Trophic ecology in an anchialine cave: A stable isotope study

Brenda Durán; Fernando Álvarez

doi:10.1371/journal.pone.0315821

. 2025 Jan 3;20(1):e0315821. doi: 10.1371/journal.pone.0315821

Trophic ecology in an anchialine cave: A stable isotope study

Brenda Durán ^1,^2,^#, Fernando Álvarez ^2,^*,^#

Editor: Giorgio Mancinelli³

PMCID: PMC11698404 PMID: 39752427

Abstract

The analysis of carbon and nitrogen stable isotopes (δ¹³C and δ¹⁵N) has been widely used in ecology since it allows to identify the circulation of energy in a trophic network. The anchialine ecosystem is one of the less explored aquatic ecosystems in the world and stable isotope analysis represents a useful tool to identify the routes through which energy flows and to define the trophic niches of species. Sampling and data recording was conducted in one anchialine cave, Cenote Vaca Ha, near the town of Tulum, Quintana Roo, Mexico, where seven stygobitic species endemic to the anchialine caves of the Yucatan Peninsula, plus sediment, water and vegetation samples were analyzed to determine what the main nutrient sources are. We compared our results with two previous studies, one conducted in the same cave and another one from a cave in the same area, both based on the same seven species which are widely distributed in the area. Our study revealed: a) that despite a certain amount of variation in the δ¹³C and δ¹⁵N values of the species through time, both seasonally and interannually, the anchialine isotopic niche is much conserved; b) through contribution models we propose what are the most probable food sources for the studied species and the results confirm previous trophic classifications; and c) that the shrimp Typhlatya pearsei presents very negative δ¹³C values, suggesting their consumption of bacterial sources consistent with a chemosynthetic origin of organic matter. The implications of the new findings show a very stable ecosystem with the shrimp Typhlatya pearsei, as the key species to link chemosynthetic microbial production of organic matter to the anchialine trophic web.

Introduction

Stable isotope analysis (SIA) has significantly contributed to identify the major routes through which energy flows in different ecosystems and has been an important tool to define trophic structure and to identify the number of trophic levels in a given ecosystem [1, 2]. In hard to access ecosystems where sampling is technically difficult, such as the flooded caves of the anchialine ecosystem [3], SIA offers a way to explore the structure of the trophic web through the analysis of samples of sediment, water, organisms, and the surrounding vegetation. The data obtained through this approach can be complemented with occasional and/or opportunistic observations, or through laboratory experiments [4]. In order to follow a logical sequence, we need to know what species occur in the anchialine habitat and what their likely trophic roles are, how nutrients are entering the caves and what the possible sources are, and finally under what degree of variation–spatial and temporal–is this structure operating?

Anchialine ecosystems have a rich and endemic fauna around the world that typically thrive in low nutrient conditions [5]. With a reduced rate of transport of nutrients into the flooded caves [6], the recurrent question has been, how can a diverse fauna with some locally abundant species develop and survive under these conditions? Specially in the Yucatan Peninsula (YP), where flooded caves can form networks spanning hundreds of kilometers [7], the conditions and mechanisms that shape the trophic web operate at various scales. There could be a short distance nutrient input that is present in the cave area adjacent to openings, such as the cenote pool, or cracks that connect the exterior to the aquifer; however, in remote areas inside the caves, hundreds or even thousands of meters from an opening to the exterior, other mechanisms may be operating differently from the direct entry of organic material from the exterior. Thus, identifying the main nutrient sources and the major paths through which energy flow becomes of primary importance.

Anchialine caves around the world are characterized as containing oligotrophic to ultraoligotrophic water masses, a condition that has promoted the appearance of both morphological and physiological adaptations in the fauna to cope with low nutrient concentrations [5, 8]. Nutrient input to the flooded caves of the YP has been associated to rainfall regimes and to the distance of the particular cave section to the shoreline [9]. However, even when seasonal fluctuations occur these cave systems remain nutrient poor [10].

Previous studies of the trophic structure of anchialine communities using SIA [11–16] identified a common pattern where anchialine fauna have δ¹³C and δ¹⁵N values that reflect the influence of the vegetation surrounding the cave entrance, in the YP these dolines or exterior connections to the aquifer are known as “cenotes”. Derived from these data short trophic webs have been described, with a first trophic level composed of filter feeders that consume particulate organic matter or grazers that feed on the biofilm and a second level composed of predatory species [11]. In the caves of the YP the first trophic level includes species such as the mysid shrimp Antromysis cenotensis which typically occurs near the caves’ entrances or even in the twilight zone and feeds on finely particulate organic matter [17] or the shrimps of the genus Typhlatya which possess a feeding apparatus made up of specialized setae to graze on the bacterial mats [18]. The species that compose the second trophic level are opportunistic predators such as the remipedes of the genus Xibalbanus, the brotulid fish Typhlias pearsei or the palaemonid shrimp Creaseria morleyi which have been observed preying on Typhlatya shrimps [5, pers. obs.]. Another significant component of the trophic web in the anchialine caves of the YP is the isopod Creaseriella anops, one of the most abundant organisms in this ecosystem, which acts as a scavenger that rapidly consumes all dead animals and decaying debris [19].

Despite the entry of organic matter to the flooded caves from the outside is evident, at least in passageways adjacent to connections to the exterior, in several instances direct observations and SIA have identified a second possible source of nutrients. In this case the degradation of organic matter by nitrifying bacteria and, depending on the type of cave and available dissolved compounds, the use of methane by chemoautotrophic bacteria to produce organic matter can supply additional nutrients [11, 12]. An important bacterial and archaea diversity has been identified in cenotes and caves of the YP with a clear zonation according to the distance to the coastline and thus the influence of saltwater penetration that creates different geochemical conditions [20]. The overall contribution of bacterial activity to the anchialine trophic web is becoming increasingly relevant as exploration advances and the enormous lengths of the caves in the Yucatan (in the hundreds of kilometers) pose doubts about the effectiveness of water circulation to spread nutrients throughout.

In a few instances, very negative δ¹³C values (<-36‰) have been recorded in the YP caves obtained from crustaceans in general or shrimp that are consistent with their feeding on nitrifying bacteria [13, 15]. Building on these ideas other authors [21] showed that a chemosynthetic route can operate under particular conditions inside anchialine caves, providing a possible explanation for the impoverished δ¹³C values that have been obtained [16]. Previously [11] it was hypothesized that one thermosbaenacean, Tulumella unidens, and the atyid shrimp Typhlatya mitchelli, could be feeding on sulfide-oxidizing and methanotrophic bacteria.

Recent studies using a variety of techniques have identified a chemosynthetic route through the degradation of methane by bacteria to produce organic matter in an anchialine cave near Tulum, Quintana Roo, Mexico [21, 22]. With these important contributions to the understanding of the anchialine trophic web, several critical questions arise. Firstly, considering that there are thousands of flooded caves in the YP, how widespread is this mechanism? That is, the very negative δ¹³C values can be found in all caves in the YP? Secondly, what species are involved in this process and how can their distribution affect such biogeochemical process? Third, do these processes influence the distribution of species creating, where they operate, hotspots of diversity?

Another relevant question regarding the stability of the anchialine trophic web is if it is influenced by seasonal (rainy vs. dry seasons) or interannual changes due, for example, to the large-scale impacts of deforestation or the occurrence of hurricanes. Since SIA reflects what organisms have incorporated in the previous weeks or months, it should be possible to detect these changes to elucidate how the anchialine ecosystem reacts to them. In this study, based in a cenote and its associated flooded cave that is representative of the cave area that develops along the Caribbean coast of Mexico (Fig 1), we compare the δ¹³C and δ¹⁵N values of seven anchialine species (Antromysis cenotensis, Stygiomysis cokei, Tuluweckelia cernua, Creaseriella anops, Typhlatya mitchelli, T. pearsei, Xibalbanus tulumensis), sediment and organic matter in one cave in two seasons (rainy and dry). To explore variation among years, we compare also the new results with the values of a previous study [11], obtained from a cave very close to our study site and based on the same species, and from the cave identical with the present study, obtained 2 years before the recent study [23].

Fig 1 — The map image was obtained at Natural Earth (https://www.naturalearthdata.com/) which is in the public domain. The layer with cenotes is taken from GeoComunes portal, and is in the public domain (http://132.248.14.102/layers/CapaBase:cenotes_completo2).

Materials and methods

The study was conducted in Cenote Vaca Ha and its associated cave, near the town of Tulum in the State of Quintana Roo, Mexico. Cenote Vaca Ha (20º16’14" N, 87º28’49" W) is located 6.67 km from the coastline, the pool is completely exposed and has an area of ~25 m² (Fig 1). Cenote Vaca Ha is surrounded by vegetation, mostly grasses and perennial trees, some palm trees are found in the surrounding areas far from the cenote’s water body, in the rainy season a large swamp area develops that connects to the cenote pool. The site is only used by divers. The cenote was named “Vaca Ha” (cow water) because the land where it was discovered was used as a livestock maintenance site, taking advantage of the cenote pool as a watering hole. Currently the land is private, and divers pay an entry fee. The interior of the cave contains wide passages, enormous columns and a rich decoration of speleothems, the passage reaches a depth of 24 m where it is possible to observe the halocline.

Despite the continuous exploration of flooded caves in the area, and the presence of two of the largest cave systems in the world (Ox Bel Ha and Sac Actun) at a very short distance (< 10 km), Cenote Vaca Ha and its associated cave remains as an unconnected independent system. Notwithstanding the absence of known connections, there is obvious water circulation inside the cave.

Field work

Sediment samples were collected in 50 ml Falcon tubes, water in collapsible 5-liter plastic bags, and stygobitic fauna individually in small vials. Collection of organisms was conducted inside the cave, starting 15 m from the cenote in complete darkness, under permit SAGARPA PPF/DGOPA-084/22. In addition, vegetation samples (leaves) from the rainforest floor in a radius of 50 m around the cenote were also collected randomly with tweezers. The common plants and trees in the area were: Erythrina standleyana, Gliricidia sepium, Brosimum alicastrum, Ceiba aesculifolia and Casearia emarginata. All samples were placed in sterile plastic vials. A mixture of 50 gr of leaves was dried to obtain one sample. Water column profiles were obtained with a YSI EXO 3 sonde programmed to take readings every 15 seconds of: depth (m), temperature (ºC), pH, conductivity (mSc), salinity (ppt) and dissolved oxygen concentration (mg/L).

Samplings were conducted by two cave divers in July 2022 for the rainy season and in March 2023 for the dry season. Every dive lasted approximately 1 h; in case some of the samples or data were missed, a second dive was done the next day to complete the sampling.

Sample processing for carbon content analysis in water and sediments

To estimate the total dissolved carbon, organic and inorganic, 600 ml of water were filtered using pretreated (ignited at 500 ºC for 4 h) glass fiber filters (0.7 μm); from these three 100 ml water subsamples were collected in amber vials. Water samples were taken in the cenote pool and in the cave, in the cave two types of samples were taken, one in the freshwater layer at 8 m depth and one in salt water at a depth of 23 m. These samples were analyzed at the Laboratorio Universitario de Nanotecnología Ambiental (LUNA), of the Instituto de Ciencias Aplicadas y Tecnología (ICAT), UNAM using a Total Organic Carbon Analyzer (Shimadzu TOC-L CSH/CSN).

To obtain the amount of total carbon (organic and inorganic) present in the sediment of the cenote pool and in the cave´s interior, samples were taken with Falcon tubes. Five cave sediment samples were taken, each one 15 m apart starting the first sample in the cenote pool (Fig 2). The samples were dried at 60 ºC for 48 h and analyzed at LUNA of ICAT-UNAM using the same Total Organic Carbon Analyzer as above but with the adapter for solid samples SSM-5000a.

Sample processing for SIA

Sediment samples were treated with hydrochloric acid (HCl) before they were analyzed to eliminate all carbonates following a conventional technique [24]. The sediment and vegetation samples were dried at 60 ºC for 48 h, and then grinded until a fine powder was obtained.

Seven species of crustaceans collected were maintained live in 500 ml plastic containers with filtered (0.45 μm) cave water and aeration for 24 h. During this period the organisms would empty their digestive tracts, and water was changed once to avoid coprophagy [25]. The organisms were then placed in 50 ml sterile vials and frozen at -4°C; the samples were taken to the laboratory, defrosted at room temperature and dried at 60 ºC for 48 h, and then grinded until a fine powder was obtained. When organisms were small (e.g., mysid shrimps, amphipods) more than one organism was used to comply with the minimum weight required for one measurement of the isotopic analysis. With larger organisms (isopods, atyid shrimps, palaemonid shrimps, remipedes) it was possible to prepare one sample from each individual.

All samples, placed in tin capsules, were analyzed in a mass spectrometer (Isotopic Ratio Spectrometer, Isoprime 100TM, CF-EA) at the Stable Isotopes Laboratory of the Geotop Center at UQAM (Université du Québec à Montréal, Montréal, Québec, Canada). Carbon and nitrogen isotope ratios are expressed as δ¹³C to Vienna Pee Dee Belemnite standard and as δ¹⁵N to air.

Data analysis

The δ¹³C and δ¹⁵N values, that is the means for every species, for the dry and rainy seasons were compared directly for each species. To graphically represent the isotopic values of the proposed sources as well as those obtained in the tissue of the consumer species (average values and their standard deviation), we constructed Bayesian mixing models (BMMs) with the MixSIAR R package using δ¹³C and δ¹⁵N as isotopic tracers to estimate the proportional contribution of potential food sources to their diets [26], this package is included in the R software [27]. We used the classic TDF 0.5‰ for δ¹³C and 3.4‰ for δ¹⁵N. The models obtained estimate the proportional contribution of the proposed sources considering the values of the isotopic signal of the consumers and the existing isotopic fractionation. The results are presented as credibility intervals (50, 75 and 95) that describe the range of possible contributions from a particular source (see Fig 5). In this study the use of BMMs is particularly advantageous since the number of species involved in the anchialine trophic web is relatively small and there are one or two trophic levels [28].

To explore how the isotopic niche of the studied species changes through time we used the package SIBER in R to create scatter diagrams of the carbon and nitrogen data and independent ellipses, representing the isotopic niche [29], for each of the analyzed datasets: one from 1997 [11], another one from 2020 [23], and the rainy and dry seasons presented herein for 2022–2023. We used the standard ellipse area (SEA) method to compare between the analyzed groups which shows the 50, 75 and 95% credibility intervals (see Fig 6).

Results

Physico-chemical characterization of the water column

The physico-chemical sonde profiles show a stratified water column with a well-defined halocline at around 21 m, overlapping values are due to the presence of a shallow dome inside the cave which is shallower than the bottom of the cenote’s pool. At the halocline salinity changed from 2 to 35 in less than a meter (Fig 3A). Dissolved oxygen concentration was close to 4 mg/L at the pool’s surface, it dropped to less than 0.5 mg/L inside the cave’s dome, was constant around 3 mg/L in the freshwater layer and then dropped again to less than 1 mg/L in the saltwater (Fig 3B). Temperature was near 25.9 ºC at the pool’s surface, slightly decreasing to 25.7 at the pool’s bottom, it increased about have a degree inside the cave´s dome and then remained constant down to the halocline where it slightly increased (Fig 3C). Finally, pH was about neutral throughout the freshwater layer, except in the cave´s dome where it slightly increased 3 tenths of a unit, it decreased within the halocline and recovered in the saltwater layer (Fig 3D). In general, no important variations are observed between the rainy and dry seasons in any of the four variables measured.

Fig 3 — (A) salinity, (B) dissolved oxygen concentration, (C) temperature, (D) pH.

Total dissolved carbon was highest in the cave’s freshwater layer, decreasing slightly towards the cenote pool and decreasing sharply in the saltwater layer (Table 1). Organic carbon represented 90.5% of all the dissolved carbon, and both organic and inorganic carbon had the same behavior regarding concentration as the total organic carbon (Table 1). Total carbon in sediments decreased steadily along the transect that started in the jungle floor, through the cenote pool and inside the cave to a penetration of 75 m. The amount of total carbon decreased to 38.7% of what was available in the exterior and the organic and inorganic fractions varied inversely, with the former being more abundant in the exterior and cenote pool, and the latter comprising almost all the available carbon inside the cave (Table 2).

Table 1. Dissolved carbon in three sections of the aquifer in Cenote Vaca Ha, the samples had a volume of 100 ml.

TC = total dissolved carbon, OC = dissolved organic carbon, IC = dissolved inorganic carbon.

	Cenote pool	Cave (fresh water)	Cave (salt water)
TC	81.82 mg/L	96.01 mg/L	41.53 mg/L
OC	74.04 mg/L	87.43 mg/L	36.46 mg/L
IC	7.78 mg/L	8.58 mg/L	5.07 mg/L

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Table 2. Total carbon in sediments (organic and inorganic fraction) through a transect inside the aquifer, this transect begins at the cenote pool and is directed to the cave region.

Sampling was carried out every 15 m until completing a distance of 75 m.

	Total carbon in sediments (%)	Organic carbon in sediments (%)	Inorganic carbon in sediments (%)
Tropical forest floor	27.01	26.7	0.24
Cenote pool sediment	20.94	19.68	1.26
15 m	11.71	4.32	7.39
30 m	11.6	0.33	11.3
45 m	11.39	2.86	8.53
60 m	11.05	1.04	10.01
75 m	10.46	0.44	10.02

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SIA analysis

Stable isotope values were obtained for seven anchialine species, all crustaceans, for the dry and rainy seasons. Among the species analyzed are primary consumers (atyid shrimps of the genus Typhlatya, the mysid Antromysis cenotensis), predators (the remipede Xibalbanus tulumensis, the amphipod Tuluweckelia cernua, the mysid Stygiomysis cokei) and a scavenger (the isopod Creaseriella anops). Six of the seven species had δ¹³C values that ranged between -37.8±3.1132 for Typhlatya mitchelli in the rainy season, to -24.0±1.264 for Creaseriella anops also in the rainy season (Table 3). These values are consistent with the feeding on organic matter from the outside that enters the cave. The second atyid species T. pearsei had very negative δ¹³C values (-53.2±1.9629 to -49.4±0.6) which can be explained by the consumption of organic matter derived from bacterial activity, specially methanotrophic bacteria (see Table 4). The δ¹⁵N values for six of the seven species ranged from 8.1±1.4882 obtained for Typhlatya mitchelli in the rainy season, to 14.7±0.3345 for Xibalbanus tulumensis in the dry season (Table 3). Clearly separated from this group is Typhlatya pearsei with extremely low values of -0.3±0.2376 and 1.4±0.4825, for the rainy and dry seasons, respectively (Table 3, Fig 4).

Table 3. Mean values (± SD) of δ¹³C and δ¹⁵N of seven anchialine species collected in Cenote Vaca Ha, first value corresponds to the rainy season and the second one to the dry season, n is the number of samples analyzed.

Species	δ¹³C	δ¹⁵N	n
Antromysis cenotensis	-33.5±0.0565	13.0±1.1234	6
Antromysis cenotensis	-31.37±0.3240	11.2±0.0012	6
Stygiomysis cokei	-28.9±0.1565	13.5±0.0565	2
Stygiomysis cokei	-31.1±0.0587	14.6±0.1873	4
Typhlatya mitchelli	-37.8±3.1132	8.1±1.4882	3
Typhlatya mitchelli	-33.60±0.4	11.50±0.053	10
Typhlatya pearsei	-53.2±1.9629	-0.3±0.2376	2
Typhlatya pearsei	-49.4±0.6	1.4±0.4825	3
Creaseriella anops	-24.0±1.264	10.5±0.7758	2
Creaseriella anops	-32.8±1.2	8.2±0.4305	2
Tuluweckelia cernua	-30.4±0.9627	12.7±0.2157	3
Tuluweckelia cernua	-30.6±0.2341	11.7±0.3468	4
Xibalbanus tulumensis	-35.9±0.3223	12.8±0.0234	2
Xibalbanus tulumensis	-30.9±0.1987	14.7±0.3345	2

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Table 4. Mean δ ¹³C values ± standard deviation for additional possible sources of contribution to the diet of consumers in Cenote Vaca Ha.

POM = particulate organic matter.

	Dry Season	Rainy Season
Source	δ ¹³C	δ ¹³C
POM in the cave	-22.23±0.1915	-22.419±0.5066
Cave sediment	-13.7564±0.3456	-27.6632±0.2916
Nitrifying bacteria	-39.8333±3.4302	-39.8333±3.4302
¹ Methane oxidizing bacteria	-64.3633±5.4242	-64.3633±5.4242
² Methane oxidizing bacteria	-68.3666±5.4271	-68.3666±5.4271
AC (Antromysis cenotensis)	-31.3157±1.2211	-32.8092±0.6088
CA (Creaseriella anops)	-32.8±1.2211	-24.0±1.2647
TM (Typhlatya mitchelli)	-33.60±0.4	-37.8±3.1132

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¹ Reference value taken in Cenote Bang ~ 5 km away from Cenote Vaca Ha [30].

² Reference value from a compilation presented by [31].

Fig 4 — Shaded areas indicate the possible food source for the anchialine fauna.

The δ¹³C values differed among some species, between the rainy and dry seasons and between seasons. Two primary consumers (Typhlatya mitchelli, T. pearsei), one predator (Xibalbanus tulumensis) and one scavenger (Creaseriella anops) showed marked differences between seasons with values differing from -4 to -5 in δ ¹³C; whereas the species that had a similar behavior in both seasons (< -3 in δ ¹³C) were one primary consumer (Antromysis cenotensis) and two predators (Stygiomysis cokei, Tuluweckelia cernua).

The Bayesian mixing model results show the relative contribution of the most likely food sources to the diet of seven species considering the values of the isotopic signal and the isotopic fractionation (Fig 5, Table 4). The diet of the remipede X. tulumensis was largely based on the consumption of the mysid shrimp A. cenotensis and in a lesser proportion of the isopod C. anops and the atyid shrimp T. mitchelli in the rainy season; whereas in the dry season its diet was almost completely composed of T. mitchelli (Fig 5A); this is consistent with the differences observed in δ ¹³C between seasons. The mysid shrimp Stygiomysis cokei also shows a change in the diet between seasons, it consumed about 70% of A. cenotensis, 25% T. mitchelli, and 5% of C. anops in the rainy season, and about 95% the amphipod T. cernua and 5% A. cenotensis in the dry season (Fig 5B); however, the variation in δ ¹³C between seasons is only of -2.2 in δ ¹³C. The amphipod Tuluweckelia cernua shows a similar diet in both seasons consuming preferentially T. mitchelli and secondarily C. anops (Fig 5C), in this case the δ ¹³C values varied only -0.2. The diet of T. mitchelli is composed mainly of nitrifying bacteria in both seasons with a reduced contribution of particulate organic matter and cave sediment (Fig 5D), but its δ ¹³C values varied -4.2. In the case of T. pearsei the feeding on methanotrophic bacteria could explain their low δ¹³C values, with a variation of -3.8 between seasons (Fig 5E). The diet of the mysid A. cenotensis is consistent with the consumption of nitrifying bacteria and particulate organic matter in both seasons (Fig 5F). Finally, the cirolanid Creaseriella anops, shows a preference for cave sediment in the rainy season and a split preference for plant matter and the mysid A. cenotensis in the dry season (Fig 5G), which is reflected in a -8.8 difference in the δ¹³C values between seasons.

Fig 5 — (A) the remipede *Xibalbanus tulumensis*, (B) the mysid shrimp *Stygiomysis cokei*, (C) the amphipod *Tuluweckelia cernua*, (D) the atyid shrimp *Typhlatya mitchelli*, (E) the atyid shrimp *Typhlatya pearsei*, (F) the mysid shrimp *Antromysis cenotensis*, (G) the isopod *Creaseriella anops*. Abbreviations for the probable food sources are: AC, *Antromysis cenotensis*; CA, *Creaseriella anops*; TM, *Typhlatya mitchelli*; POM, particulate organic matter; NB, nitrifying bacteria; MB-BP, methanotrophic bacteria, data from [30]; MB-WF, methanotrophic bacteria, data from [31].

To analyze the seasonal and interannual variation in the isotopic niche displayed by anchialine species we compared the results of this study with another three datasets using Bayesian ellipses (Fig 6). The four datasets show a considerable overlap (> 40% on average) for all species except for T. pearsei which due to very negative δ¹³C values and very low δ¹⁵N values lies outside the cluster formed by the rest of the anchialine species analyzed.

Fig 6 — (A) Bayesian standard ellipses comparing the isotopic niche of anchialine species based on four data sets [11, 23] and from the rainy and dry seasons. (B) density plots of Bayesian estimates of Standard Ellipse Areas (SEA) for the same datasets. Black dots indicate the modes of SEA, and boxed areas—Bayesian 50, 75 and 95% credible intervals; the red “x” indicates the mean.

The trophic level analysis was not conducted as no reliable values of δ¹⁵N could be obtained from the particulate organic matter in the water column or from the sediment in the cave, thus no primary production values were available to conduct the calculations. The N concentrations were below detection by the mass spectrometer.

Discussion

Environmental conditions

The water column conditions inside the cave were similar between the rainy and dry seasons showing little variation. The stratification remained without variation, with a distinct layer inside the dome of the cave, similar to what has been called a shallow halocline [22], which in our case shows changes in temperature, dissolved oxygen concentration and pH, but not in salinity; a homogeneous freshwater layer, from 5 to 21 m depth; and a well-defined halocline at 21–22 m depth. Dissolved carbon in the water column and total carbon in sediments showed a clear inverse pattern in the freshwater layer where organic carbon decreases advancing inside the cave and inorganic carbon increases, while small changes can be seen in the marine water layer due to the different dynamics that operate there. In lowland areas close to the shoreline the YP the aquifer is influenced by high permeability of the substrate and thus high infiltration rates of meteoric water and by the tidally influenced marine water intrusion below the freshwater lens. Conditions are generally stable, but can reflect meteorological events such as strong storms or hurricanes with short-period peaks in water level and flow velocity [22, 32].

Food web structure

Six of the seven anchialine species assessed from Cenote Vaca Ha form a well-defined cloud with δ¹³C values that reflect the consumption of organic matter transported to the cenote from the rainforest floor. Both, the POM in the cave and the cave sediment have isotopic values consistent with the input of organic matter from the exterior. Several authors have obtained similar isotopic values form anchialine fauna in the YP [16, 33]. Interestingly, the comparison of the four datasets considered, from 1993 through 1995 [11], 2019 [23] and this study including the 2022 rainy and 2023 dry seasons, that is a span of 30 years, shows a substantial overlap of the anchialine species isotopic niches. Temporal variation in isotopic niches has been widely documented at various scales, from weekly intervals to multi-year periods [34]. Being that stable isotopes reflect the organism’s diet in relatively short periods of time [35] it is remarkable that the anchialine animal community in two nearby caves, Cenote Mayan Blue [11] and Cenote Vaca Ha ([23] and this study) show such a reduced amount of variation through time. Some species, like the shrimp T. mitchelli, a grazer and primary consumer, remained within a narrow range of variation in both isotopic proportions (-33.0 to -37.8 δ¹³C, 8.1–11.5 δ¹⁵N) through time, whereas others, such as the predatory palaemonid shrimp Creaseria morleyi showed a wider range of variation (-25.13 to -36.4 δ¹³C, 9.24–11.7 δ¹⁵N).

The case of the atyid shrimp Typhlatya pearsei, which has very negative δ¹³C values (-49.4 to -53.2‰), is interesting as these results place the species separate from the main isotopic space where the rest of the species overlap (Fig 6). Clearly, T. pearsei has a different diet, possibly composed, at least partially, of methanotrophic bacteria (Fig 5E and 5K), than those of the rest of the anchialine species which could be related to a spatial segregation or a different feeding behavior. In contrast to T. mitchelli, T. pearsei occurs in more internal sections of the cave where nutrient input from the exterior is limited and consequently could be consuming a biofilm composed by methanotrophic bacteria which have extremely low δ¹³C values [30, 31]. This scenario is consistent with the chemoautotrophic route proposed described from the nearby Ox Bel Ha cave system [21]. Although Typhlatya shrimp, due to their filtering/grazing feeding mode, were the key link assumed to be responsible for the introduction of chemoautotrophically derived organic matter into the food web, the question remained as to which one of the three possible species of Typhlatya was involved in this process. Existing low δ¹³C values for T. pearsei (-35.0 ± 1 to -41 ± 1‰) and T. dzilamensis (-31.0 ± 1 to -44 ± 0.6‰) recorded from different caves and times [16], lack a context to allow for a comparison. The values obtained for T. dzilamensis [16], which generally occurs below the halocline, were not explained fully since the methane concentrations as well as methanotrophic bacteria are present preferentially in the freshwater lens [21].

Although any of the three possible species of Typhlatya present in the area could be feeding on methanotrophic bacteria, since they all have similar feeding apparatuses [18], here we propose, based on our findings and previously reported values of δ¹³C for T. mitchelli, that it is T. pearsei the key species that links the microbial processing of methane with the rest of the anchialine food web. Typhlatya pearsei is relatively rare, despite its wide distribution throughout the YP it always occurs in reduced numbers. In contrast, T. mitchelli is very common occurring from the surface to ~18 m depths in most caves that have been explored [36]. The rareness of T. pearsei could be very significant if it in fact represents one of the necessary components for the chemosynthesis-based food web to operate. The other necessary conditions are: rainfall, a well preserved rainforest on the surface to produce the organic matter that will decompose in the carbonate rock matrix, a cave conformation that allows for the formation of an anoxic freshwater layer next to the cave´s ceiling with methanogenic bacteria, the presence of methanotrophic bacteria in a contiguous oxygenic layer that will use methane to synthesize organic matter, and the presence of Typhlatya shrimp to feed on this microbial mat introducing new organic matter into the food web. As we pointed out, the shrimp responsible for the last crucial step is Typhlatya pearsei, at least in the Caribbean cave area of Mexico [37]. We consider only T. pearsei for this role based on the very negative δ¹³C values it shows and on the fact that this species is morphologically suited to graze on the bacterial mat that forms inside the cave. When more similar data become available, we will be able to determine if there are other species which could be occupying this role.

The tropic level of species was not determined since no reliable δ¹⁵N base values could be obtained. Typically, δ¹⁵N of the particulate organic matter in the water column or from the sediment is used as the base value, where bacteria, protists, algae, decaying plant material, and other small animals constitute a proxy to what could be considered the product of primary production. However, in this case N levels were too low to be detected. Nitrogen sources can be hard to identify as they depend on complex biogeochemical processes, microbial activity, amount of rainfall, and other direct sources [38, 39] and individuals of the same population may even show significant variation [40]. In this case the trophic roles of the studied species can be inferred from their morphology with support from observations in the field. Typhlatya shrimp have modified pereopods to filter feed or graze on the biofilm [18]; the mysid shrimp Antromysis cenotensis is usually seen at shallow depths or near the surface feeding on small POM particles; the remipede Xibalbanus tulumensis is a carnivore, it was recognized as the first venomous crustacean in the world [41]; the palaemonid shrimp Creaseria morleyi is also a predator although it can be an opportunistic omnivore [4]; the cirolanid isopod Creaseriella anops is a scavenger [19]; and both the amphipod Tuluweckelia cernua and the mysid shrimp Stygiomysis cokei due to their size and type of buccal appendages are clearly carnivores. Thus, we can infer the trophic role of these species based on their morpho-functional characteristics and δ¹³C signatures.

Seasonal variation in isotopic niche

SIA has been used to determine changes through time in the diet or trophic position since organisms incorporate, in relatively short periods of time, the isotopic signature of their food items [42, 43]. In various instances differences in isotopic values are the result of migrations or marked changes in the physical conditions of the habitat. We observed significant differences in the δ¹³C values between the rainy and dry seasons in four out of the seven species analyzed. Water movement inside caves can be significant as freshwater slides towards the sea on top of a saline water intrusion that penetrates inland. Seasonal changes due to rainfall or strong tides create a very dynamic system specially in open conduits, as caves are, and more so in areas near the coastline [44]. Changes in salinity, temperature, conductivity, water level and other variables that describe the water column have been recorded in several sites along the Caribbean cave area [22, 32, 45]. However, despite the variations in the physical environment anchialine species seem, through time, to be feeding on the same sources producing similar isotopic signatures.

The small variations observed in this study of the isotopic values suggest the stability of the anchialine ecosystem at least in terms of nutrient input. Seasonal changes in the hydrological conditions of a cave due to rainfall in the same area as that of our study site were reported [22] showing that nutrient concentrations would decrease after strong rainfall due to a higher flow that would disperse them. So even if fluctuations in nutrient concentrations are weather-driven, their origin and composition remain relatively stable.

The obtained results show an anchialine ecosystem that depends on the nutrients generated by the rainforest that develops on the surface influence area of the cave. Nutrients can enter via POM that collects in the cenote pool or through bacterial activity that transforms a variety of compounds into nutrients. In either case, the decaying organic matter from the rainforest is the primary food source for the anchialine species. Our results also show that the isotopic niches have only small variations with a substantial overlap of the community’s isotopic space over time. In a sense, the predictability of the trophic behavior of the anchialine community renders it a very vulnerable one that could collapse as the surface environment is modified. We also foresee that the disappearance of key species, such as the shrimps of the genus Typhlatya, could draw with them complete microbial loops that are unique to this ecosystem, including the described chemosynthetic processing of methane. Refinements on the description of the anchialine trophic web are forthcoming with the use of modern sampling and analysis techniques.

Supporting information

S1 Table. Vertical profile Vaca Ha (dry-rainy season).

Water column physico-chemical variables taken with a multiprobe water-quality sonde for the two seasons.

(XLSX)

pone.0315821.s001.xlsx^{(17KB, xlsx)}

S2 Table. Isotopic values Vaca Ha (dry-rainy season).

Values for δ¹³C and δ¹⁵N of all collected species for the two seasons.

(XLSX)

pone.0315821.s002.xlsx^{(13.6KB, xlsx)}

Acknowledgments

Field work was possible thanks to the help and assistance of O. Cortés, A. Mora, A. Ceballos, B. Espinosa, M. Vázquez, J.L. Villalobos and P. Kovac. We thank D. Planas, A. Adamowics and J.F. Hélie for their assistance during the stable isotope analysis at the Centre de recherche Geotop, Université du Québec à Montréal. We thank J.C. Durán for his help with the elemental analyses of carbon and nitrogen in sediment and water at the Institute of Applied Science and Technology, UNAM.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

BD CONAHCYT graduate scholarship FA grant PAPIIT IN206523 (DGAPA-UNAM) FA grant CONAHCYT Ciencia Básica A1-S-32846. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0315821.r001

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PONE-D-24-24608Trophic ecology in an anchialine cave: a stable isotope studyPLOS ONE

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Additional Editor Comments:

While two of the three reviewers found the quality of the manuscript generally good and required only a minor revision in order to be accepted for publication, a third reviewer highlighted a number of crucial flaws suggesting for a rejection. Personally, I find most of the criticisms made by rev #3 appropriate, even though my opinion is that they do not motivate a rejection. Accordingly, I recommend the authors a major revision of the ms integrating all the minor points raised by rev#1 and #2, and thoroughly considering and accounting for most, if not all the criticisms made by rev#3.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript explores the trophic relationships among species occurring in an anchialine cave. The topic is surely of interest as very little is known. However, the manuscript has several lacks that hamper its further consideration. First, I would suggest the authors to rewrite it and pay attention to the linearity of their writing and to do not forget important information and concepts. Secondly, although the data is limited (a single cave, two seasons) it is anyway of high importance. Unfortunately, data are not analyzed in a proper way and also authors aim to answer unappropriated hypotheses. For example, collecting data from two different seasons is not enough to obtain information on year variability in carbon availability. This lack cannot be fixed using data from a different cave, which may be characterized by different dynamics. I feel that the data collected by the authors has potential, but the manuscript should be surely rewritten narrowing the focus on what can be clearly done.

Reviewer #2: Reviewer comments

The authors present an interesting study on the trophic network of cave fauna in the Yucatan Peninsula using stable isotopes, and the possible sources of energy going into the system. They evaluate seasonal changes in delta-carbon 13 and compare their results with those of two previous studies carried out in the same area. Their findings confirm the importance of the tropical forest surrounding the entrance to the caves, together with that of Typhlatya pearsei as key participants introducing organic matter into the short and oligotrophic webs that characterise flooded caves. The study highlights the use of stable isotopes in subterranean ecological studies and its conclusions contribute to the awareness of these fragile ecosystems.

The aims and hypotheses stated by the authors are clear; the methods used robust and the discussion is interesting and fully and adequately referenced. There are, however, some sections where the English writing could be improved (see some examples below), and I have only a few minor comments that might increase the clarity of procedures and enhance the visual communication of the results:

1) Some basic information on samples and sampling procedure is lacking.

• Line 173, provides the description of the way in which sediment and vegetation samples were processed to determine SIA. However, there is no previous mention of which and how vegetation samples were taken.

• It is not clear how many individuals of each species conformed a single sample: whether it was a single individual in every case, or individuals were pooled to conform a sample.

• It is not clear how many water samples were taken.

2) Table 1. It is unclear why there are no dispersion measures if there were several samples taken. Or was it only one water sample in each section of the aquifer?

3) Table 2. Use “tropical forest” instead of “jungle”. According to the Mat Met, there were more than one sample of sediment taken, but there are no dispersion values.

4) Table 3: Is n the number of individuals of each species in each season? or is it the number of samples with pooling of several individuals, particularly the smallest ones? This information needs to be provided, not only for the sake of repeatability, but because results should be interpreted in the light of that information.

5) Figure 4. A suggestion would be to change the colouring of the different samples in Figure 4: use letters to identify names of authors or studies (only have four different ones: Pohlman, Duran, rainy and dry seasons (present study); use colours for different species or groups of species according to feeding habits. This will make it easier to visualize samples pertaining to similar trophic levels and food sources.

6) Table 4 shows that the two-way ANOVA applied to the delta carbon data was strongly unbalanced; most surely it has high heterogeneity of variance. When there is a violation of the ANOVA assumptions, the results of the statistical tests are unreliable. Rather than going into trying to conform to these assumptions, I would recommend asking whether all of these tests are needed (7 Tukey tests + at least 1 F-test for the interaction term) only to conclude that delta-carbon 13 values differ between seasons in some species, but not in others? Furthermore, there is no ecologically relevant pattern to such differences (e.g. filter feeders differ between seasons, but predators don’t). Wouldn’t it be equally informative to merely describe differences in terms of the statistical parameters of the various samples (means, medians, dispersions) and avoid insisting on a statistical test that produces unreliable results? Take for example the p value of 1 for the Tukey test on Tuluweckelia cernua…such a result means that the null hypothesis is 100% certain… that makes no sense!

7) Line 298: One would expect that the species in which delta-carbon values changed among seasons (ANOVA results), would also show differences in the contribution of the various food sources from the rainy to the dry season in the Bayesian mixing models.... Was that the case? If not, why? To briefly compare these results would help consistency and integration of all statistical methods used in the study.

8) Figure 5. Legends and axis information is too small and the graphs are extremely difficult to read. Also, I would suggest the graphs to be ordered in such a way to facilitate the comparisons that are described in the text: between seasons, within each species. So, maybe show graphs of the two seasons for each species side by side.

9) Because Bayesian mixing models (as Bayesian statistics in general) are uncommon, a brief description of what these models attempts to achieve would improve the clarity of this section and allow the readers to better interpret these results.

10) From the Results section onward, there is a bit of confusion between the results obtained in the present study and those from previous works. This is particularly true in Table 6. This is an important aspect that needs to be attended, since it involves the analysis and discussion of changes in delta carbon values through different scales, and the conclusions drawn from them. For instance, were the previous and present studies carried out in the same seasons? If not, would the marked differences between seasons invalidate the comparison amongst years?

11) Line 421: Is T. pearsei the one and only species in that role? Are there no other candidates? It would be interesting to present and discuss other possible candidates. Maybe there is one that was not considered or sampled by the authors in the present study.

Some examples of language use that can be improved:

• Line 18: It should say “flows”

• Line 80: Use “Despite” instead of “Although”

• Line 107: It should say “Secondly, what species are involved in this process and how can their distribution affect such biogeochemical process…

• Line 115: Maybe change the phrase to “…possible to detect these changes to elucidate how the anchialine ecosystem reacts to them.”

• Line 153: Use approximately instead of “about”, and separate this phrase from the next using a semicolon.

• Line 220: Temperature was “near” 25.9 ºC.

• Line 225: Because Profiles are not statistically analysed, I should be better to use a word different from “significant”, given its strong statistical connotation.

• Line 360-365: This sentence is too long and difficult to read and understand. Consider re-writing it.

Reviewer #3: The manuscript presents a very valuable study focused on the identification the routes through which energy flow and to define the trophic network and niches of seven stygobiotic crustaceans in an anchialine cave Cenote Vaca Ha near Tulum in Quintana Roo, Mexico. It is based on analysis of carbon and nitrogen stable isotopes (δ13C and δ15N). Since the anchialine ecosystem is one of the less explored aquatic ecosystems in the world, this study is thus very original. Especially cenotes in Yucatán Peninsula connected with sea water are very suitable for such investigations. In addition to stygobiotic crustaceans, sediment, water and vegetation samples were analysed. The recent data were compared with two previous studies, both based on the identical species. This important contribution presents results on trophic niches of the seven crustaceans and observed that the shrimp Typhlatya pearsei is the key species to link chemosynthetic microbial production of organic matter to the anchialine trophic web. The results also showed that the isotopic niches had only small variations with a substantial

overlap of the community’s isotopic space over time. The authors observed substantial differences in animal isotope data between rainy and dry seasons.

The manuscript is very well written and concise. The study goals are clearly defined, used methods of data collection and analysis are appropriate. Combination of recent, new data with the previous observation allowed evaluation of the community trophic niches across several years.

I found two shortcomings to deal with:

1) low quality of uploaded pictures and so it was difficult to follow their meaningful value, especially Fig. 3 and Fig. 5,

2) it is necessary to stress in Introduction that nutrient scarcity is characteristic of deep cave zones and served as natural force for evolution of troglo-/stygobiotic animals, using relevant source(s), such as Encyclopedia of Caves, Cave Ecology (2018) or other basic cave biology monographs.

I found also several corrections to deal with further, all listed below.

I consider this manuscript as suitable for publication in PLoS ONE after minor revision.

Corrections

Abstract, line 17-18

Correct „energy flow“ to „energy flows“

The same in Introduction, line 37

Introduction, lines 80-85

Reformulate this text, since hardly to read. Try to divide the long sentence to make text more understandable.

Introduction, lines 108-109

Reformulate the text: “Third, do these processes influence the distribution of species creating.....“

Introduction, line 120

Reformulate the text: “….from the cave identical with the present study, obtained two years before the recent study (Durán 2020).“

Materials and Methods, lines 131-132

Reformulate the text: “The interior of the cave contains wide passages, enormous columns...“

Materials and Methods, lines 201-203

The following sentence should be associated to a respective figure or a table: “The results are presented as credibility intervals (50, 75 and 95) that describe the range of possible contributions from a particular source.

The same for the following paragraph of text.

Discussion, lines 368-369

Reformulate text: “…the consumption of organic matter transported to cenote from the rainforest floor.“

Line 370 – change “entering” with “input”

Line 471 – It would be better to write as: “…is the primary food source for the anchialine species.“

Table 1, lines 244-245

Reformulate the text: „the samples had a volume of 100 ml“

Table 2, lines 249-250

Formulate text in past tense.

Table 2, lines 251-252

Abbreviations were not used in the table, thus it is incorrect to provide them (TC, OC, IC) – erase this part table caption.

Figures 6, line 343

Correct text to „the red “x” indicates the mean“

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Ľubomír Kováč

**********

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Attachment

Submitted filename: PONE-D-24-24608_rev.pdf

pone.0315821.s003.pdf^{(1.4MB, pdf)}

PLoS One. 2025 Jan 3;20(1):e0315821. doi: 10.1371/journal.pone.0315821.r002

Author response to Decision Letter 0

25 Oct 2024

Response to reviewers

Journal requirements:

1. We followed the style templates for “main body” and title, authors, affiliations”.

2. We have stated in the cover letter that “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript”.

3. We removed all funding-related text from the “Acknowledgements” section. We have now included the funding information in the cover letter.

4. We have uploaded two supporting information files to complete the analyzed in this study.

5. – (this was skipped in the editor’s letter).

6. An improved version of Figure 5 has been uploaded.

7. We have created a new map for Figure 1, which now contains only elements that can be used freely.

Reviewer 1 (marked copy).

Line 20. The term “unique” was changed for “endemic”.

Line 21. We added at the end of the sentence “analyzed to determine what the main nutrient sources are”, to complete the idea.

Line 23. We now specify that the seven species we studied are the same that were studied in the other two previous studies (line 33 in the new version).

Line 25. The reviewer asks what we mean by “anchialine niche”, now it reads “anchialine isotopic niche”, clarifying the idea.

Lines 25-27. Subsection “b” of this sentence now reads “. . . . confirm previous trophic classifications; . . . “. The idea is that based on morphology alone the studied species, which are endemic and rare, were assigned by different authors a hypothetical trophic role, our study contributes with the contribution models additional evidence of what the trophic role is for each one.

Line 30. The reviewer asks what is original about our findings, I will refer to lines 23-30 of the marked copy. Subsection “a”, no previous study have compared the δ13C and δ15N values of anchialine fauna through time, which is a central point in our study; “b”, as mentioned above, anchialine species are rare and their trophic roles have been inferred from anatomical attributes and a few observations at the community level, now we analyze their trophic roles through contribution models based on stable isotope data; “c”, no previous study have proposed which of the anchialine species in the YP could be the main link in a microbial based food web, including methanotrophic bacteria, and thus here we present evidence that points to Typhlatya pearsei as the species that has this crucial role.

Line 38. The word “visualize” has been changed for “identify”.

Line 40. The suggested reference “Mammola et al., 2021” was included in the sentence.

Line 48. The reviewer assumes that because our study site is in the Yucatan Peninsula the water in the caves have more organic resources (nutrients) compared to temperate regions. Water quality in the flooded caves of the YP has been many times considered as oligotrophic or even ultraoligotrophic in preserved areas (Álvarez et al., 2023. Anchialine Fauna of the Yucatan Peninsula: Diversity and Conservation Challenges. In: Jones, R.W., Ornelas-García, C.P., Pineda-López, R., Álvarez, F. (eds) Mexican Fauna in the Anthropocene. Springer, Cham. https://doi.org/10.1007/978-3-031-17277-9_13; Torres-Talamante et al., 2011, The key role of the chemolimnion in meromictic cenotes of the Yucatan Peninsula, Mexico. Hydrobiologia 677, 107–127 https://doi.org/10.1007/s10750-011-0746-9). Of course, intensive development in the region has contaminated to different extents specific sections of the aquifer as it occurs around the world, but large portions of the flooded caves network remain in good condition with very low nutrient concentrations (e.g., Metcalfe et al., 2011 – Environmental Pollution 159:991-997). In any case, the evolution of the anchialine fauna has occurred in low nutrient conditions in all anchialine caves around the world (Iliffe Alvarez, 2018 – Research in anchialine caves in O., Kováč, Ľ., Halse, S. (eds) Cave Ecology. Ecological Studies, vol 235. Springer, Cham. https://doi.org/10.1007/978-3-319-98852-8_18).

The reviewer also states that this cave is “less divergent from the surface environment”. We do not know on what data is the reviewer founding this assertion. Our opinion and that of many authors is that the anchialine cave environment is unique and cannot be compared to the surface environment.

Line 55. The correction has been made, we changed “different” for “differently”.

Line 57. The sentence was deleted and incorporated in the last paragraph of the Introduction.

Line 64. The reviewer suggests that the definition of “cenote” here is not necessary because it was mentioned lines above. However, since we moved the sentence (line 57) where this was mentioned, now we feel that the definition is necessary.

Line 89. The reviewer comments: “you are jumping from one topic to the other and the reading is not linear”.

Paragraph from lines 36 to 44, topic – stable isotope analysis could be useful to study the anchialine trophic web

Paragraph from lines 45 to 59, topic – anchialine caves have a rich fauna and are characterized by low nutrient concentrations.

Paragraph from lines 60 to 79, topic – previous studies of anchialine fauna in the Yucatan have described the possible trophic roles of several species.

Paragraph from lines 80 to 92, topic – SIA has identified that nutrients could also enter the anchialine environment through microbial activity.

Paragraph from lines 93 to 100, topic – from SIA data it is possible to link microbial activity to the trophic role of certain species.

Paragraph from lines 101 to 110, topic – recent studies that identify the existence of chemosynthesis in the anchialine environment support these results.

Paragraph from lines 111 to 120, topic – summary of relevant questions that we are addressing in this study.

This is the sequence of ideas that are dealt with in the introduction. We go from general to particular ideas; however, to clarify this sequence we added the following sentence at the end of the first paragraph: “In order to follow a logical sequence, we need to know what species occur in the anchialine habitat and what their likely trophic roles are, how nutrients are entering the caves and what the possible sources are, and finally under what degree of variation – spatial and temporal – is this structure operating?

Line 111. We agree with the comment. However, we deem improbable that the anchialine cave environment could suffer the invasion of “allochthonous (epigean)” species due to an increased nutrient input. Rather, the most probable effect would be the loss of native species.

Line 117. We compare the results of this study with a previous one conducted in the same cave two years before and with another one conducted 18 years ago with the same species in a nearby cave. We are aware of the shortcomings of this comparison, but this the only other comparable study available.

Line 141. We are aware of the limitations of the comparison. However, using a study conducted in the same way, with the same species, in a similar cave, only a few kilometers away, represents a good opportunity to gain knowledge about the scale of temporal variation in the trophic structure of this community.

Line 145. More detail on the sampling of organisms has been added (lines 188-189 in the new version)

Line 148. Up to 50 m around the cenote, this has been included in the text.

Line 148. There are fish in the cenote pool, mainly of the genus Astyanax, that eat all insects and other small animals that fall into the pool. Whereas leaves and other plant material sink and collect in the bottom undergoing a slow decaying process. During this time is when it can be transported into the cave.

Line 190. See comment number 6 under Reviewer 2.

Line 213. As is shown later in the section and in Figure 3, there is one complete water quality profile for each one of the samplings.

Line 256. The studied species are now mentioned in the last paragraph of the Introduction.

Line 260. We are just referring to Table 3, mentioning the species that have the maximum and minimum values for each isotope.

Line 284. See comment 6 of Reviewer 2 below.

Line 299. We are referring to Figure 5A, this is a result of our study.

Reviewer 2.

1. Line 173. The reviewer asks for more details on the collecting methods and sample sizes.

Vegetation samples consisted of leaves lying on the rainforest floor coming from the more common trees around the cenote. The most common species around the cenote are listed in the manuscript.

More details of how the water samples were obtained were added. Three filtered subsamples were obtained from the pool, cave freshwater and cave salt-water.

Regarding the samples for SIA, we explain that in the case of small species (mysid shrimps and amphipods) several organisms were used to obtain the required sample for the analysis. With larger species (isopods, atyid and palaemonid shrimps, remipedes) one organism was used to obtain one sample.

2. Table 1. There are no dispersion measures because there was one water sample for each section of the system.

3. Table 2. There were 7 sediment samples taken and analyzed to cover the transect from the rainforest floor to 75 m inside the cave. The idea was to determine how the organic and inorganic carbon fractions varied from the outside to inside the cave.

4. Table 3. We now specify that “n” is the number of samples analyzed, not the number of organisms. We explained in Mat Meth that we used several organisms for one sample in case they were very small (mysid shrimps and amphipods) and that we used one organism per sample with large specimens.

5. Figure 4. We have modified the figure to improve its clarity.

6. Table 4. The reviewer questions the usefulness of the ANOVA used to test for differences in isotopic values between species and between seasons. Since the number of values is limited due to the difficult conditions to sample this environment and the terms of the ANOVA cannot be improved, we follow his/her advice and have eliminated this analysis from the manuscript. Instead, we added more detail to the direct comparison of δ13C values.

7. Line 298. Yes, this is a very pertinent comment by the reviewer. We have now improved the paragraph where the results of the contribution models are presented. We added the information that allows the reader to know in which cases a change in the diet composition occurs with a change in the δ 13C values of the species. As is now presented, in some cases the δ 13C values change with the diet and in others it doesn’t.

8. Figure 5. The figure has now been improved as suggested.

9. Bayesian mixing models are described in the “Data analysis” section of Materials and Methods. We expanded this explanation and added a reference.

10. The reviewer comments on the data presented in Table 6 (Table 4 in the new version), which might be confusing. The isotopic values from other sources that are used for comparisons need a more detailed explanation. We have added notes to Table 4 to improve the clarity.

11. This is an interesting question by the reviewer. The reviewer asks if Typhlatya pearsei is the only species that has the role of consuming methanotrophic bacteria. Our data shows that in this cave it is the only species that can have such a role. We have added a couple of sentences in the Discussion to emphasize this point (lines 492-496 of the new version).

Line 18. Correction done.

Line 80. Correction done.

Line 107. Correction done (line 126 in the new version).

Line 115. Correction done (line 134 in the new version).

Line 153. Changes done (line 185 in the new version).

Line 220. Change done (line 269 in the new version).

Line 225. Change done (line 275 in the new version).

Lines 360-365. Sentence too long. The sentence has been divided to improve clarity (lines 435-438 in the new version).

Reviewer 3.

1. Figures 3 and 5 have been modified and improved. The rest of the figures have been checked

2. We have added a paragraph in the Introduction emphasizing the nutrient-poor condition of anchialine caves in the Yucatan.

Lines 17-18. Corrections done.

Lines 80-85. The sentence has been divided as it was too long and hard to read (lines 105-111 in the new version).

Lines 108-109. The sentence was modified as suggested (lines 135-136 in the new version).

Line 120. The sentence was modified as suggested (lines 155-156 in the new version).

Lines 131-132. The sentence was modified as suggested (line 168 in the new version).

Lines 201-203. The reviewer suggests that the description of the graphs used to represent the results of the Bayesian mixing models (BMM) presented in the “Materials and methods” section should be associated to a figure. When the results of the BMM are presented they are associated to a figure (paragraph starting on line 356 of the new version). In any case, we added a call to Fig 5 in that paragraph, and a call to Fig 6 in the next paragraph.

Lines 368-369. The sentence was modified as suggested (lines 455-456 in the new version).

Line 370. Change done (line 457 in the new version).

Line 471. Correction done (line 568 in the new version).

Table 1, lines 244-245. Correction made (line 307 in the new version).

Table 2, lines 251-252. Correction made (line 315 in the new version).

Figure 6, line 343. Correction made (line 432 in the new version).

Attachment

Submitted filename: response to reviewers.docx

pone.0315821.s004.docx^{(25.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0315821.r003

Decision Letter 1

Giorgio Mancinelli

2 Dec 2024

Trophic ecology in an anchialine cave: a stable isotope study

PONE-D-24-24608R1

Dear Dr. Alvarez,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Giorgio Mancinelli, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

After a second revision round, the current version of the manuscript successfully integrates all the points raised by the reviewers and is now acceptable for publication in PLOS ONE.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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PLoS One. doi: 10.1371/journal.pone.0315821.r004

Acceptance letter

Giorgio Mancinelli

6 Dec 2024

PONE-D-24-24608R1

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Vertical profile Vaca Ha (dry-rainy season).

Water column physico-chemical variables taken with a multiprobe water-quality sonde for the two seasons.

(XLSX)

pone.0315821.s001.xlsx^{(17KB, xlsx)}

S2 Table. Isotopic values Vaca Ha (dry-rainy season).

Values for δ¹³C and δ¹⁵N of all collected species for the two seasons.

(XLSX)

pone.0315821.s002.xlsx^{(13.6KB, xlsx)}

Attachment

Submitted filename: PONE-D-24-24608_rev.pdf

pone.0315821.s003.pdf^{(1.4MB, pdf)}

Attachment

Submitted filename: response to reviewers.docx

pone.0315821.s004.docx^{(25.1KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Trophic ecology in an anchialine cave: A stable isotope study

Brenda Durán

Fernando Álvarez

Roles

Abstract

Introduction

Fig 1. Map of the Yucatan Peninsula, Mexico, with cenotes shown in blue circles, and Cenote Vaca Ha indicated by a red circle.

Materials and methods

Field work

Sample processing for carbon content analysis in water and sediments

Fig 2. Schematic representation of Cenote Vaca Ha showing the different sections of the system, the two main water masses and the sites where water samples were taken for carbon measurements.

Sample processing for SIA

Data analysis

Results

Physico-chemical characterization of the water column

Fig 3. Physico-chemical profiles of the water column in Cenote Vaca Ha for the rainy and dry seasons.

Table 1. Dissolved carbon in three sections of the aquifer in Cenote Vaca Ha, the samples had a volume of 100 ml.

Table 2. Total carbon in sediments (organic and inorganic fraction) through a transect inside the aquifer, this transect begins at the cenote pool and is directed to the cave region.

SIA analysis

Table 3. Mean values (± SD) of δ13C and δ15N of seven anchialine species collected in Cenote Vaca Ha, first value corresponds to the rainy season and the second one to the dry season, n is the number of samples analyzed.

Table 4. Mean δ 13C values ± standard deviation for additional possible sources of contribution to the diet of consumers in Cenote Vaca Ha.

Fig 4. Biplot of mean ± standard error of δ 13C and δ15N values for four datasets: In green the one conducted from 1993 to 1995 [11], in blue the one from 2019 [23], and rainy (black) and dry (red) seasons from this study in 2022.

Fig 5. Bayesian mixing model results to estimate the most probable diet sources.

Fig 6. Comparison of the isotopic niche of anchialine species.

Discussion

Environmental conditions

Food web structure

Seasonal variation in isotopic niche

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Giorgio Mancinelli

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Author response to Decision Letter 0

Decision Letter 1

Giorgio Mancinelli

Roles

Acceptance letter

Giorgio Mancinelli

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Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Table 3. Mean values (± SD) of δ¹³C and δ¹⁵N of seven anchialine species collected in Cenote Vaca Ha, first value corresponds to the rainy season and the second one to the dry season, n is the number of samples analyzed.

Table 4. Mean δ ¹³C values ± standard deviation for additional possible sources of contribution to the diet of consumers in Cenote Vaca Ha.

Fig 4. Biplot of mean ± standard error of δ ¹³C and δ¹⁵N values for four datasets: In green the one conducted from 1993 to 1995 [11], in blue the one from 2019 [23], and rainy (black) and dry (red) seasons from this study in 2022.