Biology subjects

Bilandzija, H.

Publications and source records attributed to Bilandzija, H..

8 recordsLinked to original sources

bioRxiv2026

Cave-dwelling Planariidae in Croatia exhibit differing levels of cave trait evolution

The Dinaric karst of Croatia encompasses a network of over 10,000 caves and represents one of the worlds most important subterranean biodiversity hotspots. It is inhabited by remarkably diverse and often endemic species, including planarian flatworms, which are among the rarest macroinvertebrates encountered in cave habitats. Although the presence of cave planarians has long been known, no integrative research on this group has been conducted to date, and the evolutionary relationships between these animals and their surface water counterparts are currently unresolved. To address these gaps, we combined field sampling, phylogenetic analysis based on COI and 18S genes, and phenotypic characterization. Our results show that cave planariids in Croatia belong to at least three genera and are more widespread and diverse across both Croatia, and the broader Dinaric karst, than previously assumed. We increased the number of cave records in the Dinaric karst from 26 to 37 and documented cf. Atrioplanaria and Phagocata in Croatian caves for the first time. Phylogenetic reconstructions suggest numerous independent cave colonization events, including multiple instances within the genera Crenobia and cf. Atrioplanaria. Variation in pigmentation and eye reduction, both within and between populations, further reveal heterogeneous evolutionary trajectories of cave-associated phenotypes. The biogeographical patterns and high genetic diversity we report here point to a complex evolutionary history of planariids in the Dinarides. Our newly generated molecular phylogenies and systematic documentation of trait variability establish Planariidae as a valuable model for studying mechanisms underlying convergent evolution of pigment loss and eye reduction in cave environments.

zoology↗

bioRxiv2026

Sustained darkness reveals rapid phenotypic accommodation and loss of plasticity in cavefish evolution

How environmentally induced traits become genetically stabilized during evolution remains a central question in biology. The teleost Astyanax mexicanus, comprising surface (SF) and cave (CF) morphotypes, provides a powerful system to link environmentally induced traits with evolved phenotypes. Here, we integrate morphometric, physiological, and transcriptomic analyses to investigate how sustained darkness reshapes phenotypic plasticity across generations. We show that plastic responses in SF are rapidly modified after only two generations of dark rearing (2dSF). Morphological changes in 2dSF are highly heterogeneous: some traits shift toward cavefish values, while others overshoot them, and key traits such as eye size and pigmentation respond in the opposite direction, indicating widespread maladaptive plasticity. In contrast, CF exhibit limited environmental responsiveness, consistent with canalized phenotypes. Traits exhibiting maladaptive plasticity in SF tend to be canalized in CF, suggesting that maladaptive responses are preferentially reduced during evolution. At the molecular level, transcriptional plasticity declines sharply from SF to 2dSF and is nearly absent in CF, suggesting rapid loss of environmental responsiveness. Gene expression analyses further reveal a mixture of maladaptive and adaptive trajectories. Most transcriptional changes in 2dSF deviate from cavefish expression patterns, including those related to visual function, consistent with maladaptive morphological trends. However, a subset of genes involved in lipid metabolism and heme homeostasis shows stepwise shifts from SF through 2dSF to CF, consistent with adaptive accommodation. Notably, anesthesia resistance increases in 2dSF to levels comparable to CF within only two generations, demonstrating rapid accommodation in neural traits. Together, our results show that plastic phenotypes are initially heterogeneous but are rapidly reshaped across generations, with maladaptive components reduced and adaptive components stabilized. This process bridges immediate plasticity and long-term evolutionary change, providing empirical support for phenotypic accommodation as a mechanism facilitating rapid adaptation and highlighting how plasticity can biasing evolutionary trajectories during the colonization of extreme environments.

evolutionary biology↗

bioRxiv2026

Environmental hypoxia controls the evolution of cavefish heart asymmetry

The environmental factors responsible for the evolution of novel traits and their underlying mechanisms are poorly understood. Here we use the teleost Astyanax mexicanus consisting of ancestral surface fish and derived cavefish morphotypes to address the evolution of heart asymmetry. As in other vertebrates, surface fish develop right(D)-looping heart tubes, whereas high levels of left(L)-looping heart tubes have uniquely evolved in cavefish. Cavefish cardiac L-looping is mediated by the upregulation of Sonic Hedgehog (Shh) signalling during gastrulation, which disrupts left-right organizer (LRO) function and modifies the downstream left-oriented Nodal signalling cascade. As a proxy for the original cave colonizers, we exposed contemporary surface fish to key cave-associated environmental factors, namely complete darkness, reduced electrical conductivity, low temperature, hypoxia, or combinations of these conditions. Only hypoxia induced the cavefish L-looping phenotype. Hypoxia increased expression of genes in an integrated Hypoxia Inducing Factor 1(HIF1)-Shh axis, disrupted LRO function, and modified the L-R determination program in surface fish similarly to naturally evolved changes in cavefish. In contrast to surface fish, which show high plasticity in response to hypoxia, cavefish were unable to survive acute hypoxia, suggesting evolutionary refinement and canalization of the ancestral hypoxia response. These studies establish hypoxia as the driver of heart asymmetry and reveal a mechanistic connection between the hypoxic cave environment and the evolution of a novel trait.

evolutionary biology↗

bioRxiv2025

Analysis of multi-trait evolution across independently evolved cavefish populations reveals shared and independent evolution of suites of cave-associated traits

Environmental perturbations often lead to the evolution of multiple traits. Determining whether shared genetic factors underlie multi-trait evolution is a central question in evolutionary biology. In the Mexican tetra, Astyanax mexicanus, cave-dwelling populations have repeatedly evolved multiple traits. The repeated evolution of these traits, paired the robust environmental differences between the surface and cave habitats, provide an opportunity to investigate the genetic basis of multi-trait evolution. Here, we investigate the extent to which shared genetic mechanisms underlie the repeated evolution of multiple traits in cavefish. Across cave populations, we find evidence for shared and distinct genetic mechanisms contributing to the evolution of individual traits. Further, multiple traits covary in cave-surface F2 hybrids and many of the same trait correlations are found across independently evolved cave populations. Finally, we assessed traits that differ between pigmented and albino F2 fish in surface fish with mutations in the albinism gene oculocutaneous albinism 2 (oca2). This revealed that mutations in oca2 reduce bottom-dwelling behavior in A. mexicanus. Together, these findings suggest that multi-trait evolution occurs repeatedly through shared genetic factors across A. mexicanus cave populations. These results are consistent with pleiotropy or linkage playing a large role in multi-trait evolution in this species.

evolutionary biology↗

bioRxiv2025

A common mechanism and multiple advantages of pigment loss underlie the convergent evolution of albinism in cave animals

The loss of pigmentation is a hallmark adaptation of cave-dwelling animals but the underlying mechanisms are poorly understood. This study investigates the cellular and biochemical basis of albinism, the loss of melanin pigment, which convergently evolved across a wide range of cave animals. Is albinism caused by the interruption of pigment synthesis or elimination of the pigment cell lineage? The results suggest that albinism evolved by a common mechanism, a block in the first step of the melanin biosynthesis pathway, the conversion of L-tyrosine to L-DOPA, in diverse albino cave animals ranging from annelids and mollusks to vertebrates. Pigment cells are conserved in all tested albino cave species and distributed in patterns resembling pigmentation in their close surface relatives. The cells capable of melanin synthesis when provided with L-DOPA substrate were detected at the sites of injuries in a cave annelid, cave mollusk, and cave teleost, suggesting roles in innate immunity during tissue repair, which may explain why pigment cells are conserved despite the loss of pigment production. Next, we focused on the longstanding issue of the evolutionary forces involved in the regression of pigmentation in cave animals and explored hypotheses of why losing pigmentation might be adaptive in cave environments. First, we tested the hypothesis that pigment regression may conserve energy, which is important for survival in the food-limited cave environment. Our results indicate that melanin synthesis has a marked energetic cost in the cavefish, Astyanax mexicanus. Lastly, we show that the disruption of melanin synthesis at its first biosynthetic step is correlated with increased dopamine levels in multiple depigmented cavefish populations, including populations that evolved albinism independently. This result supports a widespread tradeoff between the melanin and catecholamine synthesis pathways in A. mexicanus. We conclude that the interruption of the conversion of L-tyrosine to L-DOPA in the synthesis of melanin confers multiple advantages that could serve as targets of natural selection, supporting an adaptive hypothesis for the evolution of albinism in cave animals.

evolutionary biology↗

bioRxiv2025

An integrative taxonomic approach reveals unexplored diversity in Croatian planarians

BackgroundFreshwater ecosystems are among the most endangered habitats on Earth, with approximately one-fourth of aquatic species at risk of extinction. Effective conservation efforts require comprehensive monitoring and accurate species identification, including often overlooked groups. DNA barcoding promises rapid and accessible species identification but requires the availability of "universal" primer pairs and robust, taxonomically curated reference libraries. Planarian flatworms are one such group for which these resources are currently lacking, even though they are common constituents of freshwater ecosystems worldwide. As a result, the true extent of planarian diversity remains underappreciated in many areas. ResultsMotivated by the highly skewed representation of planarian diversity in current GenBank records of the barcoding gene mitochondrial cytochrome c oxidase subunit I (COI), we optimised existing barcoding primers and tested them on planarians collected in a species-rich but, so far, poorly surveyed region of Croatia. Using an integrative approach combining the new primers, traditional taxonomy and karyology, we generated new taxonomically curated COI barcode sequences for European dugesiids, dendrocoelids and planariids, which, in the case of the latter two groups, represent a substantial increase of the number of entries for those families. Our efforts resulted in several significant findings, including the description of a new pigmented Dendrocoelum species, Dendrocoelum pigmentatum sp. nov, the discovery of two highly differentiated haplotypic clades in Schmidtea lugubris, and the rediscovery of Polycladodes alba in Croatia after a century. ConclusionsOverall, our work extends the utility of DNA barcoding for species-level identification of planarians to previously inaccessible groups. Additionally, it integrates Croatia as an underexplored but species-rich region into the endeavour to systematically describe the planarian fauna of Europe. The expansion of a known number of Croatian planarian species from eight to sixteen and the discovery of a new, large planarian species in continental Europe, Dendrocoelum pigmentatum, demonstrate the effectiveness of our integrative approach. Overall, our work highlights the underappreciated diversity of planarians, even in continental Europe and supports practical conservation efforts to preserve aquatic biodiversity.

zoology↗

bioRxiv2019

Phenotypic plasticity as an important mechanism of cave colonization and adaptation in Astyanax cavefish

A widely accepted model for the evolution of cave animals posits colonization by surface ancestors followed by the acquisition of adaptations over many generations. However, the speed of cave adaptation in some species suggests mechanisms operating over shorter timescales. To address these mechanisms, we used Astyanax mexicanus, a teleost with ancestral surface morphs (surface fish, SF) and derived cave morphs (cavefish, CF). We exposed SF to completely dark conditions and identified numerous altered traits at both the gene expression and phenotypic levels. Remarkably, most of these alterations mimicked CF phenotypes. Our results indicate that cave-related traits can appear within a single generation by phenotypic plasticity. In the next generation, plasticity can be further refined. The initial plastic responses are random in adaptive outcome but may determine the subsequent course of evolution. Our study suggests that phenotypic plasticity contributes to the rapid evolution of cave-related traits in A. mexicanus.

evolutionary biology↗

bioRxiv2018

The role of gene flow in rapid and repeated evolution of cave related traits in Mexican tetra, Astyanax mexicanus

Understanding the molecular basis of repeated evolved phenotypes can yield key insights into the evolutionary process. Quantifying the amount of gene flow between populations is especially important in interpreting mechanisms of repeated phenotypic evolution, and genomic analyses have revealed that admixture is more common between diverging lineages than previously thought. In this study, we resequenced and analyzed nearly 50 whole genomes of the Mexican tetra from three blind cave populations, two surface populations, and outgroup samples. We confirmed that cave populations are polyphyletic and two Astyanax mexicanus lineages are present in our dataset. The two lineages likely diverged [~]257k generations ago, which, assuming 1 generation per year, is substantially younger than previous mitochondrial estimates of 5-7mya. Divergence of cave populations from their phylogenetically closest surface population likely occurred between [~]161k - 191k generations ago. The favored demographic model for most population pairs accounts for divergence with secondary contact and heterogeneous gene flow across the genome, and we rigorously identified abundant gene flow between cave and surface fish, between caves, and between separate lineages of cave and surface fish. Therefore, the evolution of cave-related traits occurred more rapidly than previously thought, and trogolomorphic traits are maintained despite substantial gene flow with surface populations. After incorporating these new demographic estimates, our models support that selection may drive the evolution of cave-derived traits, as opposed to the classic hypothesis of disuse and drift. Finally, we show that a key QTL is enriched for genomic regions with very low divergence between caves, suggesting that regions important for cave phenotypes may be transferred between caves via gene flow. In sum, our study shows that shared evolutionary history via gene flow must be considered in studies of independent, repeated trait evolution.

evolutionary biology↗