Biological Journal of the Linnean Society

Knowledge of species ages and their distribution enhance our understanding of processes that create and maintain species diversity at both local and regional levels. The largest family of freshwater mussels (Unionidae), reach their highest species diversity in drainages of the southeastern united states. By sequencing multiple loci from mussel species distributed throughout the drainages in this region, we attempt to uncover historical patterns of divergence and determine the role of vicariance events on the species formation in mussels and extend our hypothesis to freshwater animals in general. We analyzed 346 sequences from five genera encompassing 37 species. Species were sampled across 12 distinct drainages ending either in the Atlantic Ocean or the Gulf of Mexico. Overall the topologies of the different genera returned phylogenetic trees that were congruent with geographically contiguous drainages. The most common pattern was the grouping between the Atlantic slope and gulf coast drainages, however the Tennessee drainage was often the exception to this pattern grouping with the Atlantic slope. Most mussel species find a most recent common ancestor within a drainage before finding an ancestor between drainages. This supports the hypothesis of allopatric divergence followed by later burst of speciation within a drainage. Our estimated divergence times for the Atlantic-Gulf split agree with other studies estimating vicariance in fish species of the Atlantic and gulf coast.

Venom is a metabolically expensive secretion used sparingly in a variety of ecological contexts, most notably predation and defense. Accordingly, few animals use their toxins in ranged attacks, and venom-squirting behaviour is only known from select taxa. In scorpions, species belonging to two genera are known to spray venom when threatened, and previous work in Parabuthus transvaalicus shows that venom delivery depends on perceived levels of threat. Here, I describe Tityus (Tityus) XXX sp. nov., a new species of Buthid scorpion from Cundinamarca, Colombia. Remarkably, this species is capable of venom spraying, a first for both the genus and the South American continent. Using frame-by-frame video analysis and ballistic equations, I show that T. XXX sp. nov. employs not one, but two types of airborne attacks with dramatic differences in range and venom expenditure. Further, the new species uses an unusually large reserve of prevenom-like secretion for spraying, as opposed to the costly venom used by other spitting scorpions. In light of these key specializations, I propose that venom spraying convergently evolved in response to different selection pressures, laying the groundwork for future investigation.

Hypanus brevis (Garman, 1880) and Hypanus dipterurus (Jordan & Gilbert, 1880) are currently considered as a conspecific lineage of the "diamond stingray" from the Eastern Pacific. This taxonomic group has been the subject of nomenclatural disputes for about 145 years. To clarify the historical confusion surrounding this lineage, we employed an integrative taxonomic approach using specimens from the Eastern North and Eastern South Pacific (ENP and ESP). The genetic results, based on single and multilocus mitochondrial analyses, revealed a distinct evolutionary unit in the ESP. While morphological analyses detected subtle differences between ENP and ESP specimens, most characters exhibited significant overlap (e.g., disc shape, dentition patterns, body coloration), suggesting low evolutionary divergence. A calibrated molecular clock analysis estimated this divergence at approximately 3.09 Ma. In accordance with Garmans (1880) original description based on specimens from Paita (northern Peru), we formally resurrect H. brevis from synonymy with H. dipterurus. Our findings suggest an anti-tropical speciation pathway, with core populations of H. brevis and H. dipterurus restricted to the temperate waters of the ESP and ENP, respectively. Notably, a single, fixed COI haplotype was detected in all H. brevis specimens from the north-central Peruvian coast. This result may indicate a severe bottleneck event, raising concerns about the genetic health and long-term viability of this vulnerable species. Finally, we analyzed historical fishery data of H. brevis to infer its current population status, suggesting targeted conservation measures and precautionary management to prevent further loss of genetic diversity.

Arrhenotoky is typical mode of reproduction for Hymenoptera - females originate from fertilized eggs, males from unfertilized eggs. However, some lineages of Hymenoptera have switched to thelytoky, where diploid females originate instead from unfertilized diploid eggs. In the contras with some other hymenopteran lineages, thelytoky is generally very rare in bees. Here, we examined reproduction in the small carpenter bee Ceratina dallatoreana, which is assumed to be thelytokous. We compared genotype of microsatellite loci between mothers and their offspring. Offspring were genetically identical with mother in all cases. We did not detect any male offspring. Therefore, we conclude that parthenogeny is the prevailing, and perhaps obligate, mode of reproduction in C. dallatoreana. Offspring were clones of their mother with no observed decrease of heterozygosity. Thus the cytological mode of parthenogenesis is apomixis, or automimic with central fusion and extremely reduced or non-existing recombination. Ceratina bees are originally facultatively eusocial, therefore thelytoky may influence social evolution by causing extremely high within-colony relatedness. However, to date no multifemale nests have been recorded in C. dallatoreana.

Convergence is a hallmark of adaptive evolution, yet its extent across different taxa remains unclear. Snakes, with over 4,100 species worldwide, provide a unique model to explore how habitat use influences morphology in a group with an at first sight uniform body plan. We here quantified body, head, and tail shape in over 400 species ([~]10% of the global snake diversity) to assess the role of habitat use in shaping morphological variation. Our results reveal significant differences across ecological groups, with habitat use acting as a key driver of phenotypic diversity. Terrestrial species display the highest morphological diversity in contrast to other habitat groups which appear morphologically specialized. For example, whereas arboreal and semi-arboreal species exhibit elongated heads and slender necks, aquatic and semi-aquatic snakes share streamlined bodies and narrow heads. Fossorial and semi-fossorial species, on the other hand, have compact bodies. Surprisingly, morphological convergence remains limited to arboreal, semi-arboreal, and terrestrial habitat groups. Thus, despite strong functional constraints, evolutionary convergence in fossorial and aquatic species is weak, indicating multiple adaptive solutions rather than a single morphological trajectory. Morphological disparity patterns show that non-specialist ecologies generally exhibit greater disparity than highly specialized ones, in accordance with the need of these species to move in different habitats. Our findings underscore the role of ecological constraints in shaping snake morphology and highlight the complexity of adaptation beyond strict convergent evolution. Lay summaryThe living environment of a species has been suggested to play a key role in shaping its morphology. Our study aimed to understand whether and how habitat use has influenced the evolution of external body and head shape in snakes. To do so, we gathered a comprehensive dataset of morphological traits from 436 snake species. Our findings reveal that species living in different habitats are morphologically distinct with species living in similar habitats displaying similar morphologies despite the conserved morphology of snakes.

Batesian mimicry imposes several challenges to mimics and evokes adaptations in multiple sensory modalities. Myrmecomorphy, morphological and behavioral resemblance to ants, is seen in over 2000 arthropod species. Ant-like resemblance is observed in at least 13 spider families despite spiders having a distinct body plan compared to ants. Quantifying the extent to which spiders shape, size, and behavior resemble model ants will allow us to comprehend the evolutionary pressures that have facilitated myrmecomorphy. Myrmaplata plataleoides are accurate mimics of the weaver ants, Oecophylla smaragdina. In this study, we quantify the speed of movement of model, mimic, and non-mimetic jumping spiders. We use traditional and geometric morphometrics to quantify traits such as foreleg and hindleg size, body shape between the model ant, mimic, and non-mimics. Our results suggest that while the mimics closely resemble the model ants in speed of movement, they occupy an intermediate morphological space compared to the model ants and non-mimics. We suggest that ant-mimicking spiders are better at mimicking ants locomotory movement than morphology and overall body shape. Our study provides a framework to understand the multimodal nature of mimicry and helps discern the relative contributions of such traits that drive mimetic accuracy in ant-mimicking spiders.

Understanding the evolutionary history of diversifying lineages and the delineation of species remain major challenges for evolutionary biology. Here we use single nucleotide polymorphisms (SNPs) and sequence fragment presence-absence (SilicoDArT) data to combine phylogenetics and population genetics to assess species boundaries with a focus on diagnosability. We challenge current and proposed taxonomies in a genus of Australian freshwater turtles (Chelidae: Emydura) from northern Australia and southern New Guinea. In a six-step process, we combine phylogeny with the concept of diagnosability based on fixed allelic differences to select diagnosable lineages as candidate species. Four taxa are supported as diagnosable lineages, two of which we elevate to species status. The nuclear and mitochondrial phylogenies differed in important respects, which we attribute to recent or contemporary lateral transfer of mitochondria during hybridization events, deeper historical hybridization or possibly incomplete lineage sorting of the mitochondrial genome. Taxonomic decisions in cases of allopatry require subjective judgement. Our six-step strategy and the necessary (but not sufficient) criterion of diagnosability adds an additional level of objectivity before that subjectivity is applied, and so reduces the risk of taxonomic inflation that can accompany lineage approaches to species delimitation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/664252v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1b89bc8org.highwire.dtl.DTLVardef@fb892dorg.highwire.dtl.DTLVardef@1eb09a8org.highwire.dtl.DTLVardef@1c7300c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Modification of phenotypic traits induced by character displacements allow species to coexist by reducing interspecific competition. This study explores if and how shorebirds partition feeding ressources by foraging at different water depths outside the breeding season. During this period, communities of up to dozen shorebird species coexist on suitable habitats at land-water interfaces. By video recording 22 species of migrating and wintering shorebirds in different sites, we show that their distribution according to the water height at which they forage is not random but follows a gradient of increasing water height according to increasing species body size. Species-specific beak and tarsus lengths are positively correlated with the water height at which the different species forage. Our data do not support a trade-off in investment between beak length and tarsus length in our study species set. Congeneric species that are expected to be morphologically and ecologically more similar forage at contrasted water heights. Altogether, our findings supports the idea of resource partitioning, and consequently of competition relaxation, by morphological and/or by behavioral character displacements.

The sweat bee Halictus rubicundus is an important pollinator with a large latitudinal range and many potential barriers to gene flow. Alongside typical physical barriers, including mountain ranges and oceans, the climate may also impose restrictions on gene flow in this species. The climate influences voltinism and sociality in H. rubicundus, which is bivoltine and can nest socially at lower latitudes but can be univoltine and solitary in the north of its range and at higher altitudes where the climate is cooler. Variation in voltinism due to the climate may result in differences in phenology between populations across this species geographical range. Differences in phenology could limit gene flow, rendering populations at extreme latitudes genetically isolated and potentially more vulnerable to environmental stressors. A previous study found that the Irish Sea restricts gene flow in this species, but there was no evidence that differences in phenology had a similar effect as there was no genetic differentiation between H. rubicundus populations on mainland Britain. Here we extend the previous study to consider populations of H. rubicundus at extreme northern and southern latitudes in the UK. Using 12 microsatellite markers for genotyping, we found that bees from a population in the far north of Scotland were genetically differentiated from bees collected in Cornwall in the south-west of England. In contrast, bees collected across the Irish Sea in Northern Ireland showed genetic overlap with both the Scottish and Cornish bees. Our results suggest that when populations at extreme latitudes are considered, phenology and the climate may act alongside physical barriers such as the Scottish Highlands and the Irish Sea to restrict gene flow in H. rubicundus. We discuss the implications of our results for local adaptation in the face of rapidly changing selection pressures which are likely under climate change.

Mitochondrial DNA (mtDNA) has long been used to date the divergence between species, and to explore the time when species effective population sizes changed. The idea that mitochondrial DNA is useful for molecular dating rests on the premise that its evolution is neutral. This premise was questionable to begin with, and even though it has long been challenged, the evidence against clock-like evolution of mtDNA is usually ignored. Here, we present a particularly clear and simple example to illustrate the implications of violations of the assumption of selective neutrality. DNA sequences were generated for the mtDNA COI gene and the nuclear 28S rRNA of two closely related and widely distributed rocky shore snails whose geographical ranges are defined by different thermal preferences. To our knowledge, this is the first study to use nuclear rRNA sequence for studying species-level genealogies instead of phylogenetics, presumably because this marker is considered to be uninformative at this taxonomic level. Even though the COI gene evolves at least an order of magnitude faster, which was reflected in high inter-specific divergence, intraspecific genetic variation was similar for both markers. As a result, estimates of population expansion times based on mismatch distributions were completely different for the two markers. Assuming that 28S evolves effectively clock-like, these findings likely illustrate variation-reducing purifying selection in mtDNA at the species level, and an elevated divergence rate caused by divergent selection between the two species. Although these two selective forces together make mtDNA suitable as a DNA barcoding marker because they create a barcoding gap, estimates of demographic change can be expected to be highly unreliable. Our study contributes to the growing evidence that the utility of mtDNA beyond DNA barcoding is limited.

Reptiles have repeatedly invaded and thrived in aquatic environments throughout history, however fewer than 8% of the 6000 extant species are primarily aquatic. The Galapagos Marine Iguana (Amblyrhynchus cristatus), the worlds only marine lizard, may have had one of the most unique and challenging transitions to aquatic life. Curiously, previous studies have identified relatively few physiological adaptations in Marine Iguanas, however, little is known about the extent of morphological specialisation and performance trade-offs associated with the marine environment. By examining the morphology and locomotory performance of the Marine Iguana in comparison to their closely related mainland ancestors, the Black Spiny-tailed iguana (Ctenosaura similis) and Green Iguana (Iguana iguana), we found variation reflected specialisation to ecological niches. However, variation was more pronounced among subspecies of Marine Iguana, suggesting that little morphological or performance modification is required for iguanids to successfully invade aquatic environments, thus raising the question why there are so few extant aquatic reptilian lineages. We found that specialisation for the marine environment resulted in a trade-off in sprint speed in a terrestrial environment, similar to that seen in extant crocodilians. Reduced performance in a terrestrial environment likely poses little risk to large-bodied apex predators, whereas in iguanids, a performance trade-off would likely incur increased predation. As such, we suggest that this may explain why iguanids and other ancestral lineages have not undergone transitions to aquatic life. Additionally, we found that the magnitude of morphological and performance variation was more pronounced between subspecies of Marine Iguana than between iguanid species. Summary StatementThe Marine Iguana has undergone a unique evolutionary transition to aquatic behaviour, we explore the extent of morphological and performance specialisation required and why there are so few extant marine reptiles. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

We describe the antipredator armor of a unique population of threespine stickleback (Gasterosteus aculeatus) from Narrows Pond in western Newfoundland and compare traits for this population to nearby populations from marine and freshwater systems. After standardizing for length, Narrows Pond stickleback are shallower bodied and have shorter dorsal spines than stickleback from the other populations. Also, though the number of armor plates for Narrows Pond stickleback is greater than for typical low-plate morphs, the size of the lateral plates for Narrows Pond stickleback is much smaller. Finally, most (nearly 75% of sampled individuals) Narrows Pond stickleback do not have a pelvic structure (bilateral pelvic plate, ascending process, and ventral spine) and the remaining individuals have greatly reduced pelvic girdle whereas all individuals from the other populations possessed complete pelvic structures.

AO_SCPLOWBSTRACTC_SCPLOWEnvironmental heterogeneity across freshwater systems often promotes phenotypic variation, yet disentangling environmentally induced variation from heritable differentiation remains a central goal in evolutionary ecology. We investigated the geographic distribution and morphological differentiation, and heritability of shell traits among populations of the freshwater lymnaeid snail Pectinidens diaphanus in Patagonia. Extensive field surveys across 196 freshwater sites revealed that the species occupies a broad range of lentic and lotic habitats and constitutes the only lymnaeid inhabiting southern Patagonia. While reproductive anatomical structures were conserved across populations, shell shape differed markedly among populations from contrasting habitat types, with population identity explaining nearly 50% of total shape variation. Populations from hydrologically unstable habitats (ponds and streams) exhibited more elongated shells and relatively smaller apertures, a pattern consistent with functional responses to hydroperiod variability and desiccation risk. To assess the heritability of this differentiation, we conducted a common-garden experiment across two generations. Shell shape differences between permanent- (lagoon) and temporary- (pond) habitat-derived populations persisted into the G2 generation reared under standardized laboratory conditions, indicating that the observed variation is not solely a response to local environmental conditions but includes a heritable component. Together, our findings demonstrate that P. diaphanus constitutes the sole lymnaeid across southern Patagonia, occupying a broader range than previously documented, and that populations show heritable shell differentiation potentially associated with contrasting freshwater habitats. By integrating large-scale biogeographic surveys with morphometric and experimental approaches, this study provides new insight into how habitat variation may contribute to ecological and evolutionary differentiation in freshwater gastropods.

O_LIDispersal evolution and social evolution are interlinked. Dispersal is necessary for avoiding kin competition and inbreeding, but limited dispersal also allows beneficial social interactions with kin. In ants, a correlation between poor dispersal and complex societies, where a big proportion of queens are philopatric, is well documented, but the underlying causal mechanisms are not clear. C_LIO_LIIn this study we investigate the dispersal ability of six Formica species that vary in their colony queen number and nest founding mode, from three different subgenera. Investigating resource allocation in the bodies of young queens and males allows us to analyze the evolutionary causalities between dispersal ability and social organization. We measured the body ratios including wing-muscle ratio; glycogen, triglyceride and protein resources with colorimetric assays; and microscopic wing muscle structures with transmission electron microscopy, with an overall sample size of 1515 individual males and queens. C_LIO_LIOur results suggest that the physical condition of individuals does not strongly correlate with the dispersal patterns of the species, contrary to assumptions based on earlier studies in both ants and other insects. There was still a minor effect of social organization on male wing-muscle ratio, which is an interesting case of sexual coevolution of dispersal traits: it is likely that the queen philopatric behavior is reflected in male behavior and consequently in male morphology - even when our overall results support the hypothesis that ant dispersal is male biased regardless of social organization. Further, our intraspecific analyses in two of the six species reveal different patterns in their flight abilities in connection to their social organization, further pointing towards a mismatch between dispersal behavior and ability. C_LIO_LIIn Formica queens, philopatry seems to be a behavioral trait of the individuals rather than a consequence of colony-level resource allocation into dispersal ability, pointing towards a "behavior first" evolutionary route. The queens may selfishly choose not to disperse even when their society provides them with the resources for it. This raises new questions about conflicts over dispersal in these highly social systems. C_LI

Understanding morphological variation is crucial for the study of speciation and for conservation as it helps in assessing biodiversity and predicting responses to environmental changes. These approaches are broadly applicable but are especially valuable in marine environments, where species are often elusive, difficult to study, and face heightened threats from rapid environmental shifts. The marine snail Littorina saxatilis is notable for its extensive polymorphism in shell shape, size, and colour, with ecotypes that evolve in response to environmental forces including wave exposure and crab predation. Morphometric tools have been central to investigating the mechanisms driving this phenotypic divergence; yet, a direct comparison of their methodological efficacy is lacking. Here, we took advantage of L. saxatilis ecotypes to contrast three morphometric approaches: elliptical Fourier analysis (EFA), landmarks-based geometric morphometrics (GM), and the growth-based model implemented in the ShellShaper software (SS). We assessed their clustering power, biological interpretability, robustness to measurement error and transferability among datasets. Our findings provide insights to guide method selection in studies aimed at exploring morphological variation: EFA is better suited for high-throughput screening and describing intermediate shapes; SS offers superior clustering power with highly interpretable growth parameters; and GM is best for detailed anatomical studies but is less efficient for large datasets. We provide guidelines to align method selection with specific research goals, balancing analytical efficiency with the required morphological and biological insight. By following this framework, researchers can ensure that robust morphological analysis is achieved, which is essential not only for elucidating mechanisms of adaptation and speciation but also for effective management and conservation of marine biodiversity.

We quantitatively tested the riverine barrier hypothesis and its influence on biogeographical distributions and molecular variation in New World monkeys (Parvorder: Platyrrhini). Using mitochondrial markers (cytochrome oxidase subunit II and cytochrome b), we analyzed taxonomic differences and the effects of geographical barriers on molecular patterns across Central and South America. Nearly 80% of described species are separated by geographical barriers. River width exhibited a positive correlation with molecular dissimilarity in adjacent taxa for both molecular markers. Streamflow also showed a positive association, although not statistically significant, likely due to limited sample size. Several presently described taxa were not supported based solely on these molecular phylogenetic markers, including Saimiri, Mico, Cebus, Sapajus, and Cherecebus. These taxonomic issues are far more common where river barriers do not exist. In conclusion, we found a significant correlation between river width and molecular divergence in adjacent taxa, indicating that wider rivers were associated with greater molecular dissimilarity for two commonly used mitochondrial genes. Species boundaries were predominantly found at river interfaces, and in the absence of discernable geological barriers, adjoining species were more likely to exhibit molecular similarity. Our findings suggest that both river and mountain barriers significantly restrict gene flow for the majority of neotropical taxa, with geological formation of river barriers coinciding with estimated speciation events. Additionally, river width proves to be a valuable tool for estimating molecular divergence in adjacent taxa, particularly in regions with limited sampling.

Male-biased adult sexual size dimorphism is often the result of intra-sex sexual selection driven by male-male competition. Capelin (Mallotus villosus) exhibit male-biased sexual size dimorphism but lack contests/fighting, and female mate choice, if present, is unrelated to male size. Consequently, it is hypothesized that adult sexual size dimorphism in capelin is due to a mating system that favours larger males with greater energy reserves, allowing them to compete for prolonged access to mating opportunities through endurance rivalry. To enable this, males need a continual supply of semen through the spawning season. However, they have unusually small testes and are predicted to deplete stored semen rapidly, and their unique sperm physiology (i.e. the only known external fertilizing vertebrate to release pre-activated semen) may constrain the ability to regenerate it. We found that the majority of capelin sampled on beaches in 2023 had adequate semen, but towards the end of the spawning season some fish had none. Capelin held in laboratory tanks could regenerate semen within two days, maintaining their ability to seize continual mating opportunities. These results support the endurance rivalry hypothesis.

Eusociality in insects has arisen multiple times independently in Hymenoptera (bees, wasps, ants), Blattodea (termites) and Coleoptera (beetles). In Hymenoptera and Blattodea, the evolution of eusociality led to massive species proliferation. In the hyperdiverse Coleoptera, eusociality evolved only once, in the ancient Australian ambrosia beetle species Austroplatypus incompertus (Curculionidae: Platypodinae). This species occurs in mesic eucalypt forests of eastern Australia, from Victoria to northern New South Wales. Based on few individuals collected from the southern and northern edges of the species distribution it was initially described as two distinct species; however, the names were later synonymised as no morphological differences were found in analyses of more specimens. Recent mitochondrial haplotype analyses revealed substantial latitudinal divergence across the distribution of A. incompertus. To address this apparent disparity between morphological and molecular data, we sequenced and analysed a SNP panel of over 6,656 biallelic markers from 187 individuals of 11 sites across 1000 km of this species range. Our data indicate that eusocial demographic processes such as low colony establishment success rate, limited dispersal and reliance on few reproductive individuals, together with substantial habitat fragmentation contributed to the population genetic structure of this species. We further identified that the Hunter Valley biogeographic barrier split the species into two distinct clades, with both clades in secondary close contact on the Barrington Tops plateau without any discernible admixture. Our results support the resurrection of a second species of Austroplatypus which has important consequences for the evolution eusociality in Coleoptera and the systematics of Platypodinae.

Divergent ecological selection often results in trophic niche partitioning and is one of the central processes underlying sympatric speciation. However, there are still few studies of niche partitioning in putative examples of sympatric speciation in the wild. Here we conducted the first quantitative study of dietary niche partitioning in one of the most celebrated examples of sympatric speciation, Barombi Mbo cichlids, using stomach contents and stable isotope analyses. We found little evidence for trophic niche partitioning among any Barombi Mbo cichlids, even among the nine species coexisting in sympatry in the littoral zone. Stable isotope analyses supported these conclusions of minimal dietary overlap. However, we did find extraordinary dietary specialization in some species, including spongivory and feeding on terrestrial ants, both unique feeding strategies among freshwater fishes. Stomach contents of the spongivore (Pungu maclareni) were 20% freshwater sponge, notable considering that only 0.04% of all fishes consume sponges. Overall, we conclude that while there is less trophic niche partitioning than expected among Barombi Mbo cichlids, there is evidence for dietary specialization on rare resources in support of Liems paradox.

Studies of insect mating behaviour usually focus on what happens before and during copulation. Few pay close attention to the actions needed to end copulation. However, genital separation after copulation is likely to be an important cause of mechanical stress and injuries because it often involves the withdrawal of heavily armoured male intromittent organs from membranous female reproductive tracts. Difficult and/or slow separations can also reduce male and female fitness by increasing their exposure to predation. We here report the results of a comparative study of separation behaviour in 48 species of Sepsidae (Diptera) and one outgroup. We find a surprising amount of qualitative and quantitative behavioural variability within and between species. We characterize and reconstruct three types of behaviours: 1) The sepsid ancestor likely used back-off; a gentle separation technique that does not involve any pulling or twisting (https://youtu.be/EbkJvOaubZ0). 2) This potentially gave rise to the most common pull technique where the male turns 180 degrees and pulls in an opposite direction from the female (https://youtu.be/oLf4xGpkk1s). This separation can be quick and straightforward, but in some species the pull is slow and protracted and we routinely find dead males and/or females attached to their living partners in the latter (difficult: https://youtu.be/MbYPbXN6jr0; failure: https://youtu.be/leTiXefFzCc). 3) Finally, several species use twist, a new technique where the male rotates >360 degrees from the initial mounting position (https://youtu.be/WMUXbIPyLbk). We document that species capable of using twist have shorter and less variable separation times than those using "pull". However, many species capable of twist also retain the ability to use pull ( back-off/pull= 8%; pull only= 41%; twist/ pull= 24%; twist only = 27%). Overall, our study suggests that separation behaviour can vary among closely related species and highlights the significance of studying variable behavioural traits in a phylogenetic context.