Genetic Structure and Loss of Genetic Diversity in the Savannah Side-Necked Turtle Podocnemis vogli (Testudines: Podocnemididae)
Abstract
The savannah side-necked turtle Podocnemis vogli is a species restricted to the savannas of the Orinoco basin in Colombia and Venezuela. Because of its apparent abundance, it is currently categorized as Least Concern (LC) by the IUCN Red List. However, throughout its distribution range, several populations have been extirpated or individuals in remaining populations are consumed and trafficked, and the species’ habitat has been highly degraded. To start assessing the conservation status of the species, 5 populations along their distribution range in Colombia were analyzed using 19 microsatellite loci to perform a population genetics study. Ten of those markers were useful in revealing (1) high levels of genetic diversity, (2) a marked genetic structure of 5 populations, and (3) low and asymmetric gene flow among them. However, the analyses also revealed the loss of genetic diversity (low allelic richness) and recent bottlenecks in some populations. Those identified detrimental indicators are evidencing a population decline most likely related to anthropic activities. These 5 populations correspond to 5 independent management units. The results of this research allow us to propose management and conservation guidelines for these populations in the Orinoco ecoregion.
Resumen
La tortuga sabanera Podocnemis vogli es una especie restringida a las sabanas de la cuenca del Orinoco en Colombia y Venezuela. Debido a su aparente abundancia, actualmente está categorizada como en Preocupación Menor (LC) por la Lista Roja de la UICN. Sin embargo, a lo largo de su rango de distribución, varias poblaciones han sido extirpadas o los individuos en poblaciones remanentes son consumidos y traficados, y el hábitat de la especie se ha degradado intensamente. Para comenzar a evaluar el estado de conservación de la especie, se analizaron cinco poblaciones a lo largo de su rango de distribución en Colombia utilizando 19 loci microsatélites para realizar un estudio de genética poblacional. Diez de esos marcadores fueron útiles para revelar: (1) altos niveles de diversidad genética, (2) una estructura genética marcada de cinco poblaciones y (3) flujo de genes bajo y asimétrico entre ellas. Sin embargo, los análisis también revelaron la pérdida de diversidad genética (baja riqueza alélica) y cuellos de botella recientes en algunas poblaciones. Esos indicadores perjudiciales identificados están evidenciando una disminución de las poblaciones, muy probablemente relacionada con las actividades antrópicas. Estas cinco poblaciones corresponden a cinco Unidades de Manejo Independientes. Los resultados de esta investigación nos permiten proponer lineamientos de manejo y conservación para estas poblaciones en la ecorregión del Orinoco.
Palabras Clave.- Genética de la conservación; estructura poblaciónal; flujo genético; microsatélites; test de amplificación cruzada
Turtles are ancient animals with an origin estimated around 228 million years ago in the Triassic period (Li et al. 2018), being essential organisms because of their crucial and unique ecological roles in the ecosystems they inhabit, as well as their high cultural and economic value (Mancera Rodríguez and Reyes García 2008; Lovich et al. 2018). However, today as a group, they are highly threatened because of the combination of habitat modification, exploitation, and climate change, with the aggravating situation that there is a generalized lack of information that does not allow species-based conservation programs (Lovich et al. 2018; Stanford et al. 2020). Therefore, it is urgent to increase research focused on the 357 recognized chelonian species (TTWG 2021), which are part of the evolutionary process that maintains biodiversity; the loss of an old lineage means the loss of the accumulation of historical evolution and a vital representative of the world biodiversity (May 1990; Mace et al. 2003).
The chelonian family Podocnemididae, known to have existed since the Cretaceous, includes many extinct and eight extant large freshwater turtle species from South America and Madagascar (Pritchard and Trebbau 1984; Wood 1985, 1997; de Lapparent de Broin 2000, 2001; Carvalho et al. 2002). One of these species is the savanna side-necked turtle Podocnemis vogliMüller, 1935, a small to medium-sized species with an average straight carapace length of 23 cm for adult females and 17 cm for adult males (de Lapparent de Broin 2001; Rueda-Almonacid et al. 2007). It has a restricted distribution in the Orinoco basin of Colombia and Venezuela (Rueda-Almonacid et al. 2007; TTWG 2021). This turtle mostly has an herbivorous diet and inhabits lentic and lotic water bodies (excluding big rivers) in savanna habitats, which the females use for nesting in the dry season (Ortiz-Moreno and Rodríguez-Pulido 2017). The species is currently categorized as Least Concern (LC) in Colombia because of the apparent abundance of individuals in some populations (Morales-Betancourt et al. 2015). However, it has been uplisted as Vulnerable (VU) in the recent global assessment by the Turtle Taxonomy Working Group (Páez et al., in press).
More realistically, the conservation status of P. vogli should be regarded as unknown because of the lack of knowledge on their accurate distribution, population structure, and natural history, as well as threat factors (Páez et al. 2012; Ortiz-Moreno and Rodríguez-Pulido 2017). This species has been deeply affected by habitat destruction and modification throughout its distribution range and the dramatic increase of exploitation of turtles of all age classes by humans for food and pets and as an economic resource, because of the reduction of the larger species of Podocnemis in the region (i.e., Podocnemis unifilis and Podocnemis expansa; Morales-Betancourt et al. 2015). Several populations have been extirpated in vast sectors of the departments of Meta, Casanare, and Arauca in Colombia and, especially, in the environs of urban centers (Alarcón-Pardo 1969; Ministerio de Medio Ambiente de Colombia 2002; Morales-Betancourt et al. 2015).
Conservation genetics is a discipline that uses the theory and methods of genetics to identify, evaluate, and conserve the genetic diversity in endangered species and provide information for their conservation and management (Frankham 2015). Special emphasis must be placed on the genetic conservation of endangered species, with the primary goal of preventing the loss of genetic diversity, maintaining natural gene flow, ensuring adequate population size, and avoiding artificial selection (Ferrière et al. 2004; Frankham 2010). In the context of chelonians, the assessment of conservation status has prominently employed various genetic markers, including microsatellites, single-nucleotide polymorphisms (nuclear DNA), and mitochondrial DNA sequences. These markers serve as valuable tools for genetic analyses, encompassing investigations into population differentiation, inbreeding, gene flow, bottleneck events, genetic diversity, and population assignment (Weisrock and Janzen 2000; Kuo and Janzen 2004; Pedall et al. 2011; Fritz et al. 2012; Vargas-Ramírez et al. 2012; Gallego-García et al. 2018; Michels and Vargas-Ramírez 2018). Hence, conducting genetic evaluations is imperative to gain insights into the conservation status of individual populations and to propose effective measures for their conservation and management. Consequently, using 19 microsatellite loci, the objectives of this study were 1) to quantify the genetic diversity and evaluate the presence of genetic structure in 5 natural populations of P. vogli located along a significant portion of its distribution range in Colombia, 2) to assess whether the populations have experienced size reductions by evaluating the presence of bottlenecks as well as to determine other crucial demographic parameters such as inbreeding and gene flow, and 3) to assess the utility of the microsatellite system for forensic purposes by assigning 26 confiscated individuals kept at the Estación de Biología Tropical Roberto Franco (EBTRF) in Villavicencio, Meta, Colombia, to their source populations.
METHODS
Sampling. —
For this research, 124 blood samples from adults of P. vogli were collected between April 2016 and April 2022. These samples were obtained from the Banco de Tejidos de la Biodiversidad (BTBC) at the Instituto de Genética, Universidad Nacional de Colombia. The samples originated from 5 natural populations: I) Puerto Carreño (Vichada) (16 samples), II) Paz de Ariporo (Casanare) (22 samples), III) Unillanos (Villavicencio, Meta) (20 samples), IV) Puerto López (Meta) (10 samples), and V) San Martín (Meta) (30 samples) (Fig. 1). Additionally, 26 captive individuals from the EBTRF were also included in the analysis.
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World’s Turtle and Tortoise Journal 23, 1; 10.2744/CCB-1584.1
DNA Extraction and Loci Selection. —
Genomic DNA extraction was carried out following the phenol-chloroform protocol (Green and Sambrook 2017). Cross-amplification tests using previously established primers for 19 microsatellite loci were performed using individual PCR amplifications for samples of P. vogli. Five primer pairs were originally designed for P. expansa (Pod1, Pod62, and Pod128 [Sites et al. 1999] and PE519 and PE1075 [Valenzuela 2000]) and 14 primer pairs originally designed for P. unifilis (Puni_2A9, Puni_1B2, Puni_1B10, Puni_1B11, Puni_1C3, Puni_2C11, Puni_1D11, Puni_1D12, Puni_2F6, Puni_1E1, Puni_2E7, Puni_1F10, Puni_1H9, and Puni_2D9 [Fantin et al. 2007]; Supplemental Table S1 [all supplemental material is available at http://dx.doi.org/10.2744/CCB-1584.1.s1]). The successfully amplified microsatellite loci were combined in 3 multiplex PCRs. The organization of these PCRs was determined by considering the size of each microsatellite, annealing temperature, and fluorochromes (6-FAM, HEX, PET, and NED): multiplex 1 (Ta = 60°C: Pod 128, Puni 1D12, Puni 1B11, Puni 1F10, Puni 1B10), multiplex 2 (Ta = 60°C: Puni 1B2, Puni2A9, Puni 2d9, Puni 2D9, Puni 2C11), and multiplex 3 (Ta = 63°C: Puni 1H9, Puni 1C3, Puni 1E1, Puni 2F6, Puni 1D9). The multiplex PCRs were run in an Eppendorf master cycler, with a final volume of 10 µl, containing 5 µl of My Tag HS MIX (2×), 0.2 µM of reverse primer of each locus, 0.2 µM of forward primer of each locus, 200 ng of DNA, and completed with ppH2O up to 10 µl. The thermocycling conditions were the same as reported for P. unifilis by Fantin et al. (2007). After PCR amplification, a dilution consisting of 1 μl of the PCR product and 99 μl of ultra-pure water was prepared. Subsequently, 1 μl of this dilution was combined with 8.5 μl of Hi-Di Formamide (Applied Biosystems, Norwalk, CT, USA). 0.25 μl of purified water, and 0.25 μl of GeneScan-600 LIZ Size Standard (Applied Biosystems). The determination of fragment length was carried out using an ABI 3500 Genetic Analyzer at the Servicio de Secuenciación y Análisis Molecular (SSIGMOL)-IGUN-UNAL. Genotypes were identified using GENE-MAPPER v.3.7 (Applied Biosystems) and OSIRIS v.2.13.1 (National Center for Biotechnology Information [NCBI], Bethesda, MD, USA) software.
To determine the utility of microsatellite loci for population genetic analyses, the following steps were undertaken. 1) Confirmation of the presence of the target locus in the PCR product was performed by sequencing the repeat motif. PCR products were purified through ethanol precipitation, and sequencing was carried out using the BigDye TM Terminator v.3.1 cycle sequencing kit (Applied Biosystems) on an ABI 3500 Genetic Analyzer. 2) Genotyping of all individuals for each locus was conducted through Fragment Length Analysis using an ABI 3500 Genetic Analyzer (Applied Biosystems) and the software OSIRIS v.2.12 (NCBI) and GeneMapper (Applied Biosystems). 3) Evaluation of Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) was conducted using GENEPOP (Raymond and Rousset 1995), with significance values Bonferroni-corrected (Hyphenate, composed adjective). Additionally, the presence of null alleles for each locus was assessed using the software FREENA (Chapuis and Estoup 2007).
Population Differentiation and Variability. —
The Bayesian clustering algorithm implemented in the software STRUCTURE v.2.3 was used to assess population structure. This software assumes that the loci are unlinked and at linkage equilibrium with one another within populations and optimizes the number of clusters (populations) under the assumption of Hardy–Weinberg equilibrium (Pritchard et al. 2000). Calculations were conducted using an admixture model, allowing for different ancestral populations (Hubisz et al. 2009), and allele frequencies were allowed to be correlated, which considers that the frequencies are similar to an ancestral population (Pritchard et al. 2000). The software ran with a burn-in of 20,000 and 100,000 subsequent Markov chain repetitions. In addition, population structure was modeled for Ks between 1 and 7, with an iteration of 20. The best number of K was estimated through the ΔK statistic (Evanno et al. 2005), and ‘MedMeaK’ (median of means), ‘MaxMeaK’ (maximum of means), ‘MedMedK’ (median of medians), and ‘MaxMedK’ (maximum of medians), which deal with uneven sampled size (Puechmaille 2016), implemented in the online program STRUCTURESELECTOR (Li and Liu 2018). Additionally, a principal component analysis (PCA) was also performed using the ADEGENET package (Jombart and Collins 2017) in the R software. Furthermore, analyses of molecular variance (AMOVA) were run in ARLEQUIN v.3.5 (Excoffier and Heidi 2006) and were used to detect the source of genetic variability in different hierarchical levels of population structure. This software was also used to calculate FST and RST values to assess the genetic differentiation among populations. To assess whether there was a correlation between the degree of genetic differentiation and geographical distance, a Mantel test implemented in the software IBD (Bohonak 2004) was performed.
To assess genetic diversity for each population, the software ARLEQUIN v.3.0 was used to calculate the expected heterozygosity (HE) and observed heterozygosity (HO) under Hardy–Weinberg equilibrium. Allelic richness (AR) and the number of alleles per population (NA) were determined using the software FSTAT (Goudet 2001). Additionally, GENALEX v.6.1 (Peakall and Smouse 2012) was used to calculate the number of private alleles (PA). Gene flow estimations between populations were performed by a Bayesian method implemented in the software BAYESASS, which uses individual genotypes to estimate migration rate across genetic clusters (Wilson and Rannala 2003). The software was run using the default parameters and the following settings: 10,000,000 iterations for Markov chain Monte Carlo (MCMC) and 100,000 for the burn-in, sampling the chain every 1,000 iterations. The seed was modified at random for each analysis. The possible presence of a recent bottleneck in a population was examined by the analysis of significant heterozygosity excess from the observed number of alleles using the software BOTTLENECK 1.2. (Cornuet and Luikart 1996; Piry et al. 1999) and the implemented Wilcoxon’s signed rank test. This is the most-used approach because it is powerful and robust in detecting bottlenecks with fewer than 20 polymorphic loci (Piry et al. 1999). Additionally, a second method was performed, the allele frequency test. It determines whether there is a shift mode in the distribution of allele frequencies, which evidences a bottleneck (Piry et al. 1999). Establishing the mutational model before detecting a bottleneck is essential, because it modifies the relationship between the extent of heterozygosity and the number of alleles (Cornuet and Luikart 1996). The two-phase model of mutation (TMP) and the following parameters were used: 12 in variance, 5% for multiple mutational steps, and 95% single-step mutation (Piry et al. 1999). Finally, to evaluate the presence of inbreeding per population, the value of FIS was calculated also using the software ARLEQUIN v.3.5.
Assignment Analyses. —
The most probable source population for the turtles housed in the EBTRF was determined using 3 different methods. The first method involved inferring ancestry values (Q) using the software STRUCTURE v.2.3. The other 2 assignment approaches were conducted using the software GENECLASS v.2.0 (Piry et al. 2004). The Bayesian test in GENECLASS assigns individuals based on the probability of the origin of the genotype in a population (Rannala and Mountain 1997). The third method, a frequency-based approach, utilizes the observed allele frequency in the population to assign individuals with the highest probability of belonging to a specific population (Paetkau et al. 1995). Each method employed a MCMC resampling of 1,000,000 steps.
RESULTS
Out of the 19 microsatellite loci evaluated, 12 were found to be polymorphic and thus informative for our study (Table S1). Among these, the loci Puni_1B10 and Puni_1D12 were linked with several other loci, and Puni_1D12 showed Hardy–Weinberg disequilibrium after Bonferroni correction (Table S1). Utilizing the remaining 10 unlinked polymorphic microsatellite loci, a total of 84 alleles were identified in the 98 individuals assessed, ranging from 3 to 16 alleles per locus.
Population Differentiation. —
STRUCTURESELECTOR suggested 5 populations (K = 5) based on the assessment of MedMeaK, MaxMeaK, MedMedK, MaxMedK, and 4 populations (K = 4) through the analysis of the ΔK statistic (Fig. 2). This indicates that under K = 4, two populations ([IV] Puerto López and [III] Unillanos) were grouped together, whereas they were separated under K = 5. The scenario K = 5 revealed high admixture between (IV) Puerto López and (III) Unillanos, with the localities being in close proximity, just 68 km apart, and sharing an extensive common hydrographic system. Under both scenarios, little admixture was observed for the populations (II) Paz de Ariporo and (V) San Martín, while the remaining populations showed evidence of more admixture. The high differentiation observed in the population from the locality (V) San Martín may be explained by the presence of two large rivers, the Guayuriba River and the Humadea River, which flow from west to east between San Martín and the other locations, reducing the mobility capacity of individuals from this population. The results of the PCA (Fig. 3) supported the K = 5 scenario. The scatter plot of PCA indicated the primary genetic differentiation between individuals of San Martín (Meta) and individuals from the region of Casanare (Paz de Ariporo), Meta (Unillanos, Puerto López), and Vichada (Puerto Carreño). The last group mentioned exhibited a high degree of admixture of ancestry between them (Fig. 3). The AMOVA for the 5 populations revealed that 11.71% of the variance occurred among populations (FST = 0.11708; p < 0.001) (Table S2), while 88.29% was within populations. Additionally, the amount of differentiation between all pairs of clusters for both Ks reflected in the values of FST and RST revealed a low but statistically significant differentiation (p < 0.05) (Table 1). In general, the population (V) San Martín exhibited the highest differentiation from other populations for both Ks (FST from 0.1248 to 0.1642; p < 0.001; Table 1). For K = 5, (III) Unillanos and (IV) Puerto López showed the lowest FST values (0.0541). Moreover, there was no evidence of a correlation between geographical distance and genetic distance, neither for 4 populations (Z = −3498170.73, r = −0.0184, p = 0.387) nor for 5 populations (Z = −368159.75, r = −0.1094, p = 0.490).
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World’s Turtle and Tortoise Journal 23, 1; 10.2744/CCB-1584.1
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World’s Turtle and Tortoise Journal 23, 1; 10.2744/CCB-1584.1
Bottlenecks. —
In general, the genetic clusters showed between 6 and 8 loci with an excess of heterozygosity. Wilcoxon tests were significant for the populations of (I) Puerto Carreño and (II) Paz de Ariporo (p < 0.05) for both K = 5 and K = 4, indicating a recent bottleneck (Table 2). Additionally, the allele frequency test revealed a bottleneck in all genetic populations except (III) Unillanos and (IV) Puerto López for K = 4, which may be explained by the increase in genetic diversity when these populations are combined.
Migration and Inbreeding. —
The migration rate between populations in 4 and 5 genetic clusters was low and asymmetric in most cases (Table 3). The highest migration rate, above 0.1, was directed to cluster 2 (Paz de Ariporo) for both Ks (Fig. 4), indicating unidirectional flow of individuals moving through this cluster. Furthermore, there was no evidence of inbreeding, indicating a no excess homozygosity in each population (i.e., no negative and statistically significant values of FIS; p > 0.05).
Citation: Chelonian Conservation and Biology: Celebrating 25 Years as the World’s Turtle and Tortoise Journal 23, 1; 10.2744/CCB-1584.1
Genetic Diversity. —
Allelic diversity described by the allelic richness and the number of alleles per cluster was low with means of 4.48 for the number of alleles and 3.87 for allelic richness for K = 5 and 5.07 and 4.79 for K = 4, respectively (Table 4). Furthermore, on average, the observed heterozygosity was 0.638, and the expected heterozygosity was 0.554 for both Ks, indicating high heterozygosity. The highest number of private alleles was found in the cluster of (I) Puerto Carreño with 7 alleles for K = 5 and 11 for K = 4.
Population Assignment Analyses. —
By using the methods implemented in GENECLASS and the Q value of the STRUCTURE analyses for K = 5, 17 captive turtles could be successfully assigned to their source populations. A correspondence probability higher than 70% in any method was taken as positive assignment; neither of the evaluated methods contradicted the other result (Table S3). One individual was assigned to cluster (I) Puerto Carreño, 3 individuals to cluster (II) of Paz de Ariporo, 7 individuals to cluster (III) of Unillanos, and 6 individuals to cluster (V) of San Martín.
DISCUSSION
Evidence of Population Structure. —
Our analyses revealed a clear population structure for P. vogli that largely corresponded to the 5 distinct collection sites. The observed genetic differentiation pattern is most likely caused or reinforced by one or more of the following factors, which are all not mutually exclusive. First is low dispersal capability: P. vogli seems to move only distances of 5 km per month according to radio tracking (Pinzón et al. 2017). Next are landscape features in the Orinoco region in Colombia: This region is located in eastern Colombia, from the Eastern Cordillera to the border in Venezuela. It is composed of savanna exposed to fires and floods and a water network that drains from southwest to northeast with many rivers, streams, channels, and lakes, which are the habitats of P. vogli, except for the big rivers, which are not colonized (Alarcón-Pardo 1969). The vast region features extensive livestock farming, agriculture, and human settlement, potentially acting as natural barriers that contribute to genetic differentiation and restrict gene flow. Third is the effect of geographical distance: The distribution of P. vogli shows certain populations widely separated from each other. Although our analyses did not reveal an isolation-by-distance effect, it is known that the dispersion of turtles in freshwater systems presents challenges for crossing basins, potentially reducing migration, mating, and increasing genetic differentiation among populations (e.g., Michels and Vargas-Ramírez 2018). Riverscape genetic studies in turtles have assessed connectivity factors, including cost distance of resistance from river types, slope, current, historical climatic suitability (isolation by resistance), and the presence of a main river (isolation by barrier) (Oliveira et al. 2019). For example, isolation by resistance and barrier using connectivity factors was assessed in Podocnemis erythrocephala in the Amazon basin. This species is characterized by low dispersion and tends to inhabit streams and lakes rather than the main river, as observed in P. vogli. (Oliveira et al. 2019). Olivera et al. (2019) found that connectivity variables, such as the presence of the Amazon River, were the main factors for isolation by barrier, explaining genetic differentiation in P. erythrocephala. The Orinoco region has 3 main rivers—the Guaviare River, the Meta River, and the Orinoco River—with their tributaries, including the Casanare River, the Vichada River, the Arauca River, and the Tomo River, which traverse the Arauca, Meta, Vichada, and Casanare departments. Therefore, we suggest that the hydrographic system serves as a barrier that may also explain the differentiation of the population of P. vogli in Colombia. Last is human intervention: Habitat alterations can create barriers to gene flow between populations. This, combined with the exploitation of turtles for food and other economic purposes, constitutes the primary drivers behind the current decline of P. vogli (Morales-Betancourt et al. 2015). Several populations have been extirpated in vast parts of the Colombian departments of Meta, Casanare, and Arauca and, especially in the environs of urban centers (Alarcón-Pardo 1969; Ministerio de Medio Ambiente de Colombia 2002).
Our results detected a major and asymmetrical migration rate in the genetic population of the Paz de Ariporo (cluster II) (Fig. 4). This was confirmed by the assignment test, where individuals from Paz de Ariporo showed admixture from individuals of Puerto Carreño (cluster I), Unillanos (cluster III), and Puerto López (cluster IV) (Fig. 2). Moreover, we identified individuals with a high ancestry from other geographic locations. For example, 2 individuals from Puerto Carreño showed a high percentage of ancestry from the population of Paz de Ariporo, Casanare (Fig. 2). This migration pattern may be explained by 2 models. The first is the Stepping Stone Model, in which turtles have the possibility of exchange between nearby colonies because of their extensive hydrographic area with many lakes that serve as their habitats. The second is a source-sink relationship, where the direction of migration could be influenced by human activities such as agriculture, livestock, translocations of turtles, and human settlements, which generate Paz de Ariporo as a sink for immigrants from other populations.
Regarding translocations, environmentalists, with the support of local nongovernmental and governmental institutions, have undertaken the relocation of P. vogli individuals from nature reserves to various sites in the Colombian Orinoco basin region, with the objective of bolstering populations. Recent reports highlight the release of 100 turtles in the Reserve La Esperanza, Caño Chiquito in Casanare in 2017 (SuVersión 2017), approximately 75 km from the population of Paz de Ariporo. Furthermore, in Arauca, 500 turtles were released in the Macuate Nature Reserve, Caño Negro, from Paz de Ariporo in Casanare at the end of 2020, covering a distance of 407 km from Paz de Ariporo (Violeta 2020). Unfortunately, these relocation efforts did not incorporate genetic analyses, inadvertently risking potential impacts on the genetic diversity and structure of populations. The absence of genetic considerations in such conservation activities underscores the importance of integrating genetic assessments into wildlife relocation programs to ensure the preservation of the genetic identity of populations and their evolutionary potential.
Conservation Implications. —
Conservation efforts for long-lived species such as turtles should focus on the protection of all life stages and the area of occurrence, the prioritization of species based on conservation programs, and the conservation of biodiversity hotspots that harbor the highest turtle species richness (Lovich et al. 2018; Stanford et al. 2020). Podocnemis vogli has been included in management plans for turtles aimed at protection and conservation. Examples include its incorporation into the management and conservation plan in the Tuparro Biosphere Reserve (Trujillo et al. 2008), the Colombian Continental Turtle Conservation Plan (Morales-Betancourt et al. 2015), the Comprehensive Regional Climate Change Plan for the Orinoquía (PRICCO) (CIAT 2017), and management plans for national natural parks. It is noteworthy that these programs have focused on the overall protection and management of the species, with less emphasis on specific nesting areas, and have typically addressed conservation measures for broader geographic regions.
The identification of signatures of recent population declines, indicated by bottlenecks, in the populations from (I) Puerto Carreño and (II) Paz de Ariporo suggests a significant reduction in the number of individuals. This decline is likely attributed to human interventions, including habitat alteration, destruction, and the extraction of individuals for food and economic exploitation. Additionally, the observed low allelic richness and low, asymmetric gene flow further underscore the substantial impact of human activities on these populations. Despite these challenges, the recognition of 5 genetic populations highlights the importance of considering them as independent management units (sensuMoritz 1994) for the long-term conservation of the species. This approach aims to preserve the identified genetic diversity within each unit.
Using the evaluated microsatellite loci allowed us to infer the genetic origin of 17 out of 26 individuals from the ex situ population present at the EBTRF (Table S3). Although the marker system developed for P. vogli is based on the cross-amplification of microsatellites developed for P. expansa (Sites et al. 1999; Valenzuela 2000) and P. unifilis (Fantin et al. 2007), it is acknowledged that this method may exhibit less genetic diversity (Kpatènon et al. 2020). However, despite this limitation, the method serves as a powerful tool. It not only will allow the assessment of numerous populations of P. vogli across its distribution range, but also will aid in the reintroduction efforts conducted by reception centers and environmental corporations. The ability to assign individuals to their most probable natural population enhances the effectiveness of these conservation initiatives. Although conservation programs aimed at the recovery of wild populations are crucial social initiatives and deserving of support, it is important to note that they often overlook the genetic composition of individuals. This oversight may lead to the loss of crucial genetic population dynamics. The results of this research have the potential to enhance decision-making processes in these and other conservation programs, ensuring a more comprehensive and effective approach to species recovery.
Furthermore, it is crucial to inform the environmental groups operating in the studied localities about the current situation of the species. These environmental entities, specifically the Autonomous Regional Corporation of the Orinoquia and the Corporation for the Sustainable Development of the Special Management Area of La Macarena, play a key role in managing the environment and natural resources, including the seizure and support of P. vogli individuals. In collaboration with these entities, an urgent and targeted environmental education campaign should be initiated to convey the message that populations of the species are in decline. Establishing agreements for community-based conservation initiatives can be a significant step in addressing the challenges faced by P. vogli.
Currently, the species is classified as Least Concern (LC) in Colombia (Morales-Betancourt et al. 2015), but it has been provisionally listed as Vulnerable (VU) by the Turtle Taxonomy Working Group (TTWG 2021). Moreover, the species is regulated under Appendix II of the Convention on International Trade in Endangered Species (CITES 2019), a conservation instrument that strictly regulates its commercialization to mitigate the risk of extinction. Despite these protective measures, the species in Colombia continues to face ongoing threats from illegal trafficking for purposes such as pets, food, or economic resources, thereby increasing the risk of extinction. Each year, hundreds of individuals are seized and handed over to environmental regional groups because of illegal exploitation.
Based on our findings, we strongly recommend the reconsideration of the conservation status of P. vogli, proposing a recategorization to Vulnerable (VU) on the IUCN Red List. In fact, just such a recommendation was recently made to the IUCN by the Tortoise and Freshwater Turtle Specialist Group (Páez et al., in press). This adjustment reflects the significant threats and population declines identified in our study, emphasizing the urgency of conservation efforts for this species.
The distribution range of Podocnemis vogli in gray, according to TTWG (2021). Red dots indicate the sampled sites for this study: I) Puerto Carreño (Vichada); II) Paz de Ariporo (Casanare); III) Unillanos (Villavicencio, Meta); IV) Puerto López (Meta); V) San Martín (Meta). Black square: VI) Estación de Biología Tropical Roberto Franco (Villavicencio, Meta). Inset photo: adult female P. vogli. Photo credit: Juan Manuel Vargas-Ramírez.
(a) STRUCTURE barplots based on 10 unlinked microsatellite loci for K = 5 and K = 4 for Podocnemis vogli. Each individual from the 5 collected localities is represented by a vertical bar with a subdivision into different colors corresponding to the estimated ancestry. (b) Estimated membership of the 5 and 4 genetic clusters represented by a pie chart for each locality. Gray shadow: distribution of the species according to TTWG (2021).
Principal component analysis for the genetic differentiation of Podocnemis vogli using information from 10 microsatellite loci. The clusters correspond to 5 populations: Puerto Carreño (I), Paz de Ariporo (II), Unillanos (III), Puerto López (IV), and San Martín (V).
Migration rate in Podocnemis vogli among 5 populations: Puerto Carreño, Vichada (I), Paz de Ariporo, Casanare (II), Unillanos, Meta (III), Puerto López, Meta (IV), and San Martín, Meta (V).
Contributor Notes
Handling Editor: Vivian P. Páez
