piz-t), and 003.4 (virulent to Pik-s, Pia, and Pit) (see Fig. 2b, Supplementary Table S3) against five newly introduced respective resistant KO-BLs (see Fig. 1b, Supplementary Table S1) and the annual KO-BL compositions from 2005 to 2019. The worst case (severe epidemic) simulation result (Fig. 2b, Supplementary Tables S3 and S6) showed that race 007.0 (virulent to susceptible Pik-s, Pia and Pii) became the predominant race (77.4%), and race 037.1 (virulent to Pik-s, Pia, Pii, and Pik) remained at a low frequency (21.6%) until the fifteenth year (corresponding to 2019). In addition, super-race virulent to all KO-BLs did not emerge in this simulation. These suppression of outbreaks of newly emerged virulent races, including super-race on resistant KO-BL was apparently affected by 2–3 years of change in resistant KO-BL composition, and total suppression of blast occurrence decreasing the blast population. These results indicated that almost all the epidemics analyzed reflected actual race dynamics without affecting other minor races from other susceptible cultivars grown in Niigata, especially up to 2011. Thus, our decision support system provides an evaluation of KO-BL persistence and indicates the KO-BL composition changes needed for blast race population control in large areas. In addition, our simulation model may be useful for evaluating future KO-BL composition changes.Blast occurrence drastically decreased after 2005 (Fig. 3a, Supplementary Table S4). The average occurrence of leaf and panicle blast was 46.1% and 52.9% during the 1995–2004 period and 9.5% and 9.6% during the 2005–2019 period, respectively. This resulted in a blast suppression effect by 70% of the resistant composition in KO-BL. Current seed production fields are rarely contaminated with virulent races against resistant KO-BLs. This suggests that seed sanitation contributes to the suppression of virulent pathogen epidemics in multilines. In addition, induced resistance24,25 may have no effect on the practical use of multilines. Rice plants were found to induce a resistance response when inoculated with avirulent races of blast (those that stimulate protective responses to virulent race attacks). As the detection of several races in one area is rare and blast occurrence tends to be low, conditions that induce resistance in field situations do not occur. Fungicide applications to control blast in KO-BL and other minor cultivars decreased by approximately one-third during the 2005–2019 period compared with 2004 (Fig. 3b, Supplementary Table S5). Thus, the commercial scale use of crop diversity is clearly effective for the environmentally friendly control of airborne diseases.Figure 3Leaf and panicle blast occurrence from 1994 to 2019 and blast control area from 2004 to 2019 in Niigata Prefecture. (a) A red line indicates the year (2005) when KO-BL was introduced. (b) Gross fungicide spray area decreased by approximately one-third during the 2005–2019 period compared with 2004.Full size imageThe optimum long-term solution for pathogen population control using genetic diversity includes multilines. Blast occurrence in KO-BL introduced in Niigata, and the theoretical value of blast suppression in KO-BL tested at small scales, were reduced by approximately 10% compared to that of monoculture plots26,27,28. Thirty percent of susceptible ILs in KO-BL have the potential to improve compatible races with susceptible ILs and become predominant in large areas. This would contribute to the suppression of rapid increases in new virulent races emerging in the blast population. To maintain consensus on KO-BL cultivation based on total blast suppression in Niigata, rarely detected races virulent to resistant KO-BLs in commercial fields are strictly monitored by the prefectural government. Educating Niigata farmers ensures the long-term use of KO-BL. In fact, lower blast occurrence has been attributed to careful KO-BL cultivation and seed management.The implementation of genetically diversified homogeneous seed mixtures, rotations with resistant KO-BL, restricted KO-BL cultivation, and pathogen monitoring allowed rice quality to be maintained, diseases to be suppressed, and environmentally sound agriculture to be economically viable in Niigata. Collaboration among prefectural officers, farmers, and consumers in Niigata has resulted in safer rice production with good agricultural practices (GAPs) that meet sustainable development goals (SDGs). In addition, DNA tests differentiate KO-BL from the original Koshihikari for buyers, thereby prohibiting illegal distribution. Multiline varieties have been used in small areas in two different prefectures. For example, in Miyagi pref., Sasanishiki BL consisted of Pik, Pik-m, and Piz at ratios of 4:3:3 and 3:3:4 in 1995 and 1996, respectively. This composition was changed to Pik, Pik-m, Piz, and Piz-t at a ratio of 1:1:4:4 from 1997 to 2007 to prevent an increase in race 037.1 (virulent to the BL: Pik and Pik-m). In addition, an equal mixture of seven BLs (Pib, Pik, Pik-m, Piz, Piz-t, Pita, and Pita-2) was cultivated in 300 ha areas (maximum 4000 ha) from 2008 to 2014 without any outbreaks observed. In Toyama pref., the Koshihikari Toyama BL, which consists of resistant ILs, Pita-2, Pib, and Pik-p at a ratio of 4:4:2, was cultivated in an area of 300 ha and required a 50% reduction in chemical inputs from 2003 up to the present. Our model also calculated a greater than 50-year persistence in terms of the small area effect in both prefectural cases. This result depends on an insufficient pathogen population increase in virulent mutations against resistant ILs (data not shown). In this way, the practical use of a multiline provides control without the need for as much fungicide with or without a periodic change in IL composition. Our results demonstrate that the management of crop and pathogen coevolution can control diseases at large scales and, thereby, contribute to global food security. More

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Pseudocryptic diversityThe richness was the measure of the subterranean diversity, we surveyed all data about previous records for Brazilian Collembola cave species, ecological status, lithology, and distribution from the literature, and included 11 newly found pseudocryptic species from subterranean habitats in iron and limestone rock. The pseudocryptic species were verified by comparison of chaetotaxy and “micro-morphology” through optic and scanning microscopy of disjunct populations of a widespread morphotype. The imagery was compared under hypotheses of chaetotaxic and morphologic homology, previously defined by different authors. Those populations with consistent discrete chaetotaxic and morphologic patterns were assumed to be independent species, therefore they were taxonomically diagnosed, named, and ordered in a dichotomic identification key with all Brazilian species of the genus.MicroscopySpecimens were preserved in ethanol 70% and mounted on slides following Jordana et al.31, after clearing using Nesbitt’s solution for study under phase contrast microscope, line drawings were made with help of a drawing tube. For scanning electronic microscope (SEM) study, specimens were dehydrated by ethanol, dried in a critical point dryer, and covered in gold.HomologyThe terminology used in the diagnoses for the hypotheses of homology followed: labial chaetotaxy after Gisin32 with additions of Zhang and Pan33, Fjellberg34 for labial palp papillae and maxillary palp; postlabial chaetotaxy after Chen and Christiansen35, with adaptations of Cipola et al.36 for J series; clypeal chaetotaxy after Yoshii and Suhardjono37; labral chaetotaxy after Cipola et al.38; unguiculus lamellae after Hüther39; Anterior dens chaetotaxy after Oliveira et al.40; Mari-Mutt41 for dorsal head chaetotaxy, with additions of Soto-Adames42; Szeptycki43 and Zhang and Deharveng44 for S-chaetotaxy; and Szeptycki45 for dorsal chaetotaxy, with additions and modifications provided by Soto-Adames42 and Zhang et al.46. Symbols used to depict the chaetotaxy are presented in Fig. 4A–C. Codes will be used in italics along the text to replace the morphological description of each chaeta and sensillum type. Additional information about morphology and chaetotaxy of discussed species was obtained from the literature.Abbreviations used in the diagnosesAnt–antennal segment(s); b.c.–basal chaeta(e), t.a.–terminal appendage of the maxillary palp; l.p.–lateral process of labial papilla E, lpc–labial proximal chaeta(e); Th–thoracic segment; Abd–abdominal segment(s); Omt–trochanteral organ; a.e.–antero-external lamella, a.i.–antero-internal lamella, a.t.–unguis apical tooth, b.a.–basal anterior tooth of unguis, b.p.–basal posterior tooth of unguis, m.t.–unguis median tooth, p.i.–postero-internal lamella, p.e.–postero-external lamella; mac–macrochaeta(e), mes–mesochaeta(e), mic–microchaeta(e), ms–specialized microchaeta(e), psp–pseudopore(s), sens–specialized ordinary chaeta(e) (sensillum), MSS–Mesovoid Shallow Substratum.Ecological statusTo avoid subjectivity and ambiguity to determine the ecological status of the species, we assumed to be a troglobite all the species with some degree of troglomorphism exclusively distributed in the subterranean environment, either caves, MSS, or both. Species distributed in the surface and subterranean habitats were assumed to be troglophiles.Identification Key for the known and new species of the genus Trogolaphysa recorded in Brazil
Taxonomic diagnoses and morphological platesType materials are deposited in the Coleção de Referência de Fauna de Solo, Universidade Estadual da Paraíba (CRFS-UEPB) and Museu Nacional Rio de Janeiro, Universidade Federal do Rio de Janeiro (MNRJ-UFRJ).

Additional records in Supplementary Material S1, taxonomic references in S2.

Family Paronellidae Börner, 1906

Subfamily Paronellinae Börner, 1906

Tribe Paronellini sensu Zhang et al., 2019

Genus Trogolaphysa Mills, 1938

(Figs. 3, 4, 5, 6, 7, 8, 9, 10, 11)

Figure 3Trogolaphysa sp.: habitus lateral view. (A, B) specimen fixed in ethanol. (C, D) SEM photographs.Full size imageFigure 4Trogolaphysa sp. SEM: general body chaetae. (A) Antennal chaetae, sensilla and scales: 1—macrochaeta with short ciliation, 2—macrochaeta with long ciliation, 3—microchaeta with long ciliation, 4—microchaeta with short ciliation, 5—finger-shaped sens, 6—wrinkly sens, 7—coffee bean shaped sens, 8—rod sens, 9—spine-like sens, 10—Ant IV subapical-organ, 11—lanceolate scale, 12—rounded scales. (B) Head chaetae and scales: 1—strait macrochaeta with long ciliation, 2—blunt macrochaeta, 3—smooth chaeta, 4—blunt chaeta, 5—strait microchaeta with long ciliation, 6—labial r microchaeta, 7—cephalic anterior scale, 8—cephalic posterior scale. (C) Body and appendages chaetae, sens and scales: 1—bothriotrichum, 2—blunt macrochaeta, 3—blunt mesochaeta, 4—dens external ciliate chaeta, 5—smooth microchaeta, 6—blunt microchaeta, 7—fan-shape chaeta, 8—dental spine, 9—‘al’ sens, 10—‘ms’ sens, 11—lanceolate scale, 12—intersegmental scale.Full size imageFigure 5Trogolaphysa sp. SEM: antenna: (A) Ant IV dorsal view. (B) Ant IV apex dorsal view, arrow indicates finger-shaped and wrinkly sens. (C) Ant IV apex ventral view, left arrow indicates Ant IV subapical-organ, right arrow point one sensillum type A8. (D) Ant II dorsal view, dashed line indicates rod sens. (E) Detail of the sensilla of the Ant III apical organ (red). (F) Ant I dorsal view spine like sens (arrows indicate the sensilla in red). (G) Detail of the Ant I basal, arrow indicates psp and antenobasal organ (yellow and red respectively).Full size imageFigure 6Trogolaphysa sp. SEM: head and mouthpart chaetotaxy. (A) clypeus, (B) dorsal head, (C) eyes (red) circled by dashed line, arrow indicates antenobasal organ and psp, (D) ventral head, (E) maxillary palp and sublobal plate (right side), (F) detail of maxillary palp.Full size imageFigure 7Trogolaphysa sp. SEM: thorax and abdomen dorsal chaetotaxy: (A) Th II, (B) Th III, (C) Abd I-II, (D) Abd III.Full size imageFigure 8Trogolaphysa sp. SEM: (A) Abd IV dorsal chaetotaxy, (B) Abd V dorsal chaetotaxy, (C) anal pore and male genital papilla.Full size imageFigure 9Trogolaphysa. sp. SEM: empodial complex III (A) external lamella of unguis with external teeth (pseudonychia, yellow), (B) unguis and unguiculus lateral view, unguis internal lamella with basal, medial and apical teeth (blue, red and yellow respectively), unguiculus with internal and external teeth, tenent hair capitate (white arrow), (C) lateral view, unguiculus lamellae, tenent hair acuminate (white arrow).Full size imageFigure 10Trogolaphysa sp. SEM: appendages (A) Metatrochanteral organ with pseudopores (alveoli marked in yellow, white arrows indicate pseudopores), (B) ventral tube posterior chaetae, (C) ventral tube anterior chaetae, (D) Tenaculum.Full size imageFigure 11Trogolaphysa sp. SEM: furca. (A) manubrial plate pseudopores (yellow), (B) antero-proximal chaetae of dens, (C) dens anterior view, (D) mucro.Full size imageDiagnosisHabitus typical of this genus (Fig. 3A–D), hyaline scales presents on Ant. I–II, head, body, and ventral face of furcula (Figs. 3C–D, 4A–C, 5D, F, 7, 8, 11C), Ant IV smooth or annulated and never subdivided in two (Fig. 5A); eyes 0–8 (ex. Fig. 6C); prelabral and labral formula 4/5,5,4 (prelabral smooth or ciliate, pma smooth chaetae) (Fig. 6A); antennobasal-organ present (Fig. 6C); labial chaetae L1–2 not reduced (Fig. 6E); sublobal plate of maxillary palp with 2 chaetae (Fig. 6E); Th II normally with a5 mac and p3 complex with variable number of mac, and Th III with p3 mac present or abset (Fig. 7A, B), abdominal segments II–IV with 2, 3, 3 bothriotricha (Figs. 7C, D, 8A); unguis with three external lamellae and unguiculus with p.e. lamella serrate or smooth (Fig. 9A–C); trochanteral organ with 2–4 psp (Fig. 10A) collophore anterior side with 2–3 distal mac (Fig. 10C); tenaculum with four teeth on each branch and one anterior chaeta (Fig. 10D); manubrium without spines, manubrial plate with 2–3 psp (Fig. 11A); anterior proximal dens with b.a., b.m. and i5 chaetae (Fig. 11B); dens with 1–2 rows of spines; mucro square or rectangular but relatively short, with 3–5 teeth (Fig. 11D).Trogolaphysa bellinii sp. nov. Oliveira, Lima & ZeppeliniFigures 12, 13 and 14, Tables 1 and 2Figure 12Trogolaphysa bellinii sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore; Gray cut circle pseudopore at the under surface.Full size imageFigure 13Trogolaphysa bellinii sp. nov.: Dorsal chaetotaxy: (A) Th II–III, (B) Abd I–III, (C) Abd IV–V.Full size imageFigure 14Trogolaphysa bellinii sp. nov.: (A) Trochanteral organ, (B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy.Full size imageTable 2 Trogolaphysa species of the Neotropical Region, comparative morphology.Full size tableType material. Holotype female in slide (15,482/CRFS-UEPB): Brazil, Minas Gerais State, Barão de Cocais municipality, cave MDIR-0028, next to “Mina de Brucutu”, 19°52′48.7″S, 43°26′13.6″W, 19–23.viii.2019, Carste team coll. Paratypes in slides (15,468, 15,483/CRFS-UEPB): 2 females, same data as holotype. Paratypes in slides (15,519, 15,576/CRFS-UEPB donated to MNJR): 2 females, same data as holotype. Additional records see S1.Description. Total length (head + trunk) of specimens 1.53–1.75 mm (n = 5), holotype 1.70 mm.Head. Ratio antennae: trunk = 1: 1.29–1.95 (n = 5), holotype = 1: 1.95; Ant III shorter than Ant II; Ant segments ratio as I: II, III, IV = 1: 1.80–2.24, 0.85–2.08, 0.85–2.08, holotype = 1: 1.80, 0.85, 1.34. Antennal chaetotaxy: Ant IV dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally with a longitudinal row with about eight rod sens, ventrally with one subapical-organ and several wrinkly sens (Fig. 4A); Ant III dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, about three wrinkly sens on external longitudinal row, apical organ with two mic smooth chaetae externally, two coffee bean-like sens, and one rod sens (Fig. 4A); Ant II dorsally and ventrally with several short ciliate mic and mac, dorsally with four sub-apical finger-shaped sens, one wrinkly sens and two subapical rod sens, ventrally with one apical psp, about six wrinkly sens on longitudinal external row (Fig. 4A); and Ant I dorsally and ventrally with several short ciliate mic and mac, dorsally with three basal spine-like sens, ventrally with four basal spine-like sens, about five smooth mic and several finger-shaped sens (Fig. 4A). Eyes 0 + 0, rarely 2 + 2. Head dorsal chaetotaxy (Fig. 12A) with 12 An (An1a–3), six A (A0–5), five M (M1–5), five S (S2–6), two Ps (Ps2, Ps5), four Pa (Pa1–5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; Pa5 and Pm3 as mes, An1a–3a with 10 mac plus two mes, A0 and A2 as mac; interocular p mes present. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 12B). Ventral chaetotaxy with 35–38 ciliate chaetae and one reduced lateral spine; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of four to seven mes chaetae distally (Fig. 12B). Prelabral chaetae ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and surpassing the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–3) and subequal in length (Fig. 12B). Maxillary palp with t.a. smooth and 1.23× larger than b.c.Thorax dorsal chaetotaxy (Fig. 13A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with three mac, respectively, al and ms present. Th III a, m, p series with three mic (a1–3), two mes (a6–7), three mic (m4, m6–6p), three mes (m6e, m7–7e), four mic (p1–3, p6) respectively. Ratio Th II: III = 1.04–1.36: 1 (n = 5), holotype = 1.05: 1.Abdomen dorsal chaetotaxy (Fig. 13B, C). Abd I a, m series with one (a5) and six (m2–6e) mic respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el mic and as present; a5 and m2 bothriotricha surrounded by five and four fan-shaped chaetae respectively. Abd III a, m, p series with one mic (a7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6), three mic (p6e, p7i–7), one mac (p6) chaetae respectively; a5, m2 and m5 bothriotricha with six, two and three fan-shaped chaetae respectively, as sens elongated, ms present. Abd IV A–Fe series with two mic (A1, A6), two mac (A3, A5), one mic (B1), one mes (B6), two mac (B4–5), four mic (C1–4), three mic (T1, T5–6), one mes (T7), five mic (D1–3, De3), one mes (D3p), one mic (E4p2), one mes (E4p), three mac (E1–3), one mic (Ee12), two mes (Ee10–11), one mac (Ee9), one mic (F1), two mes (F3, F3p), one mac (F2), one mic (Fe2), three mes (Fe3–5) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by five and two (T3) fan-shaped chaetae respectively; ps and as present, and at least six supernumerary sens with uncertain homology ‘s’ (Fig. 8A); Abd. IV posteriorly with four psp. Abd V a, m, p series with two mic (a1, a3), one mes (a6), one mac (a5), two mes (m5a, m5e), three mac (m2–3, m5), five mic (p3a–6ae), one mic (p6e) two mes (ap6–pp6), four mac (p1, p3–5) chaetae, respectively; as, acc.p4–5 present. Ratio Abd III: IV = 1: 3.70–4.37 (n = 5), holotype = 1: 4.37.Legs. Trochanteral organ diamond shape with about 20 spine-like chaetae, plus two psp one external and one on distal vertex of Omt (Fig. 14A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with four teeth, basal pair subequal, b.p. not reaching the m.t. apex, m.t. just after the distal half, a.t. present. Unguiculus with lamellae smooth and lanceolate (a.i., a.e., p.i.), except p.e. slightly serrate (Fig. 14B); ratio unguis: unguiculus = 1.56–1.79: 1 (n = 5), holotype = 1.56: 1. Tibiotarsal smooth chaetae about 0.9 × smaller than unguiculus; tenent hair capitate and about 0.55 × smaller than unguis outer lamella.Collophore (Fig. 14C). Anterior side with 12 ciliate, apically acuminate chaetae, five proximal, four subdistal (as mes) and three distal mac; lateral flap with 11 chaetae, five ciliate in the proximal row and six smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 14D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 24 external and 25 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.29 (holotype).Etymology. Species named after Dr. Bruno C. Bellini in recognition of his work on Brazilian Collembola.Remarks. Trogolaphysa bellinii sp. nov. resembles T. bessoni, T. epitychia sp. nov., and T. mariecurieae sp. nov. by 0 + 0 eyes (T. bellinii sp. nov. rarely with 2 + 2 eyes), Th II with 3 + 3 mac, and Th III without mac, but can be distinguished by presenting Abd IV with 4 + 4 central mac (A3, A5, B4–5); T. epitychia sp. nov. with 3 + 3 central mac on Abd IV, T. mariecurieae sp. nov. with 2 + 2 central mac on Abd IV.Trogolaphysa lacerta sp. nov. Lima, Oliveira & ZeppeliniFigures 15, 16 and 17, Tables 1 and 2Figure 15Trogolaphysa lacerta sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore; Gray cut circle pseudopore at the under surface.Full size imageFigure 16Trogolaphysa lacerta sp. nov.: Dorsal chaetotaxy. (A) Th II–III, (B) Abd I–III, (C) Abd IV–V.Full size imageFigure 17Trogolaphysa lacerta sp. nov.: (A) Trochanteral organ, (B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy.Full size imageType material. Holotype male in slide (10,311/CRFS-UEPB): Brazil, Minas Gerais State, Conceição do Rio Acima municipality, cave GAND-115, next to “Lapa do Calango”, 20°04′08.4″S, 43°40′09.9″W, 10.ii–20.iii.2014, Carste team coll. Paratypes in slides (10,312, 10,309/CRFS-UEPB): 2 males, same data as holotype. Paratypes in slides (10,313, 10,314/CRFS-UEPB donated to MNJR): 2 females, same data as holotype. Additional records see S1.Description. Total length (head + trunk) of specimens 1.31–2.43 mm (n = 5), holotype 1.86 mm.Head. Ratio antennae: trunk = 1: 1.33–1.46 (n = 2), holotype = 1: 1.46; Ant III shorter than Ant II; Ant segments ratio, I: II, III, IV = 1: 1.78–2.05: 1.5–1.64: 2.64–2.83, holotype = 1: 1.80: 1.64: 2.64. Antennal chaetotaxy (no represented): Ant IV dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally with a longitudinal row with about five rod sens, ventrally with one subapical-organ and several wrinkly sens (Fig. 4A); Ant III dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, one apical wrinkly sens on, apical organ with two coffee bean-like sens, and one rod sens (Fig. 4A); Ant II dorsally and ventrally with several short ciliate mic and mac, dorsally with three sub-apical finger-shaped sens, one wrinkly sens and two apical rod sens, ventrally with one apical psp, one longitudinal external row with two subapical wrinkly sens and two medial finger-shaped sens (Fig. 4A); and Ant I dorsally and ventrally with several short ciliate mic and mac, dorsally with three basal spine-like sens, ventrally with four basal spine-like sens, about five smooth mic and several finger-shaped sens (Fig. 4A). Eyes 0 + 0, rarely 3 + 3. Head dorsal chaetotaxy (Fig. 15A) with 15 An (An1a–3), six A (A0–5), four M (M1–4), five S (S2–6), two Ps (Ps2, Ps5), four Pa (Pa1–2, Pa4–5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; Pm3, Pa5 and Pp7 as mes, An1a–3a with 11 mac plus four meso, A0 and A2 as mac; interocular p mes present. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 15B). Ventral chaetotaxy with 36–38 ciliate chaetae and 1 reduced lateral spine; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of three to five mes chaetae distally (Fig. 15B). Prelabral chaetae ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and surpassing the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–3) and subequal in length (Fig. 15B). Maxillary palp with t.a. smooth and 1.28× larger than t.a.Thorax dorsal chaetotaxy (Fig. 16A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with six mac, respectively, al and ms present. Th III a, m, p series with three mic (a1–3), two mes (a6–7), three mic (m4, m6–6p), three mes (m6e, m7–7e), four mic (p1–3, p6) respectively. Ratio Th II: III = 1.09–1.46: 1 (n = 5), holotype = 1.09: 1.Abdomen dorsal chaetotaxy (Fig. 16B, C). Abd I m series with six (m2–6e) mic respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el mic and as present; a5 and m2 bothriotricha surrounded by four and two fan-shaped chaetae respectively. Abd III a, m, p series with one mic (a7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6), four mic (p6e, p7i–7p), one mac (p6) chaetae respectively; a5, m2 and m5 bothriotricha with seven, two and four fan-shaped chaetae respectively, as sens elongated, ms present. Abd IV A–Fe series with four mic (A1, A5–6, Ae1), one mac (A3), one mic (B1), one mes (B6), two mac (B4–5), four mic (C1–4), five mic (T1, T3, T5–7), five mic (D1–3, De3), one mes (D3p), one mic (E4p2), one mes (E4p), three mac (E1–3), one mic (Ee12), two mes (Ee10–11), one mac (Ee9), one mic (F1), two mes (F3–3p), one mac (F2), one mic (Fe2), three mes (Fe3–5) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by four and one fan-shaped chaetae respectively; ps and as present, and at least six supernumerary sens with uncertain homology ‘s’(Fig. 8A); Abd. IV posteriorly with five to six psp. Abd V a, m, p series with two mic (a1, a3), one mes (a6), one mac (a5), two mes (m5a, m5e), three mac (m2–3, m5), five mic (p3a–6ae), one mic (p6e) two mes (ap6–pp6), four mac (p1, p3–5) chaetae, respectively; as, acc.p4–5 present. Ratio Abd III: IV = 1: 3.70–4.37 (n = 5), holotype = 1: 4.37.Legs. Trochanteral organ diamond shape with about 24 spine-like chaetae, plus two psp one external and one on distal vertex of Omt (Fig. 17A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with four teeth, basal pair subequal, b.p. not reaching the m.t. apex, m.t. just after the distal half, a.t. present. Unguiculus with all lamellae smooth and lanceolate (a.i., a.e., p.i., p.e.) (Fig. 17B); ratio unguis: unguiculus = 1: 1.50–1.79 (n = 5), holotype = 1: 1.75. Tibiotarsal smooth chaetae about 0.7× smaller than unguiculus; tenent hair slightly acuminate and about 0.44× smaller than unguis outer lamella.Collophore (Fig. 17C). Anterior side with 10 ciliate, apically acuminate chaetae, five proximal (thinner); three subdistal and two distal mac; lateral flap with 11 chaetae, five ciliate in the proximal row and six smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 17D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 50 external and 37 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.31 (n = 5).Etymology. Lacerta from Latin means lizard, in allusion to the name of the cave where this species was found, Lapa do Calango (cave of the Calango), which is a small lizard common in this region.Remarks. Trogolaphysa lacerta sp. nov. The new species resembles T. caripensis, T. ernesti, T. piracurucaensis, T. formosensis and T. dandarae sp. nov. by the number of mac in Th II p3 complex (6 + 6), but is easily distinguished by the head m2 and s5 mic (T. caripensis, T. ernesti, T. formosensis, T. piracurucaensis as mac) and Th III without mac (T. dandarae sp. nov. 3 + 3).Trogolaphysa chapelensis sp. nov. Lima, Oliveira & ZeppeliniFigures 18, 19 and 20, Tables 1 and 2Figure 18Trogolaphysa chapelensis sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore; Gray cut circle pseudopore at the under surface.Full size imageFigure 19Trogolaphysa chapelensis sp. nov.: Dorsal chaetotaxy. (A) Th II–III, (B) Abd I–III, (C) Abd IV–V.Full size imageFigure 20Trogolaphysa chapelensis sp. nov.: (A) Trochanteral organ, (B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy.Full size imageType material. Holotype female in slide (4550/CRFS-UEPB): Brazil, Minas Gerais State, Rio Acima municipality, cave Gruta-2d7, next to “Morro do Chapéu” 20°07′42.1″S, 43°54′26.2″W, 02–10.viii.2011, Andrade et al. coll. Paratypes in slides (4551–4553/CRFS-UEPB): 3 females, Brazil, Minas Gerais State, Rio Acima municipality, cave Gruta-7d7, Qd7, 9d7 respectively, 20°07′42.1″S, 43°54′26.7″W, 29.iii–01.vi.2011, Andrade et al. coll. Paratype in slide (4603/CRFS-UEPB donated to MNJR): 1 female, Brazil, Minas Gerais State, Rio Acima municipality, cave Gruta Qd7, 20°09′46.1″S, 43°49′36.2″W, 925 m, 29.iii–01.vi.2011, Andrade et al. Coll. Additional records see S1.Description. Total length (head + trunk) 1.21–2.22 mm (n = 5), holotype 2.22 mm.Head. Ratio antennae: trunk = 1: 1.31–1.16 (n = 3), holotype = 1: 1.16; Ant III shorter than Ant II; Ant segments ratio, I: II, III, IV = 1: 1.66–1.85, 1.65–1.78, 2.95–3.76, holotype = 1: 1.66, 1.65, 2.95. Antennal chaetotaxy (no represented): Ant IV dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally with about six rod sens on longitudinal row, ventrally with one subapical-organ and about three subapical wrinkly sens (Fig. 4A); Ant III dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, one apical wrinkly sens, apical organ with two coffee bean-like sens, and one rod sens (Fig. 4A); Ant II dorsally and ventrally with several short ciliate mic and mac, dorsally with about three sub-apical finger-shaped sens and about three apical rod sens, ventrally with one apical psp, one longitudinal external row with four wrinkly sens (Fig. 4A); and Ant I dorsally and ventrally with several short ciliate mic and mac, dorsally with three basal spine-like sens, ventrally with four basal spine-like sens, about three smooth mic and several finger-shaped sens (Fig. 4A). Eyes 0 + 0. Head dorsal chaetotaxy (Fig. 18A) with 15 An (An1a–3), six A (A0–5), four M (M1–4), five S (S2–6), two Ps (Ps2, Ps5), four Pa (Pa1–5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; Pm3 and Pa5 as mes, An1a–3a with 13 mac plus two mes, A0 and A2 as mac; interocular p mic present. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 18B). Ventral chaetotaxy with 29 ciliate chaetae; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of six mes chaetae distally (Fig. 18B). Prelabral chaetae ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and surpassing the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–3) and subequal in length (Fig. 18B). Maxillary palp with t.a. smooth and 1.17× larger than b.c.Thorax dorsal chaetotaxy (Fig. 19A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with four mac, respectively, al and ms present. Th III a, m, p series with three mic (a1–3), two mes (a6–7), two mic (m4–6p), four mes (m6–6e, m7–7e), four mic (p1–3, p6) respectively. Ratio Th II: III = 1.10–1.31: 1 (n = 4), holotype = 1.10: 1.Abdomen dorsal chaetotaxy (Fig. 19B, C). Abd I a, m series with one (a5) and six (m2–6e) mic respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el mic and as present; a5 and m2 bothriotricha surrounded by five and four fan-shaped chaetae respectively. Abd III a, m, p series with one mic (a7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6), three mic (p6e, p7i–7), one mac (p6) chaetae respectively; a5, m2 and m5 bothriotricha with six, two and three fan-shaped chaetae respectively, as sens elongated, ms present. Abd IV A–Fe series with three mic (A1, A6, Ae1), two mac (A3, A5), one mic (B1), one mes (B6), two mac (B4–5), four mic (C1–4), three mic (T1, T5–6), one mes (T7), five mic (D1–3, De3), one mes (D3p), one mic (E4p2), one mes (E4p), three mac (E1–3), one mic (Ee12), two mes (Ee10–11), one mac (Ee9), one mic (F1), two mes (F3–3p), one mac (F2), one mic (Fe2), three mes (Fe3–5) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by four and two (T3) fan-shaped chaetae respectively; ps and as present, and at least six supernumerary sens with uncertain homology ‘s’ (Fig. 8A); Abd. IV posteriorly with nine psp. Abd V a, m, p series with two mic (a1, a3), one mes (a6), one mac (a5), two mes (m5a, m5e), three mac (m2–3, m5), five mic (p3a–6ae), one mic (p6e) two mes (ap6–pp6), four mac (p1, p3–5) chaetae, respectively; as, acc.p4–5 present. Ratio Abd III: IV = 1: 3.46–5.80 (n = 5), holotype = 1: 5.80.Legs. Trochanteral organ diamond shape with about 23 spine-like chaetae, plus two psp one external and one on distal vertex of Omt (Fig. 20A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with four teeth, basal pair subequal, b.p. not reaching the m.t. apex, m.t. just after the distal half, a.t. present. Unguiculus with lamellae smooth and lanceolate (a.i., a.e., p.i.), except p.e. slightly serrate (Fig. 20B); ratio unguis: unguiculus = 1: 1.63–1.84 (n = 5), holotype = 1: 1.79. Tibiotarsal smooth chaetae about 0.8× smaller than unguiculus; tenent hair capitate and about 0.52× smaller unguis outer lamella.Collophore (Fig. 20C). Anterior side with 13 ciliate, apically acuminate chaetae, seven proximal (thinner); four subdistal and two distal mac; lateral flap with 11 chaetae, five ciliate in the proximal row and six smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 20D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 70 external and 30 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.33 (n = 5).Etymology. Species named after Type locality Morro do Chapeu.Remarks. Trogolaphysa chapelensis sp. nov. resembles T. jacobyi, T. caripensis, T. bessoni, and T. belizeana by te absence of eyes (0 + 0 eyes) but is easily distinguished by presenting 4 + 4 mac in Th II p3 complex (2–3 + 2–3 T. jacobyi; 6 + 6 T. caripensis; 2 + 2 T. bessoni; 2–4 + 2–4 T. belizeana), and 9 + 9 psp posterior Abd IV (4 + 4T. belizeana).Trogolaphysa crystallensis sp. nov. Oliveira, Lima & ZeppeliniFigures 21, 22 and 23, Tables 1 and 2Figure 21Trogolaphysa crystallensis sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore; Gray cut circle pseudopore at the under surface.Full size imageFigure 22Trogolaphysa crystallensis sp. nov.: Dorsal chaetotaxy. (A) Th II–III, (B) Abd I–III; (C) Abd IV–V.Full size imageFigure 23Trogolaphysa crystallensis sp. nov.: (A) Trochanteral organ, (B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy.Full size imageType material. Holotype female in slide (16,252/CRFS-UEPB): Brazil, Minas Gerais State, Mariana municipality, cave LOC-0090, next to “Cachoeira Crystal”, 20°20′20.8″S, 43°23′44.3″W, 11–14.xi.2019, Carste team coll. Paratype in slide (16,251/CRFS-UEPB): female, same data as holotype. Paratype in slide (16,254/CRFS-UEPB donated to MNJR): female, same data as holotype. Additional records see S1.Description. Total length (head + trunk) of specimens 1.40–1.68 mm (n = 3), holotype 1.68 mm.Head. Ratio antennae: trunk = 1: 1.24–2.30 (n = 2), holotype = 1: 1.24; Ant III shorter than Ant II length; Ant segments ratio as I: II, III, IV = 1: 1.72–1.78, 1.58–1.64, 3.11–3.14, holotype = 1: 1.78, 1.64, 3.14. Antennal chaetotaxy (no represented): Ant IV dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally with about three rod sens on longitudinal row, ventrally with one subapical-organ and several wrinkly sens (Fig. 4A); Ant III dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, about three wrinkly sens on external longitudinal row, apical organ with two rod sens, and one finger-shaped sens (Fig. 4A); Ant II dorsally and ventrally with several short ciliate mic and mac, dorsally with three sub-apical finger-shaped sens and one wrinkly sens, ventrally with one apical psp (Fig. 4A); and Ant I dorsally and ventrally with several short ciliate mic and mac, dorsally with three basal spine-like sens, ventrally with four basal spine-like sens, about three smooth mic and several finger-shaped sens (Fig. 4A). Eyes 0 + 0. Head dorsal chaetotaxy (Fig. 21A) with 12–13 An (An1a–3), six A (A0–5), four M (M1–4), five S (S2–6), two Ps (Ps2, Ps5), four Pa (Pa1–5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; Pa5, Pm3 and Pp7 as mes, An1a–3a, A0 and A2 as mac; interocular p mes present. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 21B). Ventral chaetotaxy with 33–35 ciliate chaetae and one reduced lateral spine; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of four to six mes chaetae distally (Fig. 21B). Prelabral chaetae ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and surpassing the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–3) and subequal in length (Fig. 21B). Maxillary palp with t.a. smooth and 1.43 × larger than b.c.Thorax dorsal chaetotaxy (Fig. 22A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with five mac, respectively, al and ms present. Th III a, m, p series with two mic (a1–2), two mes (a6–7), theree mic (m4, m6–6p), three mes (m6e, m7–7e), four mic (p1–3, p6) respectively. Ratio Th II: III = 1.05–1.27: 1 (n = 3), holotype = 1.05: 1.Abdomen dorsal chaetotaxy (Fig. 22B, C). Abd I a, m series with one (a5) and six (m2–6e) mic respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el mic and as present; a5 and m2 bothriotricha surrounded by four and two fan-shaped chaetae respectively. Abd III a, m, p series with one mic (a7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6), three mic (p6e, p7i–7), one mac (p6) chaetae respectively, a5, m2 and m5 bothriotricha with six, two and three fan-shaped chaetae respectively, as sens elongated, ms present. Abd IV A–Fe series with three mic (A1, A6, Ae1), two mac (A3, A5), one mic (B1), one mes (B6), two mac (B4–5), four mic (C1–4), three mic (T1, T5–7), five mic (D1–1p, D3–3p, De3), one mes (D2), two mes (E4p–4p2), three mac (E1–3), four mes (Ee9–12), one mic (F1), three mes (F2–3p), one mic (Fe2), three mes (Fe3–5) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by three and two (T3) fan-shaped chaetae respectively; ps and as present, and at least 14 supernumerary sens with uncertain homology ‘s’(Fig. 8A); Abd. IV posteriorly with three psp. Abd V a, m, p series with two mic (a1, a3), one mes (a6), one mac (a5), two mes (m5a, m5e), three mac (m2–3, m5), five mic (p3a–P6ae), three mes (p6e–pp6), four mac (p1, p3–5) chaetae, respectively; as and acc.p4–5 present. Ratio Abd III: IV = 1: 4.06–4.51 (n = 3), holotype = 1: 4.51.Legs. Trochanteral organ diamond shape with about 18 spine-like chaetae, plus two psp one external and one on distal vertex of Omt (Fig. 23A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with three teeth, basal pair subequal, b.p. little larger, but not reaching the m.t. apex, m.t. just after the distal half, a.t. absent. Unguiculus with all lamellae smooth and lanceolate (a.i., a.e., p.i., p.e.) (Fig. 23B); ratio unguis: unguiculus = 1.48–1.79: 1 (n = 3), holotype = 1.48: 1. Tibiotarsal smooth chaetae about 0.8× smaller than unguiculus; tenent hair acuminate and about 0.5× smaller than unguis outer lamella.Collophore (Fig. 23C). Anterior side with 10 ciliate, apically acuminate chaetae, six proximal, two subdistal (as mes) and two distal mac; lateral flap with 11 chaetae, five ciliate in the proximal row and six smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 23D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 60 external and 28 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.31 (holotype).Etymology. Species named after Type locality Cachoeira Crystal (Portuguese for Crystal falls).Remarks. Trogolaphysa crystallensis sp. nov. resembles T. barroca sp. nov., T. gisbertae sp. nov., T. sotoadamesi sp. nov., T. triocelata and T. zampauloi sp. nov. by the absence of eyes (0 + 0 eyes) (T. triocelata 3 + 3 and T. zampauloi sp. nov. eventually 4 + 4), Th II with 5 + 5 mac, and Th III without mac. Can be distinguished from T. barroca sp. nov., T. gisbertae sp. nov., and T. sotoadamesi sp. nov. by the Abd IV with 4 + 4 central mac (A3, A5, B4–5); T. barroca sp. nov., T. gisbertae sp. nov., and T. triocelata, with 3 + 3 and T. sotoadamesi sp. nov. 2 + 2 central mac on Abd IV. Finally, the new species differentiates from T. zampauloi sp. nov. by unpaired lamella of unguis with one tooth, Omt with about 18 spine-like chaetae, dens external row with about 58 spines on T. crystallensis sp. nov. and unpaired lamella of unguis with two teeth, Omt with about 26 spine-like chaetae, dens external row with about 30 spines on T. zampauloi sp. nov.Trogolaphysa sotoadamesi sp. nov. Ferreira, Oliveira & ZeppeliniFigures 24, 25 and 26, Tables 1 and 2Figure 24Trogolaphysa sotoadamesi sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore, Gray cut circle pseudopore at the under surface.Full size imageFigure 25Trogolaphysa sotoadamesi sp. nov.: Dorsal chaetotaxy. (A) Th II–III, (B) Abd I–III, (C) Abd IV–V.Full size imageFigure 26Trogolaphysa sotoadamesi sp. nov.: (A) Trochanteral organ, ((B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy.Full size imageType material. Holotype male in slide (13,162/CRFS-UEPB): Brazil, Minas Gerais State, Mariana municipality, cave ALEA 0003, next to “Mina de Alegria”, 20°09′6.81″S, 43°29′13.6″W, 07.ii.2018, Bioespeloeo team coll. Paratypes in slides (13,146, 13,153/CRFS-UEPB): 2 females, same data as holotype, except 12.vi.2017. Paratype in slide (13,173, 13,186/CRFS-UEPB donated to MNJR): 2 females, same data as holotype, except 09.vi.2017. Additional records see S1.Description. Total length (head + trunk) of specimens1.50–1.81 mm (n = 5), holotype 1.50 mm.Head. Ratio antennae: trunk = 1: 1.26–1.45 (n = 3), holotype = 1: 1.38; Ant III shorter than Ant II; Ant segments ratio, I: II, III, IV = 1: 1.78–2.76, 1.52–2.22, 2.61–3.90, holotype = 1: 2.04, 1.68, 3.16. Antennal chaetotaxy (no represented): Ant IV dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally with a longitudinal row with about three rod sens, ventrally with one subapical-organ and with several wrinkly sens (Fig. 4A); Ant III dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, several wrinkly sens, apical organ with two coffee bean-like sens, one rod sens and one finger-shaped sens (Fig. 4A); Ant II dorsally and ventrally with several short ciliate mic and mac, dorsally with two sub-apical rod sens and two finger-shaped sens, ventrally with one apical psp and several finger-shaped sens (Fig. 4A); and Ant I dorsally and ventrally with several short ciliate mic and mac, dorsally with three basal spine-like sens, ventrally with about seven basal spine-like sens, about three smooth mic and several finger-shaped sens (Fig. 3A). Eyes 0 + 0. Head dorsal chaetotaxy (Fig. 24A) with 16 An (An1a–3), six A (A0–5), four M (M1–4), five S (S2–6), two Ps (Ps2, Ps5), four Pa (Pa1–5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; Pm3 as mes (rarely mic), Pa5 as mes, An1a–3a, A0 and A2 as mac; interocular p mes present. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 24B). Ventral chaetotaxy with 37 ciliate chaetae and one reduced lateral spine; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of six mes chaetae distally (Fig. 24B). Prelabral chaetae ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and surpassing the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–p3) and subequal in length (Fig. 24B). Maxillary palp with t.a. smooth and 1.28× larger than b.c.Thorax dorsal chaetotaxy (Fig. 25A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with five mac, respectively, al and ms present. Th III a, m, p series with three mic (a1–3, a6), one mes (a7), four mic (m4, m6–7, m6p), two mes (m6e, m7e), four mic (p1–3, p6) respectively. Ratio Th II: III = 1.17–1.52: 1 (n = 5), holotype = 1.03: 1.Abdomen dorsal chaetotaxy (Fig. 25B, C). Abd I a, m series with one (a5) and six (m2–6e) mic respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el mic and as present; a5 and m2 bothriotricha surrounded by five and three fan-shaped chaetae respectively. Abd III a, m, p series with one mic (a7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6), three mic (p6e, p7i–7), one mac (p6) chaetae respectively; a5, m2 and m5 bothriotricha with five, two and three fan-shaped chaetae respectively, as sens elongated, ms present. Abd IV A–Fe series with five mic (A1, A3, A5–6, Ae1), one mic (B1), one mes (B6), two mac (B4–5), four mic (C1–4), two mic (T1, T6), two mes (T5, T7), three mic (D1–2), two mes (D3p, De3), two mes (E4p–p2), three mac (E1–3), one mic (Ee12), three mes (Ee9–11), one mic (F1), two mes (F3–3p), one mac (F2), one mic (Fe2), three mes (Fe3–5) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by four and three (T3) fan-shaped chaetae respectively; ps and as present, and at least five supernumerary sens with uncertain homology ‘s’ (Fig. 8A); Abd. IV posteriorly with four psp. Abd V a, m, p series with two mic (a1, a3), one mes (a6), one mac (a5), two mes (m5a, m5e), three mac (m2–3, m5), five mic (p3a–p6ae), one mic (P6e) two mes (ap6–pp6), four mac (p1, p3–5) chaetae, respectively; as, acc.p4–5 present. Ratio Abd III: IV = 1: 5.03–4.42 (n = 5), holotype = 1: 4.42.Legs. Trochanteral organ triangular shape with about 19–21 spine-like chaetae, plus two psp one external and one on distal vertex of Omt (Fig. 26A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with two teeth, basal pair unequal, b.p. larger than b.a.; m.t. and a.t. absent. Unguiculus with all lamellae smooth and lanceolate (a.i., a.e., p.i., p.e.) (Fig. 26B); ratio unguis: unguiculus = 1: 1.46–1.91 (n = 5), holotype = 1: 1.91. Tibiotarsal smooth chaetae about 0.8 × smaller unguiculus; tenent hair acuminate and about 0.4 × smaller than unguis outer lamella.Collophore (Fig. 26C). Anterior side with seven ciliate, apically acuminate chaetae, three proximal, two subdistal and two distal mac; lateral flap with nine chaetae, four ciliate in the proximal row and five smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with five ciliate chaetae (two inner mac) and three psp (Fig. 26D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 35 external and 26 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.39 (n = 5).Etymology. Species named after Dr. Felipe N. Soto-Adames for his contribution on Collembola taxonomy and systematics.Remarks. Trogolaphysa sotoadamesi sp. nov. resembles T. barroca sp. nov., T. crystallensis sp. nov., T. gisbertae sp. nov., T. zampauloi sp. nov. by 0 + 0 eyes (T. zampauloi sp. nov. rarely with 4 + 4 eyes), Th II p3 complex with five mac, Th III without mac, manubrial plate with five ciliate chaetae and mucro with four teeth. The new species T. sotoadamesi sp. nov. with 2 + 2 central mac on Abd IV differentiates from T. barroca sp. nov., T. gisbertae sp. nov. with 3 + 3, and T. crystallensis sp. nov., T. zampauloi sp. nov. with 4 + 4 central mac.Trogolaphysa mariecurieae sp. nov. Ferreira, Oliveira & ZeppeliniFigures 27, 28 and 29, Tables 1 and 2Figure 27Trogolaphysa mariecurieae sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore, Gray cut circle pseudopore at the under surface.Full size imageFigure 28Trogolaphysa mariecurieae sp. nov.: Dorsal chaetotaxy. (A) Th II–III, (B) Abd I–III, (C) Abd IV–V.Full size imageFigure 29Trogolaphysa mariecurieae sp. nov.: (A) Trochanteral organ, (B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy.Full size imageType material. Holotype female in slide (9109/CRFS-UEPB): Brazil, Minas Gerais State, Conceição do Mato Dentro municipality, MSS 10/11, next to “Pico do Soldado” 19°00′23.86″S, 43°23′41.266″W, 11–10.ix.2015, Carste team coll. Paratypes in slides (5888, 5857/CRFS-UEPB): 2 females, same data as holotype, except,19–23.v.2014, Soares et al. coll.Paratype in slide (9222, 10,760/CRFS-UEPB donated to MNJR): 2 females, same data as holotype, except 19°00′18.72″S, 43°23′30.031″W, 14.x.2015 and 18–20.iv.2016. Additional records see S1.Description. Total length (head + trunk) of specimens 1.07–1.49 mm (n = 5), holotype 1.49 mm.Head. Ratio antennae: trunk = 1: 1.69–1.91 (n = 2), holotype = 1: 1.69; Ant III shorter than Ant II length; Ant segments ratio, I: II, III, IV = 1: 2.00–2.75, 1.69–2.55, 4.02–5.29, holotype = 1: 2.75, 1.69, 4.02. Antennal chaetotaxy (no represented): Ant IV dorsally and ventrally with several short less ciliate mic and mac, and finger-shaped sens, dorsally with one longitudinal row with about four rod sens, ventrally with one subapical-organ and several wrinkly sens (Fig. 4A); Ant III dorsally and ventrally with several short less ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, apical organ with two rod sens (Fig. 4A); Ant II dorsally and ventrally with several short less ciliate mic and mac, dorsally with five apical rod sens, ventrally with one apical psp, about five wrinkly sens on longitudinal external row (Fig. 4A); and Ant I dorsally and ventrally with several short less ciliate mic and mac, dorsally with three basal spine-like sens, ventrally with seven basal spine-like sens, about five smooth mic, and several finger-shaped sens (Fig. 4A). Eyes 0 + 0. Head dorsal chaetotaxy (Fig. 27A) with 12 An (An1a–3), six A (A0–5), four M (M1–4), five S (S2–6), two Ps (Ps2, Ps5), four Pa (Pa1–3, Pa5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; Pm3 and Pa5 as mes, An1a–3a, A0 and A2 as mac; interocular p mic present. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 27B). Ventral chaetotaxy with 34 ciliate chaetae and one reduced lateral spine; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of six mes chaetae distally (Fig. 27B). Prelabral chaetae ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and surpassing the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–3) and subequal in length (Fig. 27B). Maxillary palp with t.a. smooth and 1.13× larger than b.c.Thorax dorsal chaetotaxy (Fig. 28A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with three mac, respectively, al and ms present. Th III a, m, p series with three mic (a1–3), two mes (a6–7), three mic (m4–m6p), three mes (m6e, m7–7e), four mic (p1–3, p6) respectively. Ratio Th II: III = 0.85–1.02: 1 (n = 4), holotype = 0.89: 1. Abdomen dorsal chaetotaxy (Fig. 28B, C). Abd I a, m series with one (a5) and six (m2–6e) mic respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el mic and as present; a5 and m2 bothriotricha surrounded by four and two fan-shaped chaetae respectively. Abd III a, m, p series with one mic (a7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6), two mic (p6e, p7i), one mac (p6) chaetae respectively; a5, m2 and m5 bothriotricha with five, two and two fan-shaped chaetae respectively, as sens elongated, ms present. Abd IV A–Fe series with three mic (A1, A6, Ae1), one mac (A4), two mic (B1–2), one mes (B6), one mac (B5), four mic (C1–4), three mic (T1, T5, T7), five mic (D1–3, De3), one mes (D3p), one mic (E4p2), one mes (E4p), three mac (E1–3), one mic (Ee12), two mes (Ee10–11), one mac (Ee9), one mic (F1), three mes (F2–3p), one mic (Fe2), three mes (Fe3–5) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by four and three (T3) fan-shaped chaetae respectively; ps and as present, and at least five supernumerary sens with uncertain homology ‘s’ (Fig. 8A); Abd. IV posteriorly with four psp. Abd V a, m, p series with two mic (a1, a3), one mes (a6), one mac (a5), five mac (m2–3, m5–5e), five mic (p3a–p6ae), two mes (ap6–pp6), four mac (p1, p3–5) chaetae, respectively; as, acc.p4–5 present. Ratio Abd III: IV = 1: 4.27–5.91 (n = 5), holotype = 1: 5.02.Legs. Trochanteral organ diamond shape with about 15 spine-like chaetae, plus 2–3 psp one external, one on distal vertex and another (present or absent) on top of posterior spines row of Omt (Fig. 29A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with two teeth, basal pair subequal, b.p. larger than b.a., inner lamella with unpaired small m.t. between b.a. and b.p. and a.t. absent. Unguiculus with all lamellae smooth and truncate (a.i., a.e., p.i., p.e.) (Fig. 29B); ratio unguis: unguiculus = 1.50–1.95: 1 (n = 5), holotype = 1.95: 1. Tibiotarsal smooth chaetae about 0.9× smaller than unguiculus; tenent hair slightly capitate and about 0.6× smaller than unguis outer lamella.Collophore (Fig. 29C). Anterior side with eight ciliate, apically acuminate chaetae, six proximal and two distal mac; lateral flap with 13 chaetae, five ciliate in the proximal row and eight smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 29D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 40 external and 22 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.23 (holotype).Etymology. Species named after Dr. Marie Skłodowska-Curie for her enormous contribution to science.Remarks. Trogolaphysa mariecurieae sp. nov. resembles T. bellinii sp. nov. T. jacobyia and T. epitychia sp. nov. by the absence of eyes (T. bellinii sp. nov. rarely with 2 + 2 eyes), Th II p3 complex with three mac and with one unpaired tooth on inner lamella of unguis. The new species T. mariecurieae sp. nov. (Abd IV with 2 + 2 mac) differs from T. jacobyia, T. epitychia sp. nov. both with Abd IV 3 + 3, and T. bellinii sp. nov. with 4 + 4 central mac. T. mariecurieae sp. nov. and T. bellinii sp. nov. with capitate tenent hair, in contrast with T. jacobyia and T. epitychia sp. nov. with acuminated tenant hair.Trogolaphysa barroca sp. nov. Brito & ZeppeliniFigures 30, 31 and 32, Tables 1 and 2Figure 30Trogolaphysa barroca sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore; Gray cut circle pseudopore at the under surface.Full size imageFigure 31Trogolaphysa barroca sp. nov.: Dorsal chaetotaxy. (A) Th II–III, (B) Abd I–III, (C) Abd IV–V.Full size imageFigure 32Trogolaphysa barroca sp. nov.: (A) Trochanteral organ, (B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy.Full size imageType material. Holotype female in slide (13,167/CRFS-UEPB): Brazil, Minas Gerais State, Mariana municipality, ALFA-0003 cave, 20°09′06.8″S, 43°29′13.6″W, 07–27.ii.2018, Bioespeleo team coll. Paratype in slide (13,150/CRFS-UEPB): 1 female, same data as holotype, except 12.vi.2017. Paratype in slide (13,158/CRFS-UEPB donated to MNJR): 1 female, same data as holotype. Paratype in slide (13,197/CRFS-UEPB): 1 female, Brazil, Minas Gerais State, Mariana municipality, ALEA-0004 cave, 20°09′00.0″S, 43°29′11.8″W, 07.ii.2018, Bioespeleo team coll. Paratype in slide (13,203/CRFS-UEPB): 1 female, Brazil, Minas Gerais State, Mariana municipality, ALEA-0002 cave, 20°08′56.5″S, 43°29′09.8″W, 27.ii.2018, Bioespeleo team coll. Additional records see S1.Description. Total length (head + trunk) of specimens 1.70–2.13 mm (n = 5), holotype 1.81 mm.Head. Ratio antennae: trunk = 1: 1.27–1.60 (n = 3), holotype = 1: 1.27; Ant III shorter than Ant II; Ant segments ratio as, I: II, III, IV = 1: 1.90–2.41, 1.64–2.02, 2.69–3.64, holotype = 1: 1.90, 1.67, 2.69. Antennal chaetotaxy (no represented): Ant IV dorsally and ventrally with several short less ciliate mic and mac, and finger-shaped sens, dorsally with about four rod sens on longitudinal row, ventrally with one subapical-organ and several wrinkly sens (Fig. 4A); Ant III dorsally and ventrally with several short less ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, about nine wrinkly sens on external longitudinal row, apical organ with one finger-shaped sens, two coffee bean-like sens, and one rod sens (Fig. 4A); Ant II dorsally and ventrally with several short less ciliate mic and mac, dorsally with two sub-apical finger-shaped sens and two subapical rod sens, ventrally with one apical psp, and several wrinkly sens on longitudinal external row (Fig. 4A); and Ant I dorsally and ventrally with several short less ciliate mic and mac, dorsally with three basal spine-like sens, ventrally with about five basal spine-like sens, about five smooth mic and several finger-shaped sens (Fig. 4A). Eyes 0 + 0. Head dorsal chaetotaxy (Fig. 30A) with 14–15 An (An1a–3), six A (A0–5), five M (M1–5), six S (S1–6), two Ps (Ps2, Ps5), four Pa (Pa1–3, Pa5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; Pm3 as mic, A3 as mes, An1a–3, A0, A2 and Pa5 as mac; interocular p mic present. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 30B). Ventral chaetotaxy with 33 ciliate chaetae and one reduced lateral spine; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of five to six mes chaetae distally (Fig. 30B). Prelabral chaetae weakly ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and subequal the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–3), and subequal in length (Fig. 30B). Maxillary palp with t.a. smooth and 1.14 × larger than b.c.Thorax dorsal chaetotaxy (Fig. 31A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with five mac, respectively, al and ms present. Th III a, m, p series with three mic (a1–3), two mes (a6–7), two mic (m4, m6p), four mes (m6–6e, m7–7e), and four mic (p1–3, p6), respectively. Ratio Th II: III = 1.11–1.35: 1 (n = 5), holotype = 1.29: 1.Abdomen dorsal chaetotaxy (Fig. 31B, C). Abd I a, m series with one (a5) and six (m2–6e) mic, respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el mic and as present; a5 and m2 bothriotricha surrounded by four and three fan-shaped chaetae, respectively. Abd III a, m, p series with one mic (a7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6), three mic (p6e, p7i–7), one mac (p6) chaetae, respectively; a5, m2 and m5 bothriotricha with six, two and three fan-shaped chaetae, respectively; as sens elongated, ms present. Abd IV A–Fe series with four mic (A1, A5–6, Ae1), one mac (A3), one mic (B1), one mes (B6), two mac (B4–5), four mic (C1–4), three mic (T1, T5–6), one mes (T7), five mic (D1–3, De3), one mes (D3p), one mic (E4p2), one mes (E4p), three mac (E1–3), one mic (Ee12), three mes (Ee9–11), one mic (F1), three mes (F2–3p), one mic (Fe2), three mes (Fe3–5) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by four and two (T3) fan-shaped chaetae, respectively; ps and as present, and at least seven supernumerary sens with uncertain homology ‘s’ (Fig. 8A); Abd. IV posteriorly with four to six psp. Abd V a, m, p series with two mic (a1, a3), one mes (a6), one mac (a5), two mes (m5a–5e), three mac (m2–3, m5), five mic (p3a–6ae), one mic (p6e), two mes (ap6, pp6), four mac (p1, p3–5) chaetae, respectively; as and acc.p4–5 present. Ratio Abd III: IV = 1: 3.38–5.55 (n = 5), holotype = 1: 5.27.Legs. Trochanteral organ diamond shape with about 16–21 spine-like chaetae, plus 2–3 psp one external, and two (one of them present or absent) on top of posterior spines row of Omt (Fig. 32A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with two teeth, basal pair subequal; b.p. little larger than b.a., m.t. and a.t. absent. Unguiculus with all lamellae smooth and lanceolate (a.i., a.e., p.i., p.e.) (Fig. 32B); ratio unguis: unguiculus = 1.53–1.67: 1 (n = 5), holotype = 1.61: 1. Tibiotarsal smooth chaetae about 0.61 × smaller than unguiculus; tenent hair acuminate and about 0.4 × smaller than unguis outer lamella.Collophore (Fig. 32C). Anterior side with eight ciliate, apically acuminate chaetae, four proximal (thinner), one subdistal and three distal mac; lateral flap with 10 chaetae, five ciliate in the proximal row and five smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with five ciliate chaetae (three inner mac) and three psp (Fig. 32D). Dens posterior face with two or more longitudinal rows of spines-like chaetae about 22 external and 37–39 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.33 (holotype).Etymology. Refers to the Baroque art (which is “barroco” noun, in Portuguese) of Mariana, Minas Gerais, type locality.Remarks. Trogolaphysa barroca sp. nov. resembles T. formosensis by head Pm3 mic (mac in T. piracurucaensis, T. gisbertae sp. nov. and T. dandarae sp. nov.; mes in T. ernesti, T. sotoadamesi sp. nov. and T. mariecurieae sp. nov.); 3 + 3 head dorsal mac like T. ernesti, although in the new species it is as A0, A2 and Pa5, and in T. ernesti is A0, A2–3; unguis m.t. and a.t. teeth absent like T. sotoadamesi sp. nov. and T. dandarae sp. nov. (present in T. bellini sp. nov., T. lacerta sp. nov. and T. chapelensis sp. nov.).Trogolaphysa epitychia sp. nov. Oliveira, Lima & ZeppeliniFigures 33, 34 and 35, Tables 1 and 2Figure 33Trogolaphysa epitychia sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore; Gray cut circle pseudopore at the under surface.Full size imageFigure 34Trogolaphysa epitychia sp. nov.: Dorsal chaetotaxy. (A) Th II–III, (B) Abd I–III, (C) Abd IV–V.Full size imageFigure 35Trogolaphysa epitychia sp. nov.: (A) Trochanteral organ, (B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy.Full size imageType material. Holotype male in slide (10,578/CRFS-UEPB): Brazil, Minas Gerais State, Conceição do Mato Dentro municipality, cave CSS-0118, next to “São Sebastião do Bom Sucesso”, 18°56′14.1″S, 43°24′43.8″W, 21.xi–15.xii.2016, Carste team coll. Paratypes in slides (10,580, 10,585/CRFS-UEPB): 2 females, same data as holotype. Paratypes in slides (10,653, 10,692/CRFS-UEPB donated to MNJR): 1 female and 1 male, same data as holotype, except 22.xi–15.xii.2016 and 31.v–12.vi.2016, respectively. Additional records see S1.Description. Total length (head + trunk) 1.13–1.35 mm (n = 5), holotype 1.13 mm.Head. Ratio antennae: trunk = 1: 1.29–1.95 (n = 5), holotype = 1: 1.95; Ant III shorter than Ant II; Ant segments ratio as I: II, III, IV = 1: 1.69–2.20, 1.14–1.86, 2.37–3.52, holotype = 1: 1.71, 1.14, 2.37. Antennal chaetotaxy (no represented): Ant IV dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally with one longitudinal row with about six rod sens, ventrally with one subapical-organ and one longitudinal row with about four wrinkly sens (Fig. 4A); Ant III dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, about three wrinkly sens on external longitudinal row, apical organ with two coffee bean-like sens, one rod sens and one smooth mic (Fig. 4A); Ant II dorsally and ventrally with several short ciliate mic and mac, dorsally with about six sub-apical finger-shaped sens and one wrinkly sens, ventrally with one apical psp, about three wrinkly sens on longitudinal external row (Fig. 4A); and Ant I dorsally and ventrally with several short ciliate mic and mac, three basal spine-like sens, ventrally with four basal spine-like sens, about three smooth mic, several finger-shaped sens, and two wrinkly sens (Fig. 4A). Eyes 0 + 0. Head dorsal chaetotaxy (Fig. 33A) with 12 An (An1a–3), six A (A0–5), four M (M1–4), five S (S2–6), two Ps (Ps2, Ps5), four Pa (Pa1–5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; Pm3 and Pa5 as mes, An1a–3a, A0 and A2 as mac; interocular p mes present. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 33B). Ventral chaetotaxy with 31–32 ciliate chaetae and one reduced lateral spine; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of five to six mes chaetae distally (Fig. 33B). Prelabral chaetae ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and surpassing the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–3) and subequal in length (Fig. 33B). Maxillary palp with t.a. smooth and 1.26× larger than b.c.Thorax dorsal chaetotaxy (Fig. 34A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with three mac, respectively, al and ms present. Th III a, m, p series with three mic (a1–3), two mes (a6–a7), three mic (m4, m6–6p), three mes (m6e, m7–7e), four mic (p1–3, p6) respectively. Ratio Th II: III = 1.05–1.21: 1 (n = 5), holotype = 1.18: 1.Abdomen dorsal chaetotaxy (Fig. 34B, C). Abd I a, m series with one (a5) and six (m2–6e) mic respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el mic and as present; a5 and m2 bothriotricha surrounded by four and two fan-shaped chaetae respectively. Abd III a, m, p series with two mic (a7i–7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6), three mic (p6e, p7i–7), one mac (p6) chaetae, respectively; a5, m2 and m5 bothriotricha with five, two and one fan-shaped chaetae, respectively; as sens elongated, ms present. Abd IV A–Fe series with three mic (A1, A6, Ae1), two mac (A3, A5), two mic (B1, B4), one mes (B6), one mac (B5), four mic (C1–4), four mic (T1, T3, T5–6), one mac (T7), six mic (D1–3p, De3), two mic (E4p–4p2), three mac (E1–3), one mic (Ee11), three mes (Ee9–10, Ee12), one mic (F1), three mes (F2–3p), one mic (Fe2), three mes (Fe3–5) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by five and two fan-shaped chaetae, respectively; ps and as present, and at least seven supernumerary sens with uncertain homology ‘s’ (Fig. 8A); Abd. IV posteriorly with three psp. Abd V a, m, p series with three mic (a1, a3, a6), one mac (a5), two mic (m3, me5), three mac (m2, m5–5a), two mic (p3a–4a), one mes (p5a) two mac (p6ai–6ae), four mes (p5–pp6), three mac (p1, p3–4) chaetae, respectively; as, acc.p4–5 present. Ratio Abd III: IV = 1: 4.69–5.55 (n = 5), holotype = 1: 4.88.Legs. Trochanteral organ in V–shape with about 15 spine-like chaetae, plus 4 psp one external, one on distal vertex and another two on top of posterior spines row of Omt (Fig. 35A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with four teeth, basal pair subequal, b.p. little larger, not reaching the m.t. apex, m.t. just after the distal half, a.t. absent. Unguiculus with all lamellae smooth and slightly truncate (a.i., a.e., p.i., p.e.) (Fig. 35B); ratio unguis: unguiculus = 1.17–1.98: 1 (n = 5), holotype = 1.17: 1. Tibiotarsal smooth chaetae about 0.8× smaller than unguiculus; tenent hair acuminate and about 0.53× smaller than unguis outer lamella.Collophore (Fig. 35C). Anterior side with nine ciliate, apically acuminate chaetae, five proximal, two subdistal and two distal mac; lateral flap with 10 chaetae, five ciliate in the proximal row and five smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with five ciliate chaetae (two inner mac) and three psp (Fig. 35D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 60 external and 34 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.30 (holotype).Etymology. Epitychia from Greek means success, in allusion to the collection site where the species was found São Sebastião do Bom Sucesso.Remarks. Trogolaphysa epitychia sp. nov. resembles T. bellinii sp. nov., T. bessoni, and T. mariecurieae sp. nov. by the absence of eyes (T. bellinii sp. nov. rarely with 2 + 2 eyes), Th II with 3 + 3 mac, and Th III without mac. Differentiates from T. bellinii sp. nov. and T. mariecurieae sp. nov. by Abd IV with 3 + 3 (A3, A5, B5), 4 + 4, and 2 + 2 mac on Abd IV respectively; on T. bellinii sp. nov. and can be distinguished from T. bessoni by the absence of unpaired tooth on inner lamella of unguis, external row of dens with 25 spines, inner row of dens with 20 spines (T. epitychia sp. nov. with one unpaired tooth m.t. on inner lamella of unguis, external row of dens with about 60 spines and inner row of dens with about 34 spines).Trogolaphysa zampauloi sp. nov. Lima, Oliveira & ZeppeliniFigures 36, 37 and 38, Tables 1 and 2Figure 36Trogolaphysa zampauloi sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore; Gray cut circle pseudopore at the under surface.Full size imageFigure 37Trogolaphysa zampauloi sp. nov.: Dorsal chaetotaxy. (A) Th II–III, (B) Abd I–III, (C) Abd IV–V.Full size imageFigure 38Trogolaphysa zampauloi sp. nov.: (A) Trochanteral organ, (B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy.Full size imageType material. Holotype female in slide (11,851/CRFS-UEPB): Brazil, São Paulo State, Ribeira municipality, cave MTD-13, nexto to “Serra Pontalhão”, 24°38′47.4″S, 48°57′52.6″W, 08–20.iii.2016, Carste team coll. Paratypes in slides (11,875–11,878/CRFS-UEPB): 2 males and 2 females, Brazil, São Paulo State, Ribeira municipality, cave MTD-02, 24°37′27.3″S, 48°57′35.8″W, 08–20.iii.2016. Paratype in slide (11,876/CRFS-UEPB donated to MNJR). Additional records see S1.Description. Total length (head + trunk) of specimens 1.35–1.91 mm (n = 5), holotype 1.35 mm.Head. Ratio antennae: trunk = 1: 1.35–1.55 (n = 2), holotype = 1: 1.55; Ant III smaller than Ant II length; Ant segments ratio as I: II, III, IV = 1: 1.71–2.38, 1.60–1.88, 2.85–3.61, holotype = 1: 2.38, 1.88, 3.61. Antennal chaetotaxy (no represented): Ant IV dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally with about three rod sens on longitudinal row, ventrally with one subapical-organ, and about three wrinkly sens (Fig. 4A); Ant III dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, one apical wrinkly sens, apical organ with two coffee bean-like sens, and one rod sens (Fig. 4A); Ant II dorsally and ventrally with several short ciliate mic and mac, dorsally with about three sub-apical finger-shaped sens and two apical rod sens, ventrally with one apical psp, one longitudinal external row with two subapical finger-shaped sens and two medial wrinkly sens (Fig. 4A); and Ant I dorsally and ventrally with several short ciliate mic and mac, dorsally with three basal spine-like sens, ventrally with four basal spine-like sens, about four smooth mic and several finger-shaped sens (Fig. 4A). Eyes 0 + 0 to 4 + 4. Head dorsal chaetotaxy (Fig. 36A) with 14 An (An1a–3), six A (A0–5), four M (M1–4), five S (S2–6), two Ps (Ps2, Ps5), four Pa (Pa1–3, Pa5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; Pe4, Pe6, Pm3 and Pa5 as mes, An1a–3a as mac, A0 and A2 as mac, A3–5 as mes; interocular p mes present. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 36B). Ventral chaetotaxy with about 37 ciliate chaetae, plus one reduced lateral spine; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of eight mes chaetae distally (Fig. 36B). Prelabral chaetae ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and surpassing the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–3) and subequal in length (Fig. 36B). Maxillary palp with t.a. smooth and 1.17 × smaller than b.a.Thorax dorsal chaetotaxy (Fig. 37A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with five mac, respectively, al and ms present. Th III a, m, p series with three mic (a1–3), two mes (a6–a7), three mic (m4, m6–6p), three mes (m6e, m7–7e), four mic (p1–3, p6), respectively. Ratio Th II: III = 1.02–1.48: 1 (n = 5), holotype = 1.21: 1Abdomen dorsal chaetotaxy (Fig. 37B, C). Abd I a, m series with one (a5) and six (m2–6e) mic respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el as mic and as present; a5 and m2 bothriotricha surrounded by three and two fan-shaped chaetae respectively. Abd III a, m, p series with one mic (a7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6), three mic (p6e, p7i–7), one mac (p6) chaetae respectively; a5, m2 and m5 bothriotricha with five, two and three fan-shaped chaetae respectively, as sens elongated, ms present. Abd IV A–Fe series with three mic (A1, A6, Ae1), two mac (A3, A5), one mic (B1), one mes (B6), two mac (B4–5), four mic (C1–4), three mic (T1, T5–6), one mes (T7), five mic (D1–3, De3), one mes (D3p), one mic (E4p2), one mes (E4p), three mac (E1–3), one mic (Ee12), one mes (Ee11), two mac (Ee9–10), one mic (F1), two mes (F3–3p), one mac (F2), one mic (Fe2), two mes (Fe3, Fe5), one mac (Fe4) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by four and four (T3) fan-shaped chaetae respectively; ps and as present, and at least six supernumerary sens with uncertain homology ‘s’ (Fig. 8A); Abd. IV posteriorly with three psp. Abd V a, m, p series with two mic (a1, a3), one mes (a6), one mac (a5), two mes (m5a, m5e), three mac (m2–3, m5), five mic (p3a–6ae), one mic (p6e) two mes (ap6–pp6), four mac (p1, p3–5) chaetae, respectively; as, acc.p4–5 present. Ratio Abd III: IV = 1: 3.29–4.28 (n = 5), holotype = 1: 4.10.Legs. Trochanteral organ diamond shape with about 27 spine-like chaetae, plus 3–4 psp one external, one on distal vertex and another two (one of them present or absent) on top of posterior spines row of Omt (Fig. 38A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with four teeth, basal pair subequal, b.p. not reaching the m.t. apex, m.t. just after the distal half, a.t. present. Unguiculus with all lamellae smooth and lanceolate (a.i., a.e., p.i., p.e.) (Fig. 38B); ratio unguis: unguiculus = 1.63–1.84 (n = 5), holotype = 1: 1.79. Tibiotarsal smooth chaetae about 0.8× smaller than unguiculus; tenent hair acuminate and about 0.39× smaller than unguis outer edge.Collophore (Fig. 38C). Anterior side with five ciliate, apically acuminate chaetae, two proximal (thinner); one subdistal and two distal mac; lateral flap with 11 chaetae, five ciliate in the proximal row and six smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 38D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 30 external and 23 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.29 (n = 5).Etymology. Species named after the field biologist MSc. Robson de Almeida Zampaulo for his contribution to Brazilian biospeleology.Remarks. Trogolaphysa zampauloi sp. nov. resembles T. caripensis; T. ernesti; T. piracurucaensis by Th III without mac, and 4 + 4 central mac (A3, A5, B4–5) in Abd IV, but is easily distinguished from these species by the presence of Th II with 4 + 4 mac in p3 complex (6 + 6T. caripensis, T. ernesti, T. piracurucaensis). For more comparisons see remarks in T. crystallensis sp. nov.Trogolaphysa gisbertae sp. nov. Brito & ZeppeliniFigures 39, 40 and 41, Tables 1 and 2Figure 39Trogolaphysa gisbertae sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore; Gray cut circle pseudopore at the under surface.Full size imageFigure 40Trogolaphysa gisbertae sp. nov.: Dorsal chaetotaxy: (A) Th II–III, (B) Abd I–III, (C) Abd IV–V.Full size imageFigure 41Trogolaphysa gisbertae sp. nov.: (A) Trochanteral organ, (B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy.Full size imageType material. Holotype female in slide (6668/CRFS-UEPB): Brazil, Pará State, Parauapebas municipality, cave N1N8-N8-017, next to “Serra Norte”, 06°10′05.9″S, 50°09′25.6″W, 02–29.iv.2015, Carste team coll. Paratype in slide (6669/CRFS-UEPB donated to MNJR): 1 female, same data as holotype, except 04.ix–06.x.2014. Paratype in slide (6973/CRFS-UEPB): 1 female, same data as holotype, except 04.ix–06.x.2014. Paratypes in slides (6657, 7138/CRFS-UEPB): 2 females, Brazil, Pará State, Parauapebas municipality, N1N8-N8-020 cave, 06°10′07.8″S, 50°09′25.4″W, 17.vii–04.viii.2014, Carste team coll. Additional records see S1.Description. Total length (head + trunk) of specimens 1.10–1.23 mm (n = 5), holotype 1.15 mm.Head. Ratio antennae: trunk = 1: 1.44–1.55 (n = 3); Ant segments ratio as I: II, III, IV = 1: 1.67–2.43, 1.58–2.63, 2.91–5.46, holotype = 1: 2.03, – , 3.90. Antennal chaetotaxy (no represented): Ant IV dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally with about five rod sens in row, ventrally with one subapical-organ and several wrinkly sens row (Fig. 4A); Ant III dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, about four wrinkly sens on external longitudinal row, apical organ with one finger-shaped sens, two coffee bean-like sens, and one rod sens (Fig. 4A); Ant II dorsally and ventrally with several short ciliate mic and mac, dorsally with four finger-shapedd sens in row and two subapical rod sens, ventrally with one apical psp, and about five wrinkly sens on longitudinal external row (Fig. 4A); and Ant I dorsally and ventrally with several short ciliate mic and mac, dorsally with three basal spine-like sens, ventrally with four basal spine-like sens, about five smooth mic and several fniger-shaped sens (Fig. 4A). Eyes 0 + 0. Head dorsal chaetotaxy (Fig. 39A) with 11 An (An1a–3), six A (A0–5), four M (M1–4), five S (S1–5), two Ps (Ps2, Ps5), four Pa (Pa1–5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; An1a–3a, A0, A2–3, Pa5 and Pm3 as mac; interocular p absent. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 39B). Ventral chaetotaxy with 20 ciliate chaetae and one reduced lateral spine; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of three to four mes chaetae distally (Fig. 39B). Prelabral chaetae ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and surpassing the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–3) and subequal in length (Fig. 39B). Maxillary palp with t.a. smooth and 1.32 × larger than b.c.Thorax dorsal chaetotaxy (Fig. 40A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with five mac, respectively, al and ms presents. Th III a, m, p series with three mic (a1–3), two mes (a6–7), three mic (m4, m6–6p), three mes (m6e, m7–7e), and four mic (p1–3, p6), respectively. Ratio Th II: III = 1.00–2.60: 1 (n = 5), holotype = 1.28: 1.Abdomen dorsal chaetotaxy (Fig. 40B, C). Abd I a, m series with one (a5) and six (m2–6e) mic, respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el mic and as present; a5 and m2 bothriotricha surrounded by four and two fan-shaped chaetae, respectively. Abd III a, m, p series with one mic (a7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6), three mic (p6e, p7i–7), one mac (p6) chaetae, respectively; a5, m2 and m5 bothriotricha with six, two and three fan-shaped chaetae, respectively, as sens elongated, ms present. Abd IV A–Fe series with four mic (A1, A5–6, Ae1), one mac (A3), one mic (B1), one mes (B6), two mac (B4–5), four mic (C1–4), four mic (T1, T5–7), five mic (D1–3, De3), one mes (D3p), one mic (E4p2), one mes (E4p), three mac (E1–3), one mic (Ee12), three mes (Ee9–11), one mic (F1), three mes (F2–3p), one mic (Fe2), three mes (Fe3–5) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by four and two (T3) fan-shaped chaetae, respectively; ps and as present, and at least six supernumerary sens with uncertain homology ‘s’ (Fig. 8A); Abd. IV posteriorly with one to three psp. Abd V a, m, p series with three mic (a1, a3), one mes (a6), one mac (a5), two mes (m5a–5e), three mac (m2–3, m5), five mic (p3a–6ae), one mic (p6e), two mes (ap6, pp6), four mac (p1, p3–5) chaetae, respectively; as and acc.p4–5 present. Ratio Abd III: IV = 1: 3.29–4.90 (n = 5), holotype = 1: 3.29.Legs. Trochanteral organ diamond shape with about 25 spine-like chaetae, plus two psp one external, and one on distal vertex of Omt (Fig. 41A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with three teeth, basal pair subequal, b.p. not reaching the m.t. apex, m.t. just after the distal half, a.t. absent. Unguiculus with lamellae smooth and slightly truncate (a.i., a.e., p.i.), except p.e. slightly serrate (Fig. 41B); ratio unguis: unguiculus = 1.59–2.05: 1 (n = 5), holotype = 1.62: 1. Tibiotarsal smooth chaetae about 0.9× smaller than unguiculus; tenent hair acuminate and about 0.53× smaller than unguis outer lamella.Collophore (Fig. 41C). Anterior side with five ciliate, apically acuminate chaetae, one proximal (thinner); two subdistal and two distal mac; lateral flap with 10 chaetae, five ciliate in the proximal row and five smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with five ciliate chaetae (three inner mac) and three psp (Fig. 41D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 18 external and 24 internal, external spines larger and thinner than internal ones. Mucro with four teeth, ratio width: length = 0.26 (holotype).Etymology. Honor to Gisberta Salce Júnior, Brazilian woman, murdered in 2006 (Porto, Portugal) in a transphobia crime.Remarks. Trogolaphysa gisbertae sp. nov. differs from T. ernesti and T. formosensis (with 0 + 0 head dorsal mac), T. piracurucaensis, and T. barroca sp. nov. (1+1 head dorsal mac); and resembles T. dandarae sp. nov. (with 5+5 head dorsal mac), but it is easily distinguishable by Th II p3 complex and Th III mac (5 + 5 and 0 + 0, 6 + 6 and 3 + 3, respectively); and unguis with m.t. present (absent in T. sotoadamesi sp. nov., T. barroca sp. nov.).Trogolaphysa dandarae sp. nov. Brito & ZeppeliniFigures 42, 43 and 44, Tables 1 and 2Figure 42Trogolaphysa dandarae sp. nov.: (A) Head dorsal chaetotaxy, (B) labial proximal chaetae, basomedial and basolateral labial fields and postlabial chaetotaxy. Black cut circle, pseudopore; Gray cut circle pseudopore at the under surface.Full size imageFigure 43Trogolaphysa dandarae sp. nov.: Dorsal chaetotaxy. (A) Th II–III, (B) Abd I–III, (C) Abd IV–V.Full size imageFigure 44Trogolaphysa dandarae sp. nov.: (A) Trochanteral organ, (B) Distal tibiotarsus and empodial complex III (anterior view), (C) Manubrial plate, (D) Antero-lateral view of collophore chaetotaxy, (E) Mucro.Full size imageType material. Holotype female in slide (12,775/CRFS-UEPB): Brazil, Pará State, Parauapebas municipality, cave N4WS-0018/48, next to “Serra Norte”, 06°04′34.5″S, 50°11′37.7″W, 21–30.vii.2018, Brandt Meio Ambiente team coll. Paratype in slide (12,776/CRFS-UEPB donated to MNJR): 1 female, same data as holotype. Paratypes in slides (12,777, 12,778/CRFS-UEPB): 2 females, same data as holotype. Paratypes in slides (12,772, 12,773/CRFS-UEPB): 2 females, Brazil, Pará State, Parauapebas municipality, N4WS-0016 cave, 06°04′35.5″S, 50°11′37.1″W, 21–30.vii.2018, Brandt Meio Ambiente team coll. Additional records see S1.Description. Total length (head + trunk) of specimens 1.43–1.75 mm (n = 5), holotype 1.58 mm.Head. Ratio antennae: trunk = 1: 0.83–0.98 (n = 4), holotype = 1: 0.83; Ant III larger than Ant II; Ant segments ratio as I: II: III: IV = 1: 1.36–1.77: 1.65–2.03: 2.84–3.27, holotype = 1: 1.72: 1.99: 3.21. Antennal chaetotaxy (no represented): Ant IV dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally with about two rod sens sub-apical on longitudinal row, ventrally with one subapical-organ and about three wrinkly sens on longitudinal row (Fig. 4A); Ant III dorsally and ventrally with several short ciliate mic and mac, and finger-shaped sens, dorsally without modified sens, ventrally with one apical psp, about three wrinkly sens and three smooth mic on external longitudinal row, apical organ with one finger-shaped sens, two coffee bean-like sens, and one rod sens (Fig. 4A); Ant II dorsally and ventrally with several short ciliate mic and mac, dorsally with about four sub-apical finger-shaped sens and two subapical rod sens, ventrally with one apical psp, and several wrinkly sens on longitudinal external row (Fig. 4A); and Ant I dorsally and ventrally with several short ciliate mic and mac, dorsally with three basal spine-like sens, ventrally with four basal spine-like sens, about five smooth mic and several finger-shaped sens (Fig. 4A). Eyes 0 + 0. Head dorsal chaetotaxy (Fig. 42A) with 12 An (An1a–3), six A (A0–5), four M (M1–4), six S (S1–6), two Ps (Ps2, Ps5), four Pa (Pa1–5), two Pm (Pm1, Pm3), seven Pp (Pp1–7), and two Pe (Pe4, Pe6) chaetae; A1 as mes, An1a–3, A0, A2, S5, Pa5 and Pm3 as mac; interocular p mic present. Basomedian and basolateral labial fields with a1–5 smooth, M, Me, E and L1–2 ciliate, r reduced (Fig. 42B). Ventral chaetotaxy with 28 ciliate chaetae and one reduced lateral spine; postlabial G1–4; X, X4; H1–4; J1–2, chaetae b.c. present and a collar row of five chaetae distally (Fig. 42B). Prelabral chaetae ciliate. Labral chaetae smooth, no modifications. Labial papilla E with l.p. finger-shaped and subequal the base of apical appendage. Labial proximal chaetae smooth (an1–3, p2–3) and subequal in length (Fig. 42B). Maxillary palp with t.a. smooth and 1.58 × larger than b.c.Thorax dorsal chaetotaxy (Fig. 43A). Th II a, m, p series with two mic (a1–2), one mac (a5), three mic (m1–2, m4) and four mic (p4–6e), p3 complex with six mac, respectively, al and ms presents. Th III a, m, p series with three mic (a1–3), two mes (a6–7), two mic (m6–6p), three mes (m6e, m7–7e), and one mic (p6), respectively. Ratio Th II: III = 0.82–1.13: 1 (n = 6), holotype = 1.13: 1.Abdomen dorsal chaetotaxy (Fig. 43B, C). Abd I a, m series with one (a5) and six (m2–6e) mic, respectively, ms present. Abd II a, m, p series with two mic (a6–7), two mac (m3, m5), three mic (p5–7) respectively, el mic and as present; a5 and m2 bothriotricha surrounded by four and four fan-shaped chaetae, respectively. Abd III a, m, p series with one mic (a7), three fan-shaped chaetae (a2–3, a6), two mic (m7i–7), three mac (m3, am6, pm6) and three mic (p6e–7), one mac (p6) chaetae respectively; a5, m2 and m5 bothriotricha with five, two and two fan-shaped chaetae, respectively, as sens elongated, ms present. Abd IV A–Fe series with four mic (A1, A5–6, Ae1), one mac (A3), one mic (B1), one mes (B6), two mac (B4–5), four mic (C1–4), three mic (T1, T5–6), one mes (T7), five mic (D1–3, De3), one mes (D3p), one mic (E4p2), one mes (E4p), three mac (E1–3), one mic (Ee12), three mes (Ee9–11), one mic (F1), three mes (F2–3p), one mic (Fe2), three mes (Fe3–5) chaetae, respectively; T2, T4 and E4 bothriotricha surrounded by four and two (T3) fan-shaped chaetae, respectively; ps and as present, and at least six supernumerary sens with uncertain homology ‘s’ (Fig. 8A); Abd. IV posteriorly with three psp. Abd V a, m, p series with three mic (a1, a3), one mes (a6), one mac (a5), two mic (m5a–5e), three mac (m2–3, m5), five mic (p3a–6ae), one mic (p6e), two mes (ap6, pp6), four mac (p1, p3–5) chaetae, respectively; as and acc.p4–5 present. Ratio Abd III: IV = 1: 2.98–4.82 (n = 6), holotype = 1: 3.81.Legs. Trochanteral organ diamond shape with about 19 spine-like chaetae, plus 2–3 psp one external, one on distal vertex and another (present or absent) on top of posterior spines row of Omt (Fig. 44A). Unguis outer side with one paired tooth straight and not developed on proximal third; inner lamella wide with two teeth, basal pair subequal, m.t. and a.t. absent. Unguiculus with all lamellae smooth and lanceolate (a.i., a.e., p.i., p.e.) (Fig. 44B); ratio unguis: unguiculus = 1.49–1.80: 1 (n = 6), holotype = 1.80: 1. Tibiotarsal smooth chaetae about 1.25× smaller than unguiculus; tenent hair slightly capitate and about 0.54× smaller than unguis outer lamella.Collophore (Fig. 44C). Anterior side with 11 ciliate, apically acuminate chaetae, six proximal (thinner); two subdistal and three distal mac; lateral flap with 11 chaetae, five ciliate in the proximal row and six smooth in the distal row.Furcula. Covered with ciliate chaetae, spine-like chaetae and scales. Manubrial plate with four ciliate chaetae (two inner mac) and three psp (Fig. 44D). Dens posterior face with two or more longitudinal rows of spine-like chaetae about 31–39 external and 18–21 internal, external spines larger and thinner than internal ones. Mucro with three teeth (Fig. 44E), ratio width: length = 0,28 (holotype).Etymology. Honor to Dandara Kettley, Brazilian man, transvestite, murdered in 2017 (Ceará, Brazil) in a homophobia crime.Remarks. Trogolaphysa dandarae sp. nov. resembles T. ernesti, T. formosensis and T. piracurucaensis by chaetae head S5 mac (all other Brazilian cave species with S5 mic); head Pm3 mac as in T. gisbertae sp. nov., but they are different in terms of head ventral proximal collar mac, unguiculus, tenent hair and collophore anterior distal chaetae (5 + 5, smooth pe, capitate, 3 + 3 and 4 + 4, serrate pe, acuminate, 2 + 2, respectively); Th II P3 complex with 6 + 6 and Th III with 3 + 3 mac (6 + 6 and 0 + 0 in T. lacerta sp. nov., T. piracurucaensis, T. ernesti and T. caripensis); T. dandarae sp. nov., T. belizeana and T. jacobyi are the only cave species with 3 + 3 teeth in the mucro. See the comparison among them in remarks of the late species. More

Soil physical–chemical properties and HMs concentrationsThe soil physical–chemical properties and HMs concentrations are summarized in Table 1. The soil mean pH value was 6.17 and ranged from 4.16 to 9.04 in different sites. The samples sites of level for pH ≤ 6.0(acidic soil), 6.0  7.5(alkaline soil) were 51.5%, 36.4% and 12.1%, respectively. The average content of TN, TP and TK were 1.33 g kg−1, 1.16 g kg−1 and 23.6 g kg−1, and ranged from 0.7 g kg−1 to 2.4 g kg−1, 0.22 g kg−1 to 20.8 g kg−1 and 10.3 g k g−1 to 28 g kg−1, respectively.The mean concentration of Cd, Hg, As, Pb, Cr, Cu, Ni and Zn were 0.221, 0.155, 9.76, 32.2, 91.9, 35.2, 37.1 and 108.8 mg kg−1. Except Cd, the average concentration of Hg, As, Pb, Cr, Cu, Ni and Zn exceeded 93.8%, 7.1%, 6.3%, 17.8%, 25.3%, 10.7%, and 32.4% the soil background values for Chengdu, respectively, which indicates that HMs are enriched to a certain extent in soil. The CV of the HMs in the agricultural soils increased in the order Ni(10.1%), Cr(10.9%), Pb(15.4%), As(21.4%), Cd(31.1%), Zn(57.8%), Hg(58.1%) and Cu(59.9%). The exceptionally high variability of Cu, Hg and Zn indicates that these metals differed greatly with respect to different sites, and the existence of abnormally high values is the main reason that the CV was high. It further indicates that Cu, Hg and Zn may be affected by external interference factors. The mean concentration of all HMs in soil were below the risk screening values for soil contamination (GB 15618-2018) (MEEC, 2018), however, the results showed that in 76, 1, 1, 6 and 2 of sample sites the level of Cd, Pb, Cr, Cu and Zn exceeded the risk screening values.Assessment of heavy metal(loid)s pollutionIndex of geo-accumulationThe index of geo-accumulation of HMs in the soil in the study area are shown in Fig. 2. In a descending order of magnitude of Igeo mean value, the eight elements were as follows: Hg(0.18)  > Zn(-0.22)  > Cu(-0.30)  > Cr(-0.36)  > Ni(-0.45)  > Pb(-0.51)  > As(-0.52)  > Cd(-0.82), indicating that the soil HMs Zn, Cu, Cr, Ni, Pb, As and Cd in the study area were generally in a no contamination according to the defined classes, while Hg was in uncontaminated to moderately contaminated.Figure 2Indexes of geo-accumulation, pollution indexes and potential ecological risk indexes of HMs in study aera. Circles at the top and bottom of box plots correspond to the maximum and minimum values, respectively. The square in the box plot is the average value. Horizontal lines at the top, middle, and bottom of the box plot correspond to 75% percentile, median, and 25% percentile, respectively.Full size imageThe Igeo values for Zn, Cu, Cr, Ni, Pb, As and Cd in more than 90% of samples were less than zero, only a few outliers in the soil were classified as moderately contaminated or worse. Which meaning point pollution at those sample sites. However, the Igeo values for Hg in 47.34%, 40.61%,10.66% and 1.40% of samples were belonged to  As  > Pb  > Ni  > Cu  > Hg  > Zn  > Cd. Cr, As and Pb were the largest contributors for both adults and children, accounting for 47.33% and 42.37%, 32.68% and 39.64%, 15.95% and 13.26%, respectively. Indicating that attention should be pain to the Cr, As and Pb elements due to their noncarcinogenic risk. Which was basically consistent with the research results of Bo et al.1 and Bao et al.32. Overall, The HMs of Cr, As and Pb were the main non-carcinogenic factors in soil in the study area, and the risk control of these elements should be strengthened.Carcinogenic risk assessmentThe CR of the HMs are shown in Table 5. Three exposure pathways were considered for Cd and Hg in our study. But As and Pb were considered carcinogenic by ingestion and inhalation, and Cr was considered carcinogenic through inhalation. The TCR mean values were 3.68 × 10−5 for adults and 6.27 × 10−5 for children, obviously were in the range 1 × 10−4 from 1 × 10−6, suggesting the TCR caused by HMs in the study area was acceptable on the whole, but it still exceeds the soil treatment threshold value 10−6. The CR average values through the three exposure pathways were CRing 3.65 × 10−5, CRinh 2.53 × 10−7 and CRderm 1.07 × 10−7 for adults, CRing 6.25 × 10−5, CRinh 1.12 × 10−7 and CRderm 4.50 × 10−8 for adults. Clearly the CRing was much larger than CRinh and CRderm for both adults and children, which indicates that oral ingestion is the major exposure pathway for CR. Which was consistent with the research results of Song et al.31 and Bo et al.1. For single HM, the CR value of Pb and Hg for adults were 2.72 × 10−5 and 8.6 × 10−6, and Pb, Hg and Cd for children were 4.63 × 10−5, 1.48 × 10−5 and 1.36 × 10−6, respectively, within acceptable criterion. Overall, the longterm health effects for adults and children are not serious at current single HM level.In this study, the total contents of HMs in the soils were used to assess health risk, the bioavailability of HMs were not considered, which may have caused the assessment results to be higher than the actual local situation1,31,42. In addition, because the parameters of health risk evaluation for children were set to be more sensitive than those for adults, the non-carcinogenic risk and carcinogenic risk for children were higher than those for adults2,43,44. However, those risks were at acceptable or negligible levels. Therefore, the study area is suitable for safe and clean production of hotbed chives. More

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