Geometric morphometrics reveal a potential hybridization zone in endemic species of Hypancistrus (Siluriformes: Loricariidae) from the lower and middle rio Xingu

Franciele Fernanda Kerniske¹ , Roger Henrique Dalcin², Leandro Melo de Sousa³ and Roberto Ferreira Artoni⁴

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Abstract​

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Introduction​

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The rio Xingu, located in the Brazilian Amazon, contains habitats that support high levels of endemism and a rich biodiversity of freshwater fish (Zuanon, 1999; Goulding et al., 2003; Camargo et al., 2004; Villas-Bôas, 2012). Particularly noteworthy is the Volta Grande do Xingu in the Middle Xingu region, near the city of Altamira, where the river’s three sequential bends create distinct habitats for species highly adapted to rapids and rocky outcrops (Goulding et al., 2003; Sawakuchi et al., 2015; Silva et al., 2015). Among the ichthyofauna of the Xingu, species of Loricariidae, commonly known as plecos, stands out for its diversity and evolutionary history. Members of the Loricariidae familyare highly valued in the aquarium trade and include several endemic species from the basin, such as Hypancistrus zebra Isbrücker & Nijssen, 1991, H. seideli Sousa, Sousa, Oliveira, Sabaj Pérez, Zuanon & Rapp Py-Daniel, 2025, and H. yudja Sousa, Sousa, Oliveira, Sabaj Pérez, Zuanon & Rapp Py-Daniel, 2025 (Santos, 2019; Gonçalves, 2020; Sousa et al., 2025).

The region’s unique characteristics, combined with pressures from the ornamental fish trade and the construction of the Belo Monte Hydroelectric Plant (inaugurated in 2016), have significantly impacted these species, leading to habitat loss and changes in population dynamics (Fitzgerald et al., 2018; Jiang et al., 2018). Recent taxonomic revisions have formally described H. yudja and H. seideli, resolving previous uncertainties in their classification (Sousa et al., 2025). However, conservation concerns remain critical, particularly for H. yudja, which is restricted to a small stretch of the middle Xingu, entirely within the impact zone of the Belo Monte dam. This raises significant concerns about its vulnerability and accelerated population decline (Sousa et al., 2025). Additionally, H. zebra is classified as Endangered (EN) by the IUCN Red List (Cardoso et al., 2016; ICMBio, 2018; Santos, 2019; Sousa et al., 2021b).

Populations of Hypancistrus in the lower portion of Volta Grande do Xingu have shown signs of genetic mixing, suggesting hybridization events (Santos, 2019). Hybridization can play a significant role in speciation and diversification, potentially creatingnew genotypes that confer adaptive advantages in fragmented or transitional environments (Barton, Hewitt, 1989; Mallet, 2007), as observed in the rapids and waterfalls do rio Xingu. These findings are particularly relevant given the environmental degradation caused by dam construction, which can intensify pressure on populations, exacerbating hybridization events and extinction risks (Fitzgerald et al., 2018). Therefore, understanding these species’ evolutionary history and relationships is crucial for developing effective conservation strategies.

Geometric morphometrics has become an essential tool for studying relationships among morphologically similar groups(Adams et al., 2009). Analyzing Cartesian coordinates of anatomical landmarks allows the exploration of data and the composition of the organism’s shape, facilitating inferences about body structures (Mitteroecker, Gunz, 2009; Klingenberg, 2022). This approach is particularly relevant for studying diversification and speciation, where minor shape differences may indicate significant adaptations (Souza et al., 2018). In this study, geometric morphometrics was applied to delimit species and characterize possible hybrids of the genus Hypancistrus and their natural occurrence areas, exploring morphological variations and their evolutionary and taxonomic implications, particularly in areas affected by anthropogenic changes.

Material and methods

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Study area and sampling. A total of 55 live specimens of the genus Hypancistrus were analyzed (15 H. zebra, 15 H. seideli, 7 H. yudja and 18 Hypancistrus putative hybrids) collected from the rio Xingu (Fig. 1) and kept in aquariums in the Laboratório de Aquicultura de Peixes Ornamentais do Xingu (LAQUAX).

FIGURE 1| Map of the study area in Volta Grande do Xingu, located in the Amazon. The purple dashed line highlights the hybridization zone between Hypancistrus species. Occurrence locations are indicated by color-coded geometric shapes: yellow squares (H. zebra), black triangles (H. yudja), and orange circles (H. seideli). Dark blue areas represent the reservoirs of the Belo Monte Hydroelectric Complex, and the brown area marks the reduced flow stretch of the Xingu River.

Geometric morphometrics. The specimens were carefully transferred from their living tanks to a studio aquarium. Each individual’s dorsal, lateral, and ventral pictures were captured using a Nikon Z8 camera with a Nikkor Z 14–30 mm f/4 S lens fixed in 30 mm focal length. After capturing the photos, the specimens were immediately returned to their respective tanks, ensuring minimal stress and disruption to their natural behaviors. Only adult individuals were selected to avoid the effects of ontogenetic allometry (Frederich et al., 2008; Klingenberg, 2022). There were no deaths among the specimens during or after the procedures.

The images were converted to TPS files using tpsUtil software, and anatomical landmarks were inserted into the photographs of the individuals using TpsDIG2 software, v. 2.32 (Rohlf, 2019). A total of 12 anatomical landmarks were defined for the dorsal view, 18 for the lateral view, and 20 for the ventral view, all homologous across individuals, as adapted from Sassi et al. (2021) (Fig. 2).

FIGURE 2| Lateral, dorsal, and ventral views of Hypancistrus individuals, showing the anatomical landmarks used in the geometric morphometric analysis. A. Lateral view: 1) Anterior limit of the orbital bone; 2) Posterior limit of the orbital bone; 3) Inferior limit of the orbital bone; 4) Superior limit of the orbital bone; 5) Anterodorsal edge of the operculum bone; 6) Tip of the snout; 7) Base of the supraoccipital process; 8) Origin of the dorsal-fin spine; 9) Posterior limit of the dorsal-fin base; 10) Anterior limit of the adipose-fin base; 11) Posterior limit of the adipose-fin base; 12) Medial point of the vertical line through the distal margin of the hypurals; 13) Center of the nostril opening; 14) Insertion of the pectoral-fin spine; 15) Insertion of the pelvic-fin spine; 16) Anal-fin insertion; 17) Base of the first procurrent ray of the caudal fin (upper lobe); 18) Base of the first procurrent ray of the caudal fin (lower lobe). B. Dorsal view: 1) Left eye; 2) Right eye; 3) Center of the left nostril opening; 4) Center of the right nostril opening; 5) Tip of the snout; 6) Anterodorsal edge of the left operculum bone; 7) Anterodorsal edge of the right operculum bone; 8) Leftmost point of the body; 9) Rightmost point of the body; 10) Origin of the dorsal-fin spine; 11) Anterior limit of the adipose-fin base; 12) Caudal-fin insertion. C. Ventral view: 1) Anterior margin of the oral disc; 2) Left margin of the oral disc; 3) Right margin of the oral disc; 4) Posterior margin of the oral disc; 5) Anterodorsal edge of the left operculum bone; 6) Anterodorsal edge of the right operculum bone; 7) Insertion of the left pectoral-fin spine; 8) Insertion of the right pectoral-fin spine; 9) Tip of the left pectoral-fin spine; 10) Tip of the right pectoral-fin spine; 11) Leftmost point of the body; 12) Rightmost point of the body; 13) Origin of the left pelvic-fin spine; 14) Origin of the right pelvic-fin spine; 15) Tip of the left pelvic-fin spine; 16) Tip of the right pelvic-fin spine; 17) Anterior limit of the anal-fin base; 18) Center of the anus; 19) Posterior limit of the adipose-fin base; 20) Caudal-fin insertion. Scale bars = 1 cm.

Subsequently, the morphometric data was processed in the R programming language using the geomorph, morpho and RRPP packages (Adams, Otárola‐Castillo, 2013; Schlager, 2017; R Development Core Team, 2024). A Generalized Procrustes Analysis (GPA) was applied to align the shapes, eliminating variations in size, position, and orientation using the gpagen function, and outliers were identified and removed using the plotOutliers function (Adams, Otárola‐Castillo, 2013).

A Principal Component Analysis (PCA) was performed to identify morphological patterns of shape variation, plotting the Procrustes-aligned data in a two-dimensional morphospace (Sherratt, 2016). The analysis was conducted using the gm.prcomp function from the geomorph package, applied directly to the Procrustes coordinates without additional scaling or transformation. To statistically assess group differentiation based on shape, a Multivariate Analysis of Variance (MANOVA) was applied to the PC1 and PC2 scores from each body view, using the manova function, with Pillai’s trace as the test statistic and group identity as the independent variable.

In addition, a Canonical Variate Analysis (CVA) was conducted using the CVA function from the Morpho package (Schlager, 2017), with group identity assigned a priori. This analysis was used to maximize and visualize intergroup differentiation. The significance of Mahalanobis distances between groups was assessed via permutation tests with 10,000 iterations, and the probability of each specimen belonging to a predefined group was estimated using jackknife cross-validation. Finally, morphological disparity was calculated from the Procrustes-aligned data using the morphol.disparity function, quantifying shape variation within and among groups (Adams, Otárola‐Castillo, 2013).

Results​

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Principal Component Analysis (PCA). The PCA and the deformations observed in the landmarks revealed consistent morphological patterns among Hypancistrus zebra, H. seideli, H. yudja, and the putative hybrids. In general, putative hybrids occupied intermediate morphospaces between the parental species, suggesting morphological blending between these species. Conversely, H. zebra formed a more distinct cluster,which may indicate a certain degree of evolutionary separation.The first two principal components explained a substantial proportion of variation in all views (Dorsal: PC1 = 36.0%, PC2 = 23.9%; Lateral: PC1 = 32.9%, PC2 = 21.6%; Ventral: PC1 = 32.9%, PC2 = 23.2%) (Figs. 3A–C, 4A–C, 5A–C).

FIGURE 3| Multivariate analyses for the dorsal view in Hypancistrus species. A. Principal Component Analysis (PCA) based on Procrustes coordinates. B. Canonical Variate Analysis (CVA). C. Distribution of the landmarks overlaid on a visual grid showing the deformations found. Species are represented by color-coded geometric shapes: yellow squares (H. zebra), black triangles (H. yudja), orange circles (H. seideli), and purple diamonds (Hypancistrus putative hybrid).

FIGURE 4| Multivariate analyses for the lateral view in Hypancistrus species. A. Principal Component Analysis (PCA) based on Procrustes coordinates. B. Canonical Variate Analysis (CVA). C. Distribution of the landmarks overlaid on a visual grid showing the deformations found. Species are represented by color-coded geometric shapes: yellow squares (H. zebra), black triangles (H. yudja), orange circles (H. seideli), and purple diamonds (Hypancistrus putative hybrid).

FIGURE 5| Multivariate analyses for the ventral view in Hypancistrus species. A. Principal Component Analysis (PCA) based on Procrustes coordinates. B. Canonical Variate Analysis (CVA). C. Distribution of the landmarks overlaid on a visual grid showing the deformations found. Species are represented by color-coded geometric shapes: yellow squares (H. zebra), black triangles (H. yudja), orange circles (H. seideli), and purple diamonds (Hypancistrus putative hybrid).

In the dorsal view, visual overlap among groups was evident, and a MANOVA performed on PC1 and PC2 scores confirmed the absence of statistically significant differences (Pillai’s trace = 0.133, F = 1.12, p = 0.358), suggesting that this view captures more subtle morphological variation. In contrast, the lateral view exhibited clearer morphological separation, particularly for H. zebra, which clustered distinctly from all other groups. These visual patterns were strongly supported by MANOVA results (Pillai’s trace = 0.835, F = 11.47, p < 0.001), indicating robust shape differences. In the ventral view, although the clusters were not as visually discrete as in the lateral view, MANOVA results still indicated highly significant group differences (Pillai’s trace = 0.640, F = 7.52, p < 0.001). This suggests that, despite the apparent overlap in morphospace, important morphological distinctions exist among groups in this view as well.

Canonical Variate Analysis (CVA). The CVA highlighted a strong separation between the morphological groups. In the dorsal view (Figs. 3A–C), the first canonical axis (CV1) explained 63.3% of the variation, while the second canonical axis (CV2) explained 23.7%. The putative hybrids showed significant overlap with H. yudja, suggesting closer morphological proximity, but also displayed slight overlap with H. seideli, while H. zebra remained isolated. In the lateral analysis (CV1 = 67.4% and CV2 = 19.4%), the putative hybrids occupied an intermediate position, with more pronounced proximity to H. seideli, but without significant overlap (Figs. 4A–C). In the ventral graph, 83.2% of the variation was explained by the first axis and 10% by the second axis, with well-defined confidence ellipses indicating a clear separation between the groups (Figs. 5A–C).

Cross-validation. The cross-validation (Tabs. S1, S2 and S3) revealed variations in classification accuracy between the views. The dorsal view exhibited the lowest overall accuracy (50.9%), with putative hybrids mostly classified correctly but showing considerable confusion with other groups, suggesting moderate morphological overlap. The ventral view showed higher accuracy (67.3%), though misclassifications were more frequent between hybrids and H. yudja and H. seideli. The lateral view achieved the highest accuracy (75%), with H. zebra consistently classified correctly in all cases. The Kappa statistic reflected these trends, with values of 0.32 for the dorsal view, 0.55 for the ventral view, and 0.64 for the lateral view. Misclassifications primarily occurred between hybrids and parental species, reinforcing their morphological similarity. Despite some overlap in PCA plots, particularly in the dorsal view, H. zebra remained consistently distinct in the lateral and ventral views, supporting a stable pattern of morphological differentiation.

TABLE 1 | P-values for the disparity analysis of Hypancistrus species based on dorsal, lateral and ventral view comparisons. The significant p-value is highlighted in bold.

Comparison	Dorsal	Lateral	Ventral
Hypancistrus putative hybrid x Hypancistrus yudja	0.163	0.92	0.746
Hypancistrus putative hybrid x Hypancistrus seideli	0.565	0.369	0.297
Hypancistrus putative hybrid x Hypancistrus zebra	0.491	0.742	0.147
Hypancistrus seideli x Hypancistrus yudja	0.054	0.526	0.265
Hypancistrus seideli x Hypancistrus zebra	0.886	0.216	0.015
Hypancistrus yudja x Hypancistrus zebra	0.058	0.895	0.396

Morphological disparity. The p-value from the morphological disparity analysis between the groups indicated significant morphological variations, with H. zebra standing out for its consistently stable and distinct morphology. The putative hybrids showed greater morphological overlap with H. yudja and H. seideli, reflecting their mixed characteristics (Tab. 1).

Discussion​

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The PCA and CVA results indicate that the putative hybrids of Hypancistrus represent a morphologically intermediate group, sharing characteristics with both H. seideli and H. yudja. These results, combined with evidence of population genetic mixing (Santos, 2019) between these species, support the existence of a hybrid zone in the lower portion of the Volta Grande do Xingu. Notably, the morphological overlap was most evident between the putative hybrids and H. yudja in the dorsal view, while proximity to H. seideli was more pronounced in the lateral view. The misidentifications observed in cross-validation further reinforce the morphological proximity between putative hybrids and the parental species, suggesting that hybrids are more related to these two species than to H. zebra, which remained morphologically isolated in most analyses.

Hypancistrus zebra is a highly specialized species, with its distribution restricted to a specific 150 km stretch of the Volta Grande do Xingu. These fish inhabit crevices in large gneiss rocks in shallow waters with low to moderate currents, which makes them highly vulnerable to changes in water flow (Cardoso et al., 2016; Sousa et al., 2021b). This stable and distinct morphology of H. zebra (Sousa et al., 2024), confirmed in this study, reflects its adaptation to these exclusive habitats. However, the construction of the Belo Monte dam and the alteration of the river’s flow have drastically reduced the water flow during the dry season, compromising the rapids and rocky outcrop areas essential for the reproduction and survival of Hypancistrus species (Latrubesse et al., 2017; Fitzgerald et al., 2018). These hydrological changes may further reduce suitable habitats for H. zebra, increasing the risk of extinction for this species, which is already considered endangered(Magalhães et al., 2017; Sousa et al., 2021b; Barros et al., 2023).

In contrast, H. yudja is recognized as a habitat specialist, preferring deeper waters and areas of conglomerate rocks composed of pebbles and iron ore, primarily found above the Ananinduba waterfall on the Xingu (Santos, 2019). Its restricted distribution to a stretch of only 75 km in the Volta Grande of the Xingu River, where more than 70% of the water flow has been diverted for hydroelectric operations, exposes the species to severe habitat degradation (Sousa et al., 2025). Meanwhile, H. seideli exhibits a more generalist behavior and a wider distribution, occurring throughout the Volta Grande to the lower Xingu region, inhabiting both shallow and deep waters, and adapting to various substrates and strong currents (Cardoso et al., 2016; Santos, 2019; Sousa et al., 2021a).

Studies indicate that environmental pressure, such as habitat fragmentation caused by human activities, can force the displacement of populations into overlapping zones, promoting contact and reproduction between species that previously occupied distinct niches (Mallet, 2007; Fitzgerald et al., 2018; Santos, 2019). Climate change has contributed to a significant increase in the frequency of extreme droughts in the Amazon Basin over the last two decades, aggravating water stress in the region (Panisset et al., 2018; Tian et al., 2021). The 2024 drought, considered one of the most severe ever recorded, resulted in a sharp reduction in river levels, directly impacting water availability and compromising the region’s aquatic and terrestrial ecosystems. The hydrological alteration in the Volta Grande may have intensified the hybridization phenomenon between H. seideli and H. yudja, which could lead to a reduction in the genetic diversity of H. yudja, placing it at risk of local extinction (Todesco et al., 2016). Hybridization can dilute unique traits, weakening populations over time (Muhlfeld et al., 2009), a hypothesis that may be underway for H. yudja. Additionally, the ecological plasticity of H. seideli, which occupies a broader range of environments, gives it a significant adaptive advantage compared to the more habitat-restricted H. yudja. This difference may increase competition for habitats and resources (Todesco et al., 2016).

Thus, the niche overlap between these species may pose a significant challenge for the maintenance of H. yudja. Recent environmental monitoring and diving surveys indicate a significant decrease in the abundance of H. yudja compared to other Hypancistrus species (Sousa et al., 2025), corroborating the hypothesis that the species is losing ecological space to emerging hybrids and becoming more vulnerable in hybridization environments. The estimated population decline of more than 80% supports its possible classification as Critically Endangered (CR) (Sousa et al., 2025). Therefore, in this case, the damming of the rio Xingu not only impacts habitats but also alters interspecific interactions.

These changes in the Volta Grande ecosystem of the rio Xingu highlight the need for ongoing studies to monitor long-term impacts on Hypancistrus population structures. These studies are essential for guiding effective conservation policies, especially in a scenario of accelerated environmental degradation. This work provides a solid foundation for future research on the ongoing evolutionary processes in the Xingu River and the role of hybridization in the diversification of these species. Although our results suggest the existence of a putative hybrid between H. seideli and H. yudja, we emphasize that definitive validation of the hybridization hypothesis requires the integration of additional genetic evidence (FFK, work in progress). Finally, our findings emphasize the urgent need for targeted conservation strategies to mitigate the threats faced by these endemic species. Understanding their morphological and genetic relationships is crucial for their conservation, particularly considering the present description of a hybridization zone in the Volta Grande do Xingu.

Acknowledgments​

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We thank the members of the Laboratório de Aquicultura de Peixes ornamentais do Xingu (LAQUAX) at the Universidade Federal do Pará (UFPA), Altamira Campus, for their assistance in photographing specimens at all stages. We also thank Vanessa de Morais for illustrating the fish in the anatomical landmark representation.

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Authors

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Franciele Fernanda Kerniske¹ , Roger Henrique Dalcin², Leandro Melo de Sousa³ and Roberto Ferreira Artoni⁴

^[1]    Programa de Pós-Graduação em Genética Evolutiva e Biologia Molecular, Universidade Federal de São Carlos (UFSCar), Rodovia Washington Luis, 13565-905 São Carlos, SP, Brazil. francielekerniske17@gmail.com.

^[2]    Programa de Pós-Graduação em Ciências e Tecnologia Ambiental da UNIVALI, Universidade do Vale do Itajaí, Campus Itajaí, Rua Uruguai, 458, Centro, 88302-901 Itajaí, SC, Brazil. roger.dalcin@gmail.com.

^[3]    Universidade Federal do Pará (UFPA), Laboratório de Ictiologia de Altamira, Rua Coronel José Porfírio, 2515, 68372-040 Altamira, PA, Brazil. leandro.m.sousa@gmail.com.

^[4]    Programa de Pós-Graduação em Biologia Evolutiva, Universidade Estadual de Ponta Grossa (UEPG), Av. Carlos Cavalcanti, 4748, 84030-900 Ponta Grossa, PR, Brazil. rfartoni@gmail.com.

Authors’ Contribution

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Franciele Fernanda Kerniske: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing-original draft, Writing-review and editing.

Roger Henrique Dalcin: Conceptualization, Investigation, Methodology, Writing-original draft, Writing-review and editing.

Leandro Melo de Sousa: Data curation, Formal analysis, Investigation, Methodology, Writing-original draft, Writing-review and editing.

Roberto Ferreira Artoni: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing-original draft, Writing-review and editing.

Ethical Statement​

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The research was conducted following the guidelines of the Conselho Nacional de Controle da Experimentação Animal (CONCEA). It was authorized by the Comissão de Ética no Uso de Animais da Universidade Federal do Pará (protocol CEUA N^o. 6895300622) and the collection license of SISBIO N^o. 79124–1.

Competing Interests

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The author declares no competing interests.

How to cite this article

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Kerniske FF, Dalcin RH, Sousa LM, Artoni RF. Geometric morphometrics reveal a potential hybridization zone in endemic species of Hypancistrus (Siluriformes: Loricariidae) from the lower and middle rio Xingu. Neotrop Ichthyol. 2025; 23(2):e240107. https://doi.org/10.1590/1982-0224-2024-0107

Copyright​

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This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Distributed under

Creative Commons CC-BY 4.0

© 2025 The Authors.

Diversity and Distributions Published by SBI

Accepted March 27, 2025

Submitted October 23, 2024

Epub June 06, 2025