The fish assemblage of a Mexican protected spring system dominated by exotic fishes

Springs, as sensitive freshwater ecosystems, are particularly vulnerable to anthropogenic factors such as water extraction, pollution, and climate change. Las Estacas spring in Central Mexico, managed as a water park within a state reserve, serves as an ideal space for environmental education and conservation efforts. Snorkel surveys identified 14 fish species, with 70% being invasive and most of these come from ornamental fish. We computed multiple diversity indices and evaluated sampling completeness through rarefaction analysis. The fish assemblage was dominated by Xiphophorus helerii and Amatitlania nigrofasciata with 23% and 21% of the sample respectively, it exhibited moderate diversity (Shannon H’ = 1.955) with relatively even species distribution (Pielou’s J’ = 0.741). Rarefaction analysis confirmed adequate sampling effort, with the curve approaching asymptote at approximately 3,501 individuals (95% of observed richness). The findings provide baseline biodiversity metrics essential for conservation monitoring and ecosystem management. To address this, a rewilding project is now in place, with the goal to establish a fish assemblage dominated by native species through habitat management, exotic species control, native species introduction, and education.

Freshwater ecosystems account for less than 1% of the planet’s surface, are home to 11% of all animal species and 5% of all plant species (1), and provide important global ecosystem services that contribute to human well-being and the livelihoods of many people (2).

Springs are among the most sensitive freshwater ecosystems, and despite being abundant globally, many are disappearing due to local and global anthropogenic factors, such as water extraction, pollution, recreational use, habitat alterations, and climate change (3). This is unfortunate because springs are considered “hotspots” that allow species to remain during past climate change, and many of them will be stable hydrological refuges in the future in the face of current anthropocentric climate change (4). This has led various international organizations to call for the global protection of springs, which should be considered a distinct group of ecosystems that deserve special conservation attention (3).

Las Estacas spring is found in Central Mexico, it is managed and operated as a Water Park, that is in a State Reserve with the same name, that was decreed on June 22, 2008 (5). Due to the influx of visitors who enjoy its crystal clear waters, the Las Estacas Water Park represents an ideal space for environmental education, especially in terms of the conservation of freshwater aquatic species, which is uncommon in Mexico.

Las Estacas is not a conventional water park; for more than 20 years, it has implemented a conservation project that promotes sustainable practices, environmental protection, and the species that live there (6). Although all these efforts have managed to maintain the quality of the water and beauty of the site, the aquatic environment is dominated by exotic species that come from extensive and ornamental aquaculture.

To reverse this situation, the owners of Las Estacas Water Park, in collaboration with researchers from the Autonomous University of the State of Morelos, are developing a rewilding project that seeks to reduce the populations of exotic fish species and to reintroduce native species of the Balsas River basin to create a sanctuary for these aquatic organisms. The first part of this intervention consists of knowing the diversity of fish species that occur in spring as well as their relative abundance, which will serve as a basis for the development of a comprehensive strategy for the sustainable management of its biodiversity.

Study area

The study was carried out in a spring fed stream, localized within “Las Estacas” State Protected Area, in Morelos, Mexico. The coordinates of the spring are 18° 44’ N; 99°06’48″ W (DMS), at an altitude of 951m above sea level (7). The stream is a tributary of the Yautepec River, Amacuzac sub-basin, which is one of the runoffs that drain from the State of Morelos to the Balsas River system in Central Mexico (8).

Based on the classification by Stevens et al. (9), the spring is considered an upland hillslope type, with an average flow rate of 6,750 l/s, with minimal variations between the dry and rainy seasons (10). The water arises in the site known as “El Borbollón” (Figure 1a) that creates a stream of clear waters, which has a stretch of one kilometer within a Water Park (Las Estacas) that is mainly used for recreation, to later be channeled by the “Agro Siglo XXI” Irrigation District in southern Morelos. Due to the agricultural and recreational uses given to the water, the spring is periodically monitored by the Mexican environmental authority, so its water quality parameters are well known: water temperature ranges between 22-23.7° C, pH between 6.87-7.06, conductivity between 1,242-1,345 μS/cm and Because of its chemical composition, it is classified as having calcium-sulfated-bicarbonate water (11). An evaluation of the bathymetry of the study area (Figure 1e) showed that the maximum depth in the stream is 8.5 m, at the site known as “Poza Azul,” while the depth in most of the stream ranges from 1.5 to 3.7 m. The deepest area of “El Borbollón”, where the water emerges, has a depth of 6.4 m (12).

Over the years, seeking to promote tourist activity, a good part of the bank of the stream has been modified to stabilize slopes and retain soil through the placement of sandbags so that the littoral zone in most of the stream is vertical; however, it maintains acceptable conditions that favor the presence of aquatic species. The stream bed is composed mainly of sandy and rocky substrates (Figure 1d), although in some areas, it has silt (Figure 1b). There are dense patches of Vallisneria americana as well as some other Cabomba palaeformis (Figure 1c) that serve as a habitat for fish and macroinvertebrates. Arredondo et al. (13) reported the presence of 28 species of aquatic plants in Las Estacas, belonging to 23 genera and 19 families. As can be seen in the photographs (Figure 1) along its course, the stream is bordered by riparian vegetation.

There are few published recollections of the fishes from Las Estacas; a popular publication (6) mentions the presence of nine species, but several other invasions have been introduced in the region over the years, which are now in the spring (14).

Fish assemblage evaluation

To characterize ichthyofauna, snorkel surveys were used to study fish assemblages by quantifying the relative abundance of each species present. This technique has been proven to be effective for this type of evaluation (15–17). Diurnal fish counts were calculated from April to July 2023 in six 20 meter reaches of the spring, and due to the amplitude of the channel, the expansion estimate method (18) was employed, where four trained snorkelers with 35–15 years of experience with regional freshwater fishes counted the fish, and their observations were then summed for the total count of the reach sampled. Additionaly photographs and videos of the fish were taken using GoPro 10 & 11 cameras throughout the study, to serve as taxonomic validation for species identification, and these visual records were doposited at the fish collection of the University of Morelos. Snorkel surveys also allowed us to obtain information related to habitat use for each species.

We computed a comprehensive suite of alpha diversity metrics to capture different facets of community structure (19): 1) Species Richness, Observed species richness (S), Margalef’s richness index (d) (20), Chao1 estimator for total richness (21); 2) Evenness and Dominance, Shannon diversity index (H’) (22), Pielou’s evenness (J’) (23), Simpson’s dominance (D) and diversity (1-D) (24), Inverse Simpson index (1/D), Berger-Parker dominance index (25). All indices were calculated using standard ecological formulas. 3) Rarefaction Analysis, to assess sampling completeness, we generated individual-based analytical rarefaction curves following Hurlbert’s method (26). Expected species richness E[S(n)] was calculated for sample sizes from 1 to 4,521 individuals, with 95% confidence intervals computed using variance estimates (27). The threshold for 95% saturation was determined as the sample size at which expected richness reached 95% of observed richness. Community structure was visualized through rank-abundance curves (Whittaker plots) displaying species relative abundances on a log scale (28).

Fish diversity and relative abundance

As a result of fish surveys in Las Estacas Spring, 14 species of fish belonging to 6 families and 13 genera were recorded. Ten of these species are exotic (Table 1), and only four are native to spring (Astyanax mexicanus (De Filippi, 1853), Amphilophus istlanus (Jordan and Snyder, 1899), Ictalurus balsanus (Jordan and Snyder, 1899), and Poecilia sphenops (Valenciennes, 1846). Poecilids are represented by five species, four which are exotic to the site, and are naturally distributed in river systems along the Gulf of Mexico region (Pseudoxiphophorus bimaculatus (Heckel, 1848), Poeciliopsis gracilis (Heckel, 1848), Xiphophorus helerii Heckel, (1848), and Xiphophorus variatus (Meek, 1904)); the last Poecilid is Poecilia sphenops, which is native to the Balsas River basin. Cichlids are also represented by five species Amatitlania nigrofasciata (Günther, 1867) and Andinoacara rivulatus (Günther, 1860) from Central and South America, Oreochromis niloticus (Linnaeus, 1758) from Africa, Thorichthys maculipinnis (Steindachner, 1864) from the Papaloapan River Basin, and the native Amphilophus istlanus. The Acestrorhamphidae family is represented by one species native to the Balsas River, Astyanax mexicanus. There are two catfish species, the native Ictalurid Ictalurus balsanus and an armored catfish from South America, Pterygoplichthys disjunctivus (Weber, 1991). It is important to note that the remaining Cyprinid species (Cyprinus carpio) Linnaeus, (1758), was only found in an irrigation canal adjacent to the main spring channel.

Having found species from other regions of Mexico, Eurasia, Africa, and Central and South America, and that invasives dominate with 70% of the sample, reflects on the lack of attention that has been paid to the aquatic fauna of the spring and highlights the need to establish management actions to revert this situation.

By means of the snorkel surveys 4,521 fishes were counted, and results show that the fish assemblage was dominated by Xiphophorus helerii and Amatitlania nigrofasciata with 22.5% and 21% of the sample respectively (Figure 2), followed by two native species Poecilia sphenops and Astyanax mexicanus with 15% each. Tilapias (Oreochromis niloticus) represented only 11% of the sample, but were conspicuous due to their size. Poeciliopsis gracilis represented 7% of the sample, followed by Andinoacara rivulatus and Pseudoxiphophorus bimaculatus, with 4% each. Another group of six species (n=36) was occasionally observed, representing less than 1% of the sample (Cyprinus carpio, Pterygoplichthys disjunctivus, Ictalurus balsanus, Xiphophorus variatus, Thorichthys maculipinnis, and Amphilophus istlanus).

The rank-abundance curve revealed a moderately steep decline, indicating a community dominated by a few abundant species with several rare taxa (Figure 3). The most abundant species accounted for 22.5% of total individuals (Berger-Parker dominance = 0.225), while the rarest species occurred as singletons or doubletons.

Alpha diversity metrics

The community exhibited moderate to high diversity across multiple indices Species richness: S = 14 species (observed); Chao1 = 17.0 species (estimated); Shannon diversity: H’ = 1.955; Pielou’s evenness: J’ = 0.741; Simpson’s diversity: 1-D = 0.839; Inverse Simpson: 1/D = 6.205; Margalef’s richness: d = 1.545; Berger-Parker dominance: 0.225.

The Shannon diversity value of 1.955 indicates moderate diversity, while Pielou’s evenness of 0.741 suggests relatively balanced species abundances. The Simpson’s diversity index (1-D = 0.839) confirms high probability of encountering different species in random draws. The Chao1 estimator predicted 17 total species, suggesting 3 additional rare species may be present but undetected (21).

Sampling adequacy

The individual-based rarefaction curve approached asymptote, indicating adequate sampling effort (Figure 4). The curve reached 95% of observed richness (13.3 species) at approximately 3,501 individuals, representing 77.4% of total sampling effort. The 95% confidence interval narrowed substantially beyond 2,000 individuals, demonstrating stable richness estimates. The near-asymptotic trajectory confirms that additional sampling would yield diminishing returns in species discovery (26, 27).

Perhaps the most outstanding result of the study is the fact that most species (70%) are exotic, something that has been found in many parts of the world (30–32), which reflects the little care that has been taken regarding the movement and management of species from one place to another. This situation can seriously impact the populations of native species and, in many cases, also have economic impacts (33).

Previous studies have shown that prior to the year 2000 (6), there were nine species of fish in the spring, and now there are 14, which means that in 25 years, five species or the equivalent of one every five years were added. It is important to clarify that the fish assemblage structure present in the cuttings, which is largely dominated by exotic species, reflects what occurs in the aquatic ecosystems of the region (34) and not the specific introductions that occurred in the spring. This reflects the condition of the aquatic ecosystems of the state of Morelos and the Central region of Mexico, where 25 species of exotic fish have been documented (14, 35).

If we consider the origin of the exotic species present in the spring, it stands out that 80% comes from ornamental aquaculture, a situation that makes sense since the State of Morelos and the region near the Las Estacas Spring stand out as one of the most important points for the production of ornamental fish in Mexico (36). Most ornamental fish farms lack safeguards that prevent the escape of organisms into the natural ecosystems of the state.

The low population of the Balsas River Cichlid (Amphilophus istlanus) may be due to the presence of exotic cichlid species such as Convicts (Amatitlania nigrofasciata) that have been shown to have a detrimental effect on populations of this species in aquatic environments of the region (37). Another native species that is not very abundant is the Balsas catfish (Ictalurus balsanus), of which only one specimen was observed, even though the Park staff mentioned that many years ago, it was common to see them in the spring. This species is subject to significant fishing pressure in the rivers of the region (38) and apparently due to this same situation, overfishing, was practically eradicated from the spring.

Even though this is an “artifitial” fish assemblage, these baseline diversity metrics provide essential reference data for long-term monitoring of the Las Estacas ecosystem. The fish community exhibits moderate alpha diversity with relatively even species distribution. The Shannon diversity value (H’ = 1.955) falls within the typical range for freshwater fish assemblages in tropical and subtropical regions (39). The high evenness (J’ = 0.741) indicates that dominance is distributed among multiple species rather than concentrated in one or two taxa, suggesting a relatively stable community structure (23).

The rank-abundance pattern, characterized by a moderate slope, is consistent with log-series or log-normal distributions commonly observed in mature freshwater fish communities (28). The presence of several rare species (singletons and doubletons) is typical of natural assemblages and may represent transient individuals, seasonal migrants, or genuinely rare resident species (40). In this case, two of the rare species, the Balsas river catfish and Cichlid, are important to natural assemblages in other freshwater ecosystems of the region.

The rarefaction analysis provides strong evidence for sampling adequacy. The curve’s approach to asymptote indicates that the survey captured the majority of common and moderately rare species (Hulbert, 1971). The discrepancy between observed richness (S = 14) and Chao1-estimated richness (17 species) suggests 3 additional rare species may exist in the community but were not detected (21). This is consistent with the presence of singletons and doubletons in the dataset, which inform the Chao1 estimator.

The 95% saturation threshold at 3,501 individuals (77.4% of sampling effort) demonstrates efficient sampling design. This metric is particularly valuable for planning future surveys, as it indicates that comparable richness estimates could be obtained with approximately 20% less effort (27).

Based on the above results, work has been conducted in collaboration with the Park authorities to develop an intervention strategy that aims to “establish a fish assemblage dominated by native species in the Las Estacas Spring,” for which a Theory of Change model has been integrated that presents four themes or chains of intervention: (1) management and adaptation of aquatic habitat, (2) control of exotic species, (3) introduction of native species, and (4) education and training. This project called “Rewilding Las Estacas” has already been authorized and financed by the Park authorities, and the first results will be available soon.

This study provides a rigorous quantitative assessment of fish community diversity at Las Estacas spring, Mexico. The community comprises 14 observed species with moderate diversity (H’ = 1.955) and relatively even abundance distribution (J’ = 0.741). Rarefaction analysis confirms adequate sampling completeness, with the curve approaching asymptote at 77.4% of total effort. The Chao1 estimator suggests 3 additional rare species may be present but undetected.

These findings establish critical baseline data for conservation monitoring and ecosystem management. Future research should incorporate temporal replication, environmental covariates, and targeted surveys for rare species to enhance understanding of community dynamics and drivers of diversity patterns. The methodological framework presented here—combining multiple diversity indices with rarefaction-based sampling evaluation—provides a robust template for biodiversity assessment in freshwater ecosystems.