Mountain caves of the central region of Veracruz: A vertebrate biodiversity reservoir in a Neotropical hotspot

which receives limited sunlight during a few hours of the day, and aphotic (deep cave zone), which is characterized by the complete absence of light [25]. Two vertebrate surveys were car- ried out in each cave, one in spring and one in fall of 2021. Vertebrate surveys and sampling completeness For the survey of birds in the euphotic zone, two sites were established for fixed-radius (20 m) point counts [26]. The first one was located between 20–50 m outside of the main access of each site, and the second one at an approximate distance of 100–300 m from the first counting point outwards. The sampling time was of approximately 60–120 min during daytime hours (07:00–11:00) and 60–120 min during evening hours (17:00–21:00). Observations were made with a Vortex Diamondback 20-60x80 spotting scope and Vortex Diamondback HD 8x42 bin- oculars. Bird calls were also recorded using a Tascam Dr-05x audio recorder. Subsequently, sampling inside the caves was carried out by searching for birds on ceilings and within crevices in established sites using Fenix HM65R-T headlamps [21]. For mammals, reptiles, amphibians, and fish, timed searches of 20 min were performed within a 2 m radius in established sites in the euphotic, disphotic, and aphotic environments [21,27]. For mammals, direct observations and the presence of tracks and traces [28] were also recorded, and identifications were made using taxonomic guides [29]. For amphibians and reptiles, photographic records were obtained for their subsequent identification using herpeto-

logical guides [30–32]. The guides by Howell and Webb [33] and Sibley [34] were used for the identification of birds and the guide by Miller [35] was used for the identification of fish. Statistical analysis The richness and abundance of each vertebrate species were pooled per cave origin, altitudinal zone, and environment. We estimated species richness in each site using Hill numbers, which allow for comparisons between species richness estimates allotting more weight to rare (q = 0), common (q = 1), dominant (q = 2), and very dominant (q = 3) species [36]. Vertebrate assem- blages in karst and volcanic caves were characterized by their diversity and composition using alpha-diversity and beta-diversity. Analyses were conducted for each of the following vertebrate groups: mammals, birds, reptiles, and amphibians; fish were excluded from the analysis due to their low representativeness. Statistical analyses were run using the R analysis language [37]. In order to evaluate the completeness of the sampling process, extrapolation and interpola- tion procedures were carried out with 100 randomizations using the iNEXT software version 1.3.0 available online at https://chao.shinyapps.io/iNEXTOnline ([38]. The richness values of each vertebrate class were statistically compared between cave origins, altitudinal zones, and environments using 95% confidence intervals. Sampling completeness was variable in karst and volcanic caves, but most groups reached between 70–100% sampling completeness (Fig 1).

To determine the similarity in species richness between karst and volcanic caves, a cluster analysis with UPGMA clustering method was conducted in PAST 4.03, using Bray-Curtis dis- tance matrix and log (x + 1) transformation. To evaluate the relationship between the geo- graphical distance of karstic and volcanic caves and vertebrate composition (Bray-Curtis dissimilarities), the Mantel test [39] with 9,999 permutations was performed using the vegan R package [40]. We computed β-diversity on abundance data using the Jaccard dissimilarity coefficient. To capture the processes underlying species dissimilarity among communities, we partitioned the overall β-diversity values into their species replacement (change in biodiversity due to species turnover) and species richness (change in biodiversity due to differences in species richness) components [41]. We calculated β-diversity between karst and volcanic caves, between caves PLOS ONE Vertebrates in caves of Neotropical hotspot PLOS ONE | https://doi.org/10.1371/journal.pone.0306105 August 9, 2024 5 / 19