Genetic structure of American bullfrog populations in Brazil.

Non-native species are a major problem affecting numerous biomes around the globe. Information on their population genetics is crucial for understanding their invasion history and dynamics. We evaluated the population structure of the non-native American bullfrog, Aquarana catesbeiana, in Brazil on the basis of 324 samples collected from feral and captive groups at 38 sites in seven of the nine states where feral populations occur. We genotyped all samples using previously developed, highly polymorphic microsatellite loci and performed a discriminant analysis of principal components together with Jost's D index to quantify pairwise differentiation between populations. We then amplified 1,047 base pairs of the mitochondrial cytochrome b (cytb) gene from the most divergent samples from each genetic population and calculated their pairwise differences. Both the microsatellite and cytb data indicated that bullfrogs comprise two populations. Population grouping 1 is widespread and possesses two cytb haplotypes. Population grouping 2 is restricted to only one state and possesses only one of the haplotypes from Population grouping 1. We show that there were two imports of bullfrogs to Brazil and that there is low genetic exchange between population groupings. Also, we find that there is no genetic divergence among feral and captive populations suggesting continuous releases. The limited genetic variability present in the country is associated to the small number of introductions and founders. Feral bullfrogs are highly associated to leaks from farms, and control measures should focus on preventing escapes using other resources than genetics, as feral and captive populations do not differ.

Dose-dependent alkaloid sequestration and N-methylation of decahydroquinoline in poison frogs.

Sequestration of chemical defenses from dietary sources is dependent on the availability of compounds in the environment and the mechanism of sequestration. Previous experiments have shown that sequestration efficiency varies among alkaloids in poison frogs, but little is known about the underlying mechanism. The aim of this study was to quantify the extent to which alkaloid sequestration and modification are dependent on alkaloid availability and/or sequestration mechanism. To do this, we administered different doses of histrionicotoxin (HTX) 235A and decahydroquinoline (DHQ) to captive-bred Adelphobates galactonotus and measured alkaloid quantity in muscle, kidney, liver, and feces. HTX 235A and DHQ were detected in all organs, whereas only DHQ was present in trace amounts in feces. For both liver and skin, the quantity of alkaloid accumulated increased at higher doses for both alkaloids. Accumulation efficiency in the skin increased at higher doses for HTX 235A but remained constant for DHQ. In contrast, the efficiency of HTX 235A accumulation in the liver was inversely related to dose and a similar, albeit statistically nonsignificant, pattern was observed for DHQ. We identified and quantified the N-methylation of DHQ in A. galactonotus, which represents a previously unknown example of alkaloid modification in poison frogs. Our study suggests that variation in alkaloid composition among individuals and species can result from differences in sequestration efficiency related to the type and amount of alkaloids available in the environment.

Structure and evolution of the sexually dimorphic integumentary swelling on the hands of dendrobatid poison frogs and their relatives (Amphibia: Anura: Dendrobatoidea).

The hands of adult males of many dendrobatid poison frogs and their relatives possess swelling formed by glandular tissue hypothesized to secrete courtship pheromones delivered to the female during cephalic amplexus. Variation in the occurrence and external morphology of the swelling of finger IV has provided important evidence for dendrobatoid systematics for decades, but its underlying structure has not been investigated. We undertook a detailed comparative analysis of the integument of the hand, including both external morphology and histology, of 36 species representing the diversity of dendrobatoid frogs and several close relatives. The swelling is caused by four densely packed, hypertrophic, morpho-histochemical types of specialized mucous glands (SMGs). We observed type I SMGs on fingers II-V and the wrist, including areas that are not swollen, types II and IV exclusively on finger IV, and type III on finger IV and the wrist. Type I SMGs occur either in isolation or together with types II, III, or IV; types II, III, and IV never occur together or without type I. We delimited 15 characters to account for the variation in external morphology and the occurrence of SMGs. Our data suggest that type I SMGs are a new synapomorphy for Dendrobatoidea and that type II SMGs originated in either the most recent common ancestor (MRCA) of Dendrobatidae or independently in the MRCAs of Aromobatidae and Colostethinae, respectively, while types III and IV are restricted to Anomaloglossus. The discovery of these SMGs adds a new dimension to studies of poison frog reproductive biology, which have investigated acoustic, visual, and tactile cues in courtship, mating, and parental care across the diversity of Dendrobatoidea for decades but have almost entirely overlooked the possible role of chemical cues.

An empirical test of the relationship between the bootstrap and likelihood ratio support in maximum likelihood phylogenetic analysis.

In maximum likelihood (ML), the support for a clade can be calculated directly as the likelihood ratio (LR) or log-likelihood difference (S, LLD) of the best trees with and without the clade of interest. However, bootstrap (BS) clade frequencies are more pervasive in ML phylogenetics and are almost universally interpreted as measuring support. In addition to theoretical arguments against that interpretation, BS has several undesirable attributes for a support measure. For example, it does not vary in proportion to optimality or identify clades that are rejected by the evidence and can be overestimated due to missing data. Nevertheless, if BS is a reliable predictor of S, then it might be an efficient indirect method of measuring support-an attractive possibility, given the speed of many BS implementations. To assess the relationship between S and BS, we analyzed 106 empirical datasets retrieved from TreeBASE. Also, to evaluate the degree to which S and BS are affected by the number of replicates during suboptimal tree searches for S and pseudoreplicates during BS estimation, we randomly selected 5 of the 106 datasets and analyzed them using variable numbers of replicates and pseudoreplicates, respectively. The correlation between S and BS was extremely weak in the datasets we analyzed. Increasing the number of replicates during tree search decreased the estimated values of S for most clades, but the magnitude of change was small. In contrast, although increasing pseudoreplicates affected BS values for only approximately 40% of clades, values both increased and decreased, and they did so at much greater magnitudes. Increasing replicates/pseudoreplicates affected the rank order of clades in each tree for both S and BS. Our findings show decisively that BS is not an efficient indirect method of measuring support and suggest that even quite superficial searches to calculate S provide better estimates of support.

Dose-dependent alkaloid sequestration and N-methylation of decahydroquinoline in poison frogs

Sequestration of chemical defenses from dietary sources is dependent on the availability of compounds in the environment and the mechanism of sequestration. Previous experiments have shown that sequestration efficiency varies among alkaloids in poison frogs, but little is known about the underlying mechanism. The aim of this study was to quantify the extent to which alkaloid sequestration and modification are dependent on alkaloid availability and/or sequestration mechanism. To do this, we administered different doses of histrionicotoxin (HTX) 235A and decahydroquinoline (DHQ) to captive-bred Adelphobates galactonotus and measured alkaloid quantity in muscle, kidney, liver, and feces. HTX 235A and DHQ were detected in all organs, whereas only DHQ was present in trace amounts in feces. For both liver and skin, the quantity of alkaloid accumulated increased at higher doses for both alkaloids. Accumulation efficiency in the skin increased at higher doses for HTX 235A but remained constant for DHQ. In contrast, the efficiency of HTX 235A accumulation in the liver was inversely related to dose and a similar, albeit statistically nonsignificant, pattern was observed for DHQ. We identified and quantified the N-methylation of DHQ in A. galactonotus, which represents a previously unknown example of alkaloid modification in poison frogs. Our study suggests that variation in alkaloid composition among individuals and species can result from differences in sequestration efficiency related to the type and amount of alkaloids available in the environment.

A new species of Leucostethus (Anura, Dendrobatidae) from Gorgona Island, Colombia.

We describe a new species of Leucostethus from Gorgona Island, a small (13 km2) island located 35 km from the Pacific coast of southern Colombia. The new species most resembles L.argyrogaster and L.fugax from western Amazonia at 340-870 m elev. in Peru and Ecuador, with which it shares pale ventral coloration and orange suffusion of the axilla, groin and concealed surfaces of the hind limb, but it is most closely related to L.bilsa from ca. 340 km SW in the southern Chocó at 420-515 m elev., northwestern Ecuador. We report miniscule white spots on the posteroventral surface of the thighs of the new species and, on the basis of our preliminary assessment of their taxonomic distribution, hypothesize that their presence is a synapomorphy of Dendrobatoidea with subsequent losses in a few groups. Given the apparent restriction of the new species to Gorgona Island, it is Vulnerable according to IUCN Red List criteria. In addition to naming the new species, we also propose the following new combinations: L.alacris (Rivero and Granados-Díaz, 1990) comb. nov., L.dysprosium (Rivero and Serna, 2000) comb. nov., and L.yaguara (Rivero and Serna, 1991) comb. nov.

The phylogeny of Dendropsophini (Anura: Hylidae: Hylinae).

The relationships of the hyline tribe Dendropsophini remain poorly studied, with most published analyses dealing with few of the species groups of Dendropsophus. In order to test the monophyly of Dendropsophini, its genera, and the species groups currently recognized in Dendropsophus, we performed a total evidence phylogenetic analysis. The molecular dataset included sequences of three mitochondrial and five nuclear genes from 210 terminals, including 12 outgroup species, the two species of Xenohyla, and 93 of the 108 recognized species of Dendropsophus. The phenomic dataset includes 46 terminals, one per species (34 Dendropsophus, one Xenohyla, and 11 outgroup species). Our results corroborate the monophyly of Dendropsophini and the reciprocal monophyly of Dendropsophus and Xenohyla. Some species groups of Dendropsophus are paraphyletic (the D. microcephalus, D. minimus, and D. parviceps groups, and the D. rubicundulus clade). On the basis of our results, we recognize nine species groups; for three of them (D. leucophyllatus, D. microcephalus, and D. parviceps groups) we recognize some nominal clades to highlight specific morphology or relationships and facilitate species taxonomy. We further discuss the evolution of oviposition site selection, where our results show multiple instances of independent evolution of terrestrial egg clutches during the evolutionary history of Dendropsophus.

The phylogeny of the Casque-headed Treefrogs (Hylidae: Hylinae: Lophyohylini).

The South American and West Indian Casque-headed Treefrogs (Hylidae: Hylinae: Lophyohylini) include 85 species. These are notably diverse in morphology (e.g. disparate levels of cranial hyperossification) and life history (e.g. different reproductive modes, chemical defences), have a wide distribution, and occupy habitats from the tropical rainforests to semiarid scrubland. In this paper, we present a phylogenetic analysis of this hylid tribe based on sequence fragments of up to five mitochondrial (12S, 16S, ND1, COI, Cytb) and six nuclear genes (POMC, RAG-1, RHOD, SIAH, TNS3, TYR). We included most of its species (> 96%), in addition to a number of new species. Our results indicate: (i) the paraphyly of Trachycephalus with respect to Aparasphenodon venezolanus; (ii) the nonmonophyly of Aparasphenodon, with Argenteohyla siemersi, Corythomantis galeata and Nyctimantis rugiceps nested within it, and Ap. venezolanus nested within Trachycephalus; (iii) the polyphyly of Corythomantis; (iv) the nonmonophyly of the recognized species groups of Phyllodytes; and (v) a pervasive low support for the deep relationships among the major clades of Lophyohylini, including C. greeningi and the monotypic genera Itapotihyla and Phytotriades. To remedy the nonmonophyly of Aparasphenodon, Corythomantis, and Trachycephalus, we redefined Nyctimantis to include Aparasphenodon (with the exception of Ap. venezolanus, which we transferred to Trachycephalus), Argenteohyla, and C. galeata. Additionally, our results indicate the need for taxonomic work in the following clades: (i) Trachycephalus dibernardoi and Tr. imitatrix; (ii) Tr. atlas, Tr. mambaiensis and Tr. nigromaculatus; and (iii) Phyllodytes. On the basis of our phylogenetic results, we analyzed the evolution of skull hyperossification and reproductive biology, with emphasis on the multiple independent origins of phytotelm breeding, in the context of Anura. We also analyzed the inter-related aspects of chemical defences, venom delivery, phragmotic behaviour, co-ossification, and prevention of evaporative water loss.

Evidence of absence treated as absence of evidence: The effects of variation in the number and distribution of gaps treated as missing data on the results of standard maximum likelihood analysis.

Although numerous studies have demonstrated the theoretical and empirical importance of treating gaps as insertion/deletion (indel) events in phylogenetic analyses, the standard approach to maximum likelihood (ML) analysis employed in the vast majority of empirical studies codes gaps as nucleotides of unknown identity ("missing data"). Therefore, it is imperative to understand the empirical consequences of different numbers and distributions of gaps treated as missing data. We evaluated the effects of variation in the number and distribution of gaps (i.e., no base, coded as IUPAC "." or "-") treated as missing data (i.e., any base, coded as "?" or IUPAC "N") in standard ML analysis. We obtained alignments with variable numbers and arrangements of gaps by aligning seven diverse empirical datasets under different gap opening costs using MAFFT. We selected the optimal substitution model for each alignment using the corrected Akaike Information Criterion in jModelTest2 and searched for optimal trees using GARLI. We also employed a Monte Carlo approach to randomly replace nucleotides with gaps (treated as missing data) in an empirical dataset to understand more precisely the effects of varying their number and distribution. To compare alignments, we developed four new indices and used several existing measures to quantify the number and distribution of gaps in all alignments. Our most important finding is that ML scores correlate negatively with gap opening costs and the amount of missing data. However, this negative relationship is not due to the increase in missing data per se-which increases ML scores-but instead to the effect of gaps on nucleotide homology. These variables also cause significant but largely unpredictable effects on tree topology.

Use of whole-body cryosectioning and desorption electrospray ionization mass spectrometry imaging to visualize alkaloid distribution in poison frogs.

Ambient mass spectrometry is useful for analyzing compounds that would be affected by other chemical procedures. Poison frogs are known to sequester alkaloids from their diet, but the sequestration pathway is unknown. Here, we describe methods for whole-body cryosectioning of frogs and use desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to map the orally administered alkaloid histrionicotoxin 235A in a whole-body section of the poison frog Dendrobates tinctorius. Our results show that whole-body cryosectioning coupled with histochemical staining and DESI-MSI is an effective technique to visualize alkaloid distribution and help elucidate the mechanisms involved in alkaloid sequestration in poison frogs.

Lens transmittance shapes ultraviolet sensitivity in the eyes of frogs from diverse ecological and phylogenetic backgrounds.

The amount of short wavelength (ultraviolet (UV), violet and blue) light that reaches the retina depends on the transmittance properties of the ocular media, especially the lens, and varies greatly across species in all vertebrate groups studied previously. We measured the lens transmittance in 32 anuran amphibians with different habits, geographical distributions and phylogenetic positions and used them together with eye size and pupil shape to evaluate the relationship with diel activity pattern, elevation and latitude. We found an unusually high lens UV transmittance in the most basal species, and a cut-off range that extends into the visible spectrum for the rest of the sample, with lenses even absorbing violet light in some diurnal species. However, other diurnal frogs had lenses that transmit UV light like the nocturnal species. This unclear pattern in the segregation of ocular media transmittance and diel activity is shared with other vertebrates and is consistent with the absence of significant correlations in our statistical analyses. Although we did not detect a significant phylogenetic effect, closely related species tend to have similar transmittances, irrespective of whether they share the same diel pattern or not, suggesting that anuran ocular media transmittance properties might be related to phylogeny.

Variations in tetrodotoxin levels in populations of Taricha granulosa are expressed in the morphology of their cutaneous glands.

Tetrodotoxin (TTX), one of the most toxic substances in nature, is present in bacteria, invertebrates, fishes, and amphibians. Marine organisms seem to bioaccumulate TTX from their food or acquire it from symbiotic bacteria, but its origin in amphibians is unclear. Taricha granulosa can exhibit high TTX levels, presumably concentrated in skin poison glands, acting as an agent of selection upon predatory garter snakes (Thamnophis). This co-evolutionary arms race induces variation in T. granulosa TTX levels, from very high to undetectable. Using morphology and biochemistry, we investigated differences in toxin localization and quality between two populations at the extremes of toxicity. TTX concentration within poison glands is related to the volume of a single cell type in which TTX occurs exclusively in distinctive secretory granules, suggesting a relationship between granule structure and chemical composition. TTX was detected in mucous glands in both populations, contradicting the general understanding that these glands do not secrete defensive chemicals and expanding currently held interpretations of amphibian skin gland functionality. Skin secretions of the two populations differed in low-mass molecules and proteins. Our results demonstrate that interpopulation variation in TTX levels is related to poison gland morphology.

A dune with a view: the eyes of a neotropical fossorial lizard.

BACKGROUND: Lizards are excellent models to study the adaptations of the visual system to different scenarios, and surface-dwelling representatives have been relatively well studied. In contrast, very little is known about the functional anatomy of the eyes of fossorial lineages, and properties such as the light transmission by the ocular media have never been characterised in any fossorial species. Some lizards in the family Gymnophthalmidae endemic to the sand dunes of North Eastern Brazil have evolved sand-burrowing habits and nocturnal activity. Lizards in the sister group to Gymnophthalmidae, the family Teiidae, have decidedly diurnal and epigeal lifestyles, yet they are equally poorly known in terms of visual systems. We focussed on the eye anatomy, photoreceptor morphology and light transmittance properties of the ocular media and oil droplets in the gymnophthalmid Calyptommatus nicterus and the teiid Ameivula ocellifera. RESULTS: The general organisation of the eyes of the fossorial nocturnal C. nicterus and the epigeal diurnal A. ocellifera is remarkably similar. The lenses are highly transmissive to light well into the ultraviolet part of the spectrum. The photoreceptors have the typical cone morphology, with narrow short outer segments and oil droplets. The main difference between the two species is that C. nicterus has only colourless oil droplets, whereas A. ocellifera has colourless as well as green-yellow and pale-orange droplets. CONCLUSIONS: Our results challenge the assumption that fossorial lizards undergo loss of visual function, a claim that is usually guided by the reduced size and external morphology of their eyes. In the case of C. nicterus, the visual system is well suited for vision in bright light and shows specialisations that improve sensitivity in dim light, suggesting that they might perform some visually-guided behaviour above the surface at the beginning or the end of their daily activity period, when light levels are relatively high in their open dunes habitat. This work highlights how studies on the functional anatomy of sensory systems can provide insights into the habits of secretive species.

Differences in ocular media transmittance in classical frog and toad model species and its impact on visual sensitivity.

The transmittance properties of the cornea, lens and humours of vertebrates determine how much light across the visible spectrum reaches the retina, influencing sensitivity to visual stimuli. Amphibians are the only vertebrate class in which the light transmittance of these ocular media has not been thoroughly characterised, preventing large-scale comparative studies and precise quantification of visual stimuli in physiological and behavioural experiments. We measured the ocular media transmittance in some commonly used species of amphibians (the bufonids Bufo bufo and Rhinella ornate, and the ranids Lithobates catesbeianus and Rana temporaria) and found low transmittance of short wavelength light, with ranids having less transmissive ocular media than bufonids. Our analyses also show that these transmittance properties have a considerable impact on spectral sensitivity, highlighting the need to incorporate this type of measurement into the design of stimuli for experiments on visual function.

The poverty of adult morphology: Bioacoustics, genetics, and internal tadpole morphology reveal a new species of glassfrog (Anura: Centrolenidae: Ikakogi) from the Sierra Nevada de Santa Marta, Colombia.

Ikakogi is a behaviorally and morphologically intriguing genus of glassfrog. Using tadpole morphology, vocalizations, and DNA, a new species is described from the Sierra Nevada de Santa Marta (SNSM), an isolated mountain range in northern Colombia. The new taxon is the second known species of the genus Ikakogi and is morphologically identical to I. tayrona (except for some larval characters) but differs by its genetic distance (14.8% in mitochondrial encoded cytochrome b MT-CYB; ca. 371 bp) and by the dominant frequency of its advertisement call (2928-3273 Hz in contrast to 2650-2870 Hz in I. tayrona). They also differ in the number of lateral buccal floor papillae, and the position of the buccal roof arena papillae. Additionally, the new species is differentiated from all other species of Centrolenidae by the following traits: tympanum visible, vomerine teeth absent, humeral spines present in adult males, bones in life white with pale green in epiphyses, minute punctuations present on green skin dorsum, and flanks with lateral row of small, enameled dots that extend from below eye to just posterior to arm insertion. We describe the external and internal larval morphology of the new species and we redescribe the larval morphology of Ikakogi tayrona on the basis of field collected specimens representing several stages of development from early to late metamorphosis. We discuss the relevance of larval morphology for the taxonomy and systematics of Ikakogi and other centrolenid genera. Finally, we document intraspecific larval variation in meristic characters and ontogenetic changes in eye size, coloration, and labial tooth-rows formulas, and compare tadpoles of related species. Ikakogi tayrona has been proposed as the sister taxon of all other Centrolenidae; our observations and new species description offers insights about the ancestral character-states of adults, egg clutches, and larval features in this lineage of frogs.

A total evidence analysis of the phylogeny of hatchet-faced treefrogs (Anura: Hylidae: Sphaenorhynchus).

The Neotropical hylid genus Sphaenorhynchus includes 15 species of small, greenish treefrogs widespread in the Amazon and Orinoco basins, and in the Atlantic Forest of Brazil. Although some studies have addressed the phylogenetic relationships of the genus with other hylids using a few exemplar species, its internal relationships remain poorly understood. In order to test its monophyly and the relationships among its species, we performed a total evidence phylogenetic analysis of sequences of three mitochondrial and three nuclear genes, and 193 phenotypic characters from all species of Sphaenorhynchus. Our results support the monophyly of Sphaenorhynchus with molecular and phenotypic evidence, with S. pauloalvini as the earliest diverging taxon, followed by S. carneus, as the sister taxon of all remaining species of the genus. We recognize three species groups in Sphaenorhynchus (the S. lacteus, S. planicola and S. platycephalus groups), to facilitate its taxonomic study; only three species (S. carneus, S. pauloalvini and S. prasinus) remain unassigned to any group. Sequence data were not available for only two species (S. bromelicola and S. palustris) for which we scored phenotypic data; wildcard behaviour was detected only in S. bromelicola nested inside the S. platycephalus group. On the basis of the resulting phylogenetic hypothesis, we discuss the evolution of oviposition site and a number of phenotypic characters that could be associated with heterochronic events in the evolutionary history of this group.

Variations in tetrodotoxin levels in populations of Taricha granulosa are expressed in the morphology of their cutaneous glands

Tetrodotoxin (TTX), one of the most toxic substances in nature, is present in bacteria, invertebrates, fishes, and amphibians. Marine organisms seem to bioaccumulate TTX from their food or acquire it from symbiotic bacteria, but its origin in amphibians is unclear. Taricha granulosa can exhibit high TTX levels, presumably concentrated in skin poison glands, acting as an agent of selection upon predatory garter snakes (Thamnophis). This co-evolutionary arms race induces variation in T. granulosa TTX levels, from very high to undetectable. Using morphology and biochemistry, we investigated differences in toxin localization and quality between two populations at the extremes of toxicity. TTX concentration within poison glands is related to the volume of a single cell type in which TTX occurs exclusively in distinctive secretory granules, suggesting a relationship between granule structure and chemical composition. TTX was detected in mucous glands in both populations, contradicting the general understanding that these glands do not secrete defensive chemicals and expanding currently held interpretations of amphibian skin gland functionality. Skin secretions of the two populations differed in low-mass molecules and proteins. Our results demonstrate that interpopulation variation in TTX levels is related to poison gland morphology.

Variations in tetrodotoxin levels in populations of Taricha granulosa are expressed in the morphology of their cutaneous glands

Tetrodotoxin (TTX), one of the most toxic substances in nature, is present in bacteria, invertebrates, fishes, and amphibians. Marine organisms seem to bioaccumulate TTX from their food or acquire it from symbiotic bacteria, but its origin in amphibians is unclear. Taricha granulosa can exhibit high TTX levels, presumably concentrated in skin poison glands, acting as an agent of selection upon predatory garter snakes (Thamnophis). This co-evolutionary arms race induces variation in T. granulosa TTX levels, from very high to undetectable. Using morphology and biochemistry, we investigated differences in toxin localization and quality between two populations at the extremes of toxicity. TTX concentration within poison glands is related to the volume of a single cell type in which TTX occurs exclusively in distinctive secretory granules, suggesting a relationship between granule structure and chemical composition. TTX was detected in mucous glands in both populations, contradicting the general understanding that these glands do not secrete defensive chemicals and expanding currently held interpretations of amphibian skin gland functionality. Skin secretions of the two populations differed in low-mass molecules and proteins. Our results demonstrate that interpopulation variation in TTX levels is related to poison gland morphology.

A new strategy to infer circularity applied to four new complete frog mitogenomes.

We applied a novel strategy to infer sequence circularity and complete assembly of four mitochondrial genomes (mitogenomes) of the frog families Bufonidae (Melanophryniscus moreirae), Dendrobatidae (Hyloxalus subpunctatus and Phyllobates terribilis), and Scaphiopodidae (Scaphiopus holbrookii). These are the first complete mitogenomes of these four genera and Scaphiopodidae. We assembled mitogenomes from short genomic sequence reads using a baiting and iterative mapping strategy followed by a new ad hoc mapping strategy developed to test for assembly circularization. To assess the quality of the inferred circularization, we used Bowtie2 alignment scores and a new per-position sequence coverage value (which we named "connectivity"). Permutation tests with 400 iterations per specimen and 1% or 5% chance of mutation at the ends of the putative circular sequences showed that the proposed method is highly sensitive, with a single nucleotide insertion or deletion being sufficient for circularity to be rejected. False positives comprised only 2% of all observations and possessed significantly lower alignment scores. The size, gene content, and gene arrangement of each mitogenome differed among the species but matched the expectations for their clades. We argue that basic studies on circular sequences can benefit from the results and bioinformatics procedures introduced here, especially when closely related references are lacking.