Locating Nesting Sites for Critically Endangered Galápagos Pink Land Iguanas (Conolophus marthae).

Invasive alien species control is recognized worldwide as a priority action to preserve global biodiversity. However, a lack of general life history knowledge for threatened species can impede the effectiveness of conservation actions. Galápagos pink land iguanas (Conolophus marthae) are endemic to Wolf Volcano, Galápagos, Ecuador. These iguanas are threatened by invasive alien species, particularly feral cats, that may affect their small population size. To guarantee the long-term survival of C. marthae, the Galápagos National Park Directorate is considering, along with an ongoing campaign of feral cat control, the implementation of a head-start program. However, the success of this management strategy necessarily relies on the identification of pink iguana nesting grounds, which were still unknown at the onset of this study. We modeled the movement patterns of male and female iguanas during the reproductive season, using location data collected from custom-made remote tracking devices installed on adult pink iguanas in April 2021. We first calculated for each individual the vector of distances from its starting location, which was defined as net displacement. We then used net displacement as the response variable in a generalized additive mixed model with day of the year as the predictor. Based on the hypothesis that males and females may behaviorally differ after mating, we looked for female-specific migratory behavior suggesting females were moving toward nesting areas. The results obtained confirmed our hypothesis, as females exhibited a distinct migratory behavior, reaching a small plateau area inside of Wolf Volcano's caldera and ca. 400 m below the volcano's northern rim. Moreover, once inside the caldera, females displayed a more aggregated distribution pattern. The movement data obtained allowed Galápagos National Park rangers to locate individual pink iguana nests and subsequently to sight and collect the first observed hatchlings of the species. This work constitutes a necessary baseline to perform dedicated studies of pink iguana nests and emerging hatchling iguanas, which is an essential step toward the development of an effective head-start program.

Hand grab or noose pole? Evaluating the least stressful practice for capture of endangered Turks and Caicos Rock Iguanas Cyclura carinata.

As the analysis of blood metabolites has become more readily accessible thanks to the use of point-of-care analyzers, it is now possible to evaluate stress level of wild animals directly in the field. Lactate is receiving much attention as a good stress level proxy in individuals subjected to capture, manual restraint, and data sampling in the wild, and appropriate protocols to maintain lactate values low should be preferred. In this study we compare how two different capture methodologies, hand grab vs. noose pole, affect the variation of blood lactate values in Cyclura carinata iguanas when captured for sampling. We used blood lactate concentration, measured immediately upon- and 15 min after-capture, as a proxy for stress level. While the primary goal of this work is to determine the least stressful capture methodology to be favored when sampling this and other wild iguanas, we also evaluated additional baseline physiological parameters relevant to the health and disease monitoring for this species. Our results show that while initial lactate values level-out in sampled individuals after 15 min in captivity, regardless of the capture methodology, rock iguanas captured by noose pole showed significantly higher lactate concentration and increased heartbeat rate immediately after capture. While the overall health evaluation determined that all analyzed individuals were in good health, based on our results we recommend that, when possible, hand capture should be preferred over noose pole when sampling wild individuals.

Novel herpesvirus in the critically endangered Galapagos pink land iguana.

Virus surveillance in wildlife is important to understanding ecosystem health, taxonomy, and evolution. Nevertheless, viruses in reptiles, and specifically in squamates, continue to be understudied. Herein, we conducted a health assessment on the critically endangered Galapagos pink land iguana (Conolophus marthae) and the vulnerable Galapagos land iguana (Conolophus subcristatus). We collected oral and/or cloacal swabs from 47 clinically healthy iguanas and tested for adenovirus (cloacal swabs, n = 47) and herpesvirus (oral swabs, n = 45) using broad-spectrum PCRs. Two out of 38 (5.3 %) Galapagos pink land iguanas tested positive for herpesvirus, while no herpesvirus was detected in all Galapagos land iguanas (n = 7). Both herpesviral sequences were identical between them and divergent (61.9 % amino acid identity) when compared to the closest herpesvirus sequences available in GenBank/EMBL/DDBJ. The genetic distance between this and other herpesviruses is consistent with its classification as a novel virus species. All iguanas were negative for adenovirus. This is the first description of a herpesvirus in iguanas of the Galapagos islands, and the first report of a potential pathogen for the iconic Galapagos pink land iguana. Further research is needed to understand the implications of this virus in the conservation and management of one of the most endangered iguana species in the world.

Nitrogen and carbon stable isotope analysis sheds light on trophic competition between two syntopic land iguana species from Galápagos.

Coexistence between closely related species can lead to intense competition for resources. Stable isotope analysis (SIA) is a reliable tool to estimate the extent of species competition. We employed SIA to evaluate niche partitioning among two syntopic species of Galápagos land iguanas: Conolophus subcristatus and C. marthae. Samples were collected on Wolf Volcano, Isabela Island, where C. marthae is endemic and syntopic with C. subcristatus. We determined δ13C and δ15N ratios and described the isotopic niche of each species using corrected standard ellipse area (SEAc). We tested for differentiation between the isotopic niches, while controlling for sex, body size, spatial location of samples and mean annual primary productivity at capture points, using bivariate linear models. Despite the extensive overlap of the isotopic niches, we found species and sex to be a significant, interacting predictor of a sample's location in the δ13C, δ15N space, indicating the existence of niche partitioning mechanisms acting between species and sexes. We also found that body size and productivity at the capture points, compounded with yet undetermined spatial effects, explain ca. 75% of the differences observed between species and sexes, providing evidence for differential microhabitat and food-items usage. Our study provides essential baselines for evaluating conservation actions for C. marthae, such as the potential translocation to a sanctuary area free of competition from C. subcristatus.

25-Hydroxyvitamin D Plasma Levels in Natural Populations of Pigmented and Partially Pigmented Land Iguanas from Galápagos (Conolophus spp.).

We report the first data on 25-hydroxyvitamin D plasma levels in natural populations of three species of land iguana endemic to the Galápagos Islands (Conolophus marthae, C. subcristatus, and C. pallidus). The pigment is present throughout the whole body in the skin of C. subcristatus and C. pallidus. On the contrary, pigment is not present in the skin of an extended part of the body in C. marthae. The only existing population of C. marthae is syntopic with a population of C. subcristatus, and the two species are closely related. These circumstances would suggest that, under the assumption that the species show a similar basking behavior and in the absence of compensatory mechanisms, lighter pigmentation should favor higher vitamin D levels. Thus, C. marthae, compared with C. subcristatus in Wolf Volcano, could show higher levels of 25(OH)D plasma levels, or equal, if compensatory mechanisms exist. The three species showed levels in the range of average values for healthy iguanas. However, contrary to the expectation, C. marthae consistently exhibited the lowest 25(OH)D plasma levels. We discuss possible factors affecting vitamin concentration and hypothesize that C. marthae may use the habitat to limit exposure to the high UVB irradiation at Wolf Volcano.

Conservation of a flagship species: Health assessment of the pink land iguana, Conolophus marthae.

The pink land iguana, Conolophus marthae, is one of four species of iguanas (three terrestrial and one marine) in the Galápagos Islands, and the only one listed as critically endangered by the IUCN. The species can only be found on the north-west slopes of the highest volcano on Isabela Island and was first described to science in 2009. As part of a population telemetry study, a health assessment was authorized by the Galápagos National Park. Wild adult iguanas were captured on Wolf Volcano in September 2019 and April 2021 to record morphological and physiological parameters including body temperature, heart rate, intraocular pressures, tear formation, and infrared iris images. Blood samples were also collected and analyzed. An i-STAT portable blood analyzer was used to obtain values for base excess in the extracellular fluid compartment (BEecf), glucose (Glu), hematocrit (HctPCV), hemoglobin (Hb), ionized calcium (iCa), partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), percent oxygen saturation (sO2%), pH, potassium (K), and sodium (Na). When possible, data were compared to previously published and available data for the other Galápagos iguanas. The results reported here provide baseline values that will be useful in detecting changes in health status among pink land iguanas affected by climate change, invasive species, anthropogenic threats, or natural disturbances. The collected data also provide an invaluable resource for conservation scientists planning to implement conservation strategies, like translocations, that may temporarily alter these baseline values.

Evidence of Another Anthropic Impact on Iguana delicatissima from the Lesser Antilles: The Presence of Antibiotic Resistant Enterobacteria.

The improper use of antibiotics by humans may promote the dissemination of resistance in wildlife. The persistence and spread of acquired antibiotic resistance and human-associated bacteria in the environment, while representing a threat to wildlife, can also be exploited as a tool to monitor the extent of human impact, particularly on endangered animal species. Hence, we investigated both the associated enterobacterial species and the presence of acquired resistance traits in the cloacal microbiota of the critically endangered lesser Antillean iguana (Iguana delicatissima), by comparing two separate populations living in similar climatic conditions but exposed to different anthropic pressures. A combination of techniques, including direct plating, DNA sequencing and antimicrobial susceptibility testing allowed us to characterize the dominant enterobacterial populations, the antibiotic resistant strains and their profiles. A higher frequency of Escherichia coli was found in the samples from the more anthropized site, where multi-drug resistant strains were also isolated. These results confirm how human-associated bacteria as well as their antibiotic-resistance determinants may be transferred to wildlife, which, in turn, may act as a reservoir of antibiotic resistance.

The ultimate database to (re)set the evolutionary history of primate genital bones.

Scientific literature concerning genital bones in primates consists of both ancient works (dating back to the nineteenth century) and more recent revisions/meta-analyses, which, however, are not always so detailed or exhaustive. Based on a thorough analysis, several conflicting data, inaccurate references, and questionable claims have emerged. We generated a binary matrix of genital bone occurrence data, considering only data at the species level, based on (1) a rigorous literature search protocol, (2) raw data (collected exclusively from primary literature), (3) an updated taxonomy (often tracing back to the species taxonomic history) and (4) new occurrence data from scanned genitals of fresh and museum specimens (using micro-computed tomography-micro-CT). Thanks to this methodological approach, we almost doubled available occurrence data so far, avoiding any arbitrary extension of generic data to conspecific species. This practice, in fact, has been recently responsible for an overestimation of the occurrence data, definitively flattening the interspecific variability. We performed the ancestral state reconstruction analysis of genital bone occurrence and results were mapped onto the most updated phylogeny of primates. As for baculum, we definitively demonstrated its simplesiomorphy for the entire order. As for baubellum, we interpreted all scattered absences as losses, actually proposing (for the first time) a simplesiomorphic state for the clitoral bone as well. The occurrence data obtained, while indirectly confirming the baculum/baubellum homology (i.e., for each baubellum a baculum was invariably present), could also directly demonstrate an intra-specific variability affecting ossa genitalia occurrence. With our results, we established a radically improved and updated database about the occurrence of genital bones in primates, available for further comparative analyses.

Chemical signatures of femoral pore secretions in two syntopic but reproductively isolated species of Galápagos land iguanas (Conolophus marthae and C. subcristatus).

The only known population of Conolophus marthae (Reptilia, Iguanidae) and a population of C. subcristatus are syntopic on Wolf Volcano (Isabela Island, Galápagos). No gene flow occurs suggesting that effective reproductive isolating mechanisms exist between these two species. Chemical signature of femoral pore secretions is important for intra- and inter-specific chemical communication in squamates. As a first step towards testing the hypothesis that chemical signals could mediate reproductive isolation between C. marthae and C. subcristatus, we compared the chemical profiles of femoral gland exudate from adults caught on Wolf Volcano. We compared data from three different years and focused on two years in particular when femoral gland exudate was collected from adults during the reproductive season. Samples were processed using Gas Chromatography coupled with Mass Spectrometry (GC-MS). We identified over 100 different chemical compounds. Non-Metric Multidimensional Scaling (nMDS) was used to graphically represent the similarity among individuals based on their chemical profiles. Results from non-parametric statistical tests indicate that the separation between the two species is significant, suggesting that the chemical profile signatures of the two species may help prevent hybridization between C. marthae and C. subcristatus. Further investigation is needed to better resolve environmental influence and temporal reproductive patterns in determining the variation of biochemical profiles in both species.

Health assessment of Conolophus subcristatus, Conolophus pallidus, and C. subcristatus X Amblyrhynchus cristatus hybrid (Galápagos land iguanas).

The land iguanas, Conolophus pallidus and Conolophus subcristatu are large and charismatic lizards endemic to the Galápagos archipelago, but little information exists on their normal health parameters. The former is restricted to Santa Fe island, while C. subcristatus inhabits the islands of the central and western region of the archipelago. Both species are classified as vulnerable by the IUCN Red List of Threatened Species. As part of a population health assessment authorized by the Galápagos National Park, wild adult iguanas from three islands (North Seymour, South Plazas, and Santa Fe) were captured in July 2018. Data from a single C. subcristatus X Amblyrhynchus cristatus hybrid captured on South Plazas is also included. We analyzed blood samples drawn from 52 healthy wild adult land iguanas captured on three islands. An iSTAT portable blood analyzer was used to obtain values for pH, lactate, pO2, pCO2, HCO3-, sO2%, hematocrit, packed cell volume (PCV), hemoglobin Na, K, iCa, and glucose. Standard laboratory hematology techniques were employed for PCV determination; resulting values were also compared to the hematocrit values generated by the iSTAT. Body temperature, heart rate, respiratory rate, and body measurements were also recorded and compared to previously published data for the marine iguana (Amblyrhynchus cristatus), which shares a common ancestor with the land iguana. The data reported here provide preliminary baseline values that may be useful in comparisons between captive and wild populations, between wild populations, and in detecting changes in health status among Galápagos land iguanas affected by anthropogenic threats, climate change, or natural disturbances.

Genetic structure, phylogeography, and demography of Anadara tuberculosa (Bivalvia) from East Pacific as revealed by mtDNA: Implications to conservation.

Wild populations of the pustulose ark, Anadara tuberculosa (Bivalvia), an emblematic species of the East Pacific mangrove ecosystem declined in South American countries (Colombia, Ecuador, and Peru) mainly due to overharvesting and habitat loss or degradation. Understanding the genetic aspects of geographic variations and population structure of A. tuberculosa, currently unknown, appears as a priority to fishery authorities in order to elaborate integrated and collaborative conservation policies for fishery management, aquaculture, and stock enhancement programs. We used mtDNA sequence data to investigate haplotype diversity, genetic structure, and demography of A. tuberculosa. Results indicate genetic homogeneity of populations distributed north and south of the equator, respectively. However, statistically significant differentiation emerged between northern and southern populations with pairwise ф ST values ranging between 0.036 and 0.092. The oceanic current system acting in the area (Panama Current and Humboldt Current) might play a role in limiting the larval dispersal of the species, still poorly understood. Demography reconstruction supported recent population expansion, possibly started after last glacial maximum. Our results would suggest separate and independent management of populations north and south of the equator.

The Design of an Energy Harvesting Wireless Sensor Node for Tracking Pink Iguanas.

The design of wireless sensor nodes for animal tracking is a multidisciplinary activity that presents several research challenges both from a technical and a biological point of view. A monitoring device has to be designed accounting for all system requirements including the specific characteristics of animals and environment. In this work we present some aspects of the design of a wireless sensor node to track and monitor the pink iguana of the Galápagos: a recently discovered species living in remote locations at the Galápagos Islands. The few individuals of this species live in a relatively small area that lacks of any available communication infrastructure. We present and discuss the energy harvesting architecture and the related energy management logic. We also discuss the impact of packaging on the sensor performance and the consequences of the limited available energy on the GPS tracking.

Effects of parasitic infection and reproduction on corticosterone plasma levels in Galápagos land iguanas, Conolophus marthae and C. subcristatus.

In vertebrates, one main feature of stress response is the release of glucocorticoids (corticosterone in reptiles), steroid hormones whose synthesis is regulated by the hypothalamic-pituitary-adrenal axis (HPA). In the Galápagos Islands, populations of land iguanas are differentially impacted by a tick-transmitted apicomplexan hemoparasite of genus Hepatozoon, which could cause diseases and ultimately reduce fitness. Using competitive enzyme-linked immunosorbent assays (ELISA), we examined baseline plasma corticosterone levels of two syntopic and highly parasitized populations of the land iguana species Conolophus marthae and C. subcristatus in Wolf volcano (Isabela Island). We also used a poorly parasitized population of C. subcristatus from the same island (Bahia Urbina) as a reference. To better interpret the observed glucocorticoids patterns, we simultaneously performed the count of white blood cells (WBCs) in all individuals and investigated the reproductive status of females. We did not find evidence in support of either a positive or negative relationship between the tick load, hemoparasite infection, and glucocorticoid plasma concentration in C. marthae and C. subcristatus at Wolf volcano. The comparison between parasitized and non-parasitized sites (V. Wolf and Bahia Urbina) would instead suggest an inverse relationship between corticosterone and parasites. Our findings support association between corticosterone plasma levels and reproduction.

Plasma concentrations of progesterone and estradiol and the relation to reproduction in Galápagos land iguanas, Conolophus marthae and C. subcristatus (Squamata, Iguanidae).

In a combined approach, endocrine and ultrasonic analyses were performed to assess reproduction of two syntopic populations of terrestrial Galápagos iguanas the Conolophus marthae (the Galápagos Pink Land Iguana) and C. subcristatus on the Volcán Wolf (Isabela Island). The ELISA methods (enzyme-linked immunosorbent assay) were used to measure plasma concentrations of progesterone (P4) and 17ß-estradiol (E2) from samples collected over the course of three different seasons: July 2010, June 2012-2014. As for C. subcristatus, the large number of females with eggs in 2012 and 2014 were associated with increased plasma P4 concentrations and the corresponding absence of females with eggs in July 2010 when concentrations of both hormones levels were basal indicating reproduction was still ongoing in June and had ended in July. In C. marthae, even though there was a positive relationship between egg-development stages and hormone concentrations, P4 concentrations were basal through the three years that samples were collected, with some females having a lesser number of eggs compared with C. subcristatus. In C. marthae P4 and E2 patterns did not allow for defining a specific breeding season.

Mature Erythrocytes of Iguana iguana (Squamata, Iguanidae) Possess Functional Mitochondria.

Electron microscopy analyses of Iguana iguana blood preparations revealed the presence of mitochondria within erythrocytes with well-structured cristae. Fluorescence microscopy analyses upon incubation with phalloidin-FITC, Hoechst 33342 and mitochondrial transmembrane potential (Δψm)-sensitive probe MitoTracker Red indicated that mitochondria i) widely occur in erythrocytes, ii) are polarized, and iii) seem to be preferentially confined at a "perinuclear" region, as confirmed by electron microscopy. The analysis of NADH-dependent oxygen consumption showed that red blood cells retain the capability to consume oxygen, thereby providing compelling evidence that mitochondria of Iguana erythrocytes are functional and capable to perform oxidative phosphorylation.

Hybridization masks speciation in the evolutionary history of the Galápagos marine iguana.

The effects of the direct interaction between hybridization and speciation-two major contrasting evolutionary processes--are poorly understood. We present here the evolutionary history of the Galápagos marine iguana (Amblyrhynchus cristatus) and reveal a case of incipient within--island speciation, which is paralleled by between-island hybridization. In-depth genome-wide analyses suggest that Amblyrhynchus diverged from its sister group, the Galápagos land iguanas, around 4.5 million years ago (Ma), but divergence among extant populations is exceedingly young (less than 50,000 years). Despite Amblyrhynchus appearing as a single long-branch species phylogenetically, we find strong population structure between islands, and one case of incipient speciation of sister lineages within the same island--ostensibly initiated by volcanic events. Hybridization between both lineages is exceedingly rare, yet frequent hybridization with migrants from nearby islands is evident. The contemporary snapshot provided by highly variable markers indicates that speciation events may have occurred throughout the evolutionary history of marine iguanas, though these events are not visible in the deeper phylogenetic trees. We hypothesize that the observed interplay of speciation and hybridization might be a mechanism by which local adaptations, generated by incipient speciation, can be absorbed into a common gene pool, thereby enhancing the evolutionary potential of the species as a whole.

Characterization of Salmonella occurring at high prevalence in a population of the land iguana Conolophus subcristatus in Galápagos Islands, Ecuador.

The aim of the study was to elucidate the association between the zoonotic pathogen Salmonella and a population of land iguana, Colonophus subcristatus, endemic to Galápagos Islands in Ecuador. We assessed the presence of Salmonella subspecies and serovars and estimated the prevalence of the pathogen in that population. Additionally, we investigated the genetic relatedness among isolates and serovars utilising pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA and determined the antimicrobial susceptibility to a panel of antimicrobials. The study was carried out by sampling cloacal swabs from animals (n = 63) in their natural environment on in the island of Santa Cruz. A high prevalence (62/63, 98.4%) was observed with heterogeneity of Salmonella subspecies and serovars, all known to be associated with reptiles and with reptile-associated salomonellosis in humans. Serotyping revealed 14 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 48), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 1), and S. enterica subsp. houtenae (n = 7). Four serovars were predominant: S. Poona (n = 18), S. Pomona (n = 10), S. Abaetetuba (n = 8), and S. Newport (n = 5). The S. Poona isolates revealed nine unique XbaI PFGE patterns, with 15 isolates showing a similarity of 70%. Nine S. Pomona isolates had a similarity of 84%. One main cluster with seven (88%) indistinguishable isolates of S. Abaetetuba was observed. All the Salmonella isolates were pan-susceptible to antimicrobials representative of the most relevant therapeutic classes. The high prevalence and absence of clinical signs suggest a natural interaction of the different Salmonella serovars with the host species. The interaction may have been established before any possible exposure of the iguanas and the biocenosis to direct or indirect environmental factors influenced by the use of antimicrobials in agriculture, in human medicine or in veterinary medicine.

Tracking acquired antibiotic resistance in commensal bacteria of Galápagos land iguanas: no man, no resistance.

BACKGROUND: Antibiotic resistance, evolving and spreading among bacterial pathogens, poses a serious threat to human health. Antibiotic use for clinical, veterinary and agricultural practices provides the major selective pressure for emergence and persistence of acquired resistance determinants. However, resistance has also been found in the absence of antibiotic exposure, such as in bacteria from wildlife, raising a question about the mechanisms of emergence and persistence of resistant strains under similar conditions, and the implications for resistance control strategies. Since previous studies yielded some contrasting results, possibly due to differences in the ecological landscapes of the studied wildlife, we further investigated this issue in wildlife from a remote setting of the Galapagos archipelago. METHODOLOGY/PRINCIPAL FINDINGS: Screening for acquired antibiotic resistance was carried out in commensal enterobacteria from Conolophus pallidus, the terrestrial iguana of Isla Santa Fe, where: i) the abiotic conditions ensure to microbes good survival possibilities in the environment; ii) the animal density and their habits favour microbial circulation between individuals; and iii) there is no history of antibiotic exposure and the impact of humans and introduced animal species is minimal except for restricted areas. Results revealed that acquired antibiotic resistance traits were exceedingly rare among bacteria, occurring only as non-dominant strains from an area of minor human impact. CONCLUSIONS/SIGNIFICANCE: Where both the exposure to antibiotics and the anthropic pressure are minimal, acquired antibiotic resistance traits are not normally found in bacteria from wildlife, even if the ecological landscape is highly favourable to bacterial circulation among animals. Monitoring antibiotic resistance in wildlife from remote areas could also be a useful tool to evaluate the impact of anthropic pressure.

Uneven frequency of Vibrio alginolyticus-group isolates among different populations of Galápagos marine iguana (Amblyrhynchus cristatus).

The presence of Vibrio isolates was investigated in cloacal swabs from the Galápagos marine iguana (Amblyrhyncus cristatus). Such unique iguana is endemic to the Galápagos Archipelago, it is listed as vulnerable in the IUCN Red List (2009), and is strictly protected by CITES and Ecuador laws. Our results revealed an uneven isolation frequency of vibrios from animals living in different settings: maximal among the Santa Fe population, scarce at Bahía Tortuga but practically absent in the samples from Puerto Ayora and Plaza Sur. A 16S sequencing confirmed that the isolates belonged to the genus Vibrio, placing them within the V. alginolyticus group; the biochemical identification was, indeed, consistent with V. alginolyticus features. The reason of the observed discrepancy is not clear, but could be either linked to a higher pollution in the inhabited or more touristic places or to differential influence of chemical and physical parameters at a local scale. As V. alginolyticus is an opportunistic pathogen for man and it is known to cause disease in sea-living animals, the ability of these vibrios to enter and persist to a certain extent in the marine iguana gut should be regarded as a risk for health of both the animals and the human personnel involved in monitoring activities.

Phylogeography of Helleria brevicornis (Crustacea, Oniscidea): old and recent differentiations of an ancient lineage.

Helleria brevicornis has a disjunct, peri-Tyrrhenian distribution that mirrors that of several organisms, for which geographic vicariance is invoked, due to the geological events started with the Oligocene split of the Sardo-Corsican microplate from the Pyrenees, and successive separation between Sardinia and Corsica. Molecular phylogenetic analyses demonstrated that such a biogeographic model does not apply to Helleria. The original split of the Sardinian and Corsican lineages originated in the Early Pliocene. Further diversification occurred later. The colonization of the Tuscan archipelago, French, and Italian mainland occurred most recently, but a possible time dependency bias suggests that such colonization was driven by human-transport.