Integration of genomic and ecological methods inform management of an undescribed, yet highly exploited, sardine species.

Assessing genetic diversity within species is key for conservation strategies in the context of human-induced biotic changes. This is important in marine systems, where many species remain undescribed while being overfished, and conflicts between resource-users and conservation agencies are common. Combining niche modelling with population genomics can contribute to resolving those conflicts by identifying management units and understanding how past climatic cycles resulted in current patterns of genetic diversity. We addressed these issues on an undescribed but already overexploited species of sardine of the genus Harengula. We find that the species distribution is determined by salinity and depth, with a continuous distribution along the Brazilian mainland and two disconnected oceanic archipelagos. Genomic data indicate that such biogeographic barriers are associated with two divergent intraspecific lineages. Changes in habitat availability during the last glacial cycle led to different demographic histories among stocks. One coastal population experienced a 3.6-fold expansion, whereas an island-associated population contracted 3-fold, relative to the size of the ancestral population. Our results indicate that the island population should be managed separately from the coastal population, and that a Marine Protected Area covering part of the island population distribution can support the viability of this lineage.

Ejaculate characteristics over seasons in five species of lancehead pitvipers (Bothrops spp) kept in captivity.

Due to their major medical importance in Latin America, lancehead pitvipers are frequently kept and bred in captivity for venom extraction to the production of antivenom serums. Nevertheless, despite the great contribution given to captive breeding, much of the knowledge of Bothrops' reproductive biology derived from sporadic and insufficient data provided by zoological collections. Thus, we aimed to investigate seasonal changes in gonadosomatic index (GSI) and seminal parameters (e.g., volume, concentration, motility, viability, and acrosome integrity) of five species of lancehead pitvipers from different biomes and phylogenetic groups, maintained in the indoors serpentarium at Butantan Institute (Brazil). Patterns of variation in GSI and semen parameters differed from one species to another, suggesting that captive populations should perhaps be managed distinctly to maximize reproductive success. Furthermore, in none of the studied species did changes in GSI occur concomitantly with seminal variations. GSI remained unaltered year-round for Jararaca (Bothrops jararaca) and Brazilian lancehead (Bothrops moojeni), whereas it peaked in the autumn for Common lancehead (Bothrops atrox), Jararacussu (Bothrops jararacussu), and Whitetail lancehead (Bothrops leucurus). But surprisingly, the scenario was inverted when we estimated the total number of motile spermatozoa per season, as Jararaca and Brazilian lancehead displayed seasonal differences and the other species did not vary throughout the year. Potential ecological and evolutionary factors underlying these differences were also discussed in the present article. Together, these findings can help to better define breeding management strategies for each species in captivity, in addition to optimizing the future use of artificial insemination and semen cryopreservation.

Ejaculate characteristics over seasons in five species of lancehead pitvipers (Bothrops spp ) kept in captivity

Due to their major medical importance in Latin America, lancehead pitvipers are frequently kept and bred in captivity for venom extraction to the production of antivenom serums. Nevertheless, despite the great contribution given to captive breeding, much of the knowledge of Bothrops' reproductive biology derived from sporadic and insufficient data provided by zoological collections. Thus, we aimed to investigate seasonal changes in gonadosomatic index (GSI) and seminal parameters (e.g., volume, concentration, motility, viability, and acrosome integrity) of five species of lancehead pitvipers from different biomes and phylogenetic groups, maintained in the indoors serpentarium at Butantan Institute (Brazil). Patterns of variation in GSI and semen parameters differed from one species to another, suggesting that captive populations should perhaps be managed distinctly to maximize reproductive success. Furthermore, in none of the studied species did changes in GSI occur concomitantly with seminal variations. GSI remained unaltered year-round for Jararaca (Bothrops jararaca) and Brazilian lancehead (Bothrops moojeni), whereas it peaked in the autumn for Common lancehead (Bothrops atrox), Jararacussu (Bothrops jararacussu), and Whitetail lancehead (Bothrops leucurus). But surprisingly, the scenario was inverted when we estimated the total number of motile spermatozoa per season, as Jararaca and Brazilian lancehead displayed seasonal differences and the other species did not vary throughout the year. Potential ecological and evolutionary factors underlying these differences were also discussed in the present article. Together, these findings can help to better define breeding management strategies for each species in captivity, in addition to optimizing the future use of artificial insemination and semen cryopreservation.

Assessing different liquid-storage temperatures for rooster spermatozoa.

Although liquid-storage is extensively used in poultry, there are still questions on how sperm physiology is affected and to what extent sperm functions are disrupted by storage temperature and time. There, therefore, was investigation of storage temperature and durations on multiple semen variables. The storage at 37 °C was the most damaging, affecting values for several variables within 4 h of storage, whereas most differences occurred between 5 and 25 °C after 8 h. Progressive motility and mitochondrial function started to decrease within 2 h at 25 and 37 °C, and within 4 h at 5 °C. Acrosomal damage only occurred in samples at 37 °C. Eosin-negrosin staining indicated there was damage to the plasma membrane at 37 °C, however, with use of propidium iodide there were differences between 5 and 25 °C following 24 h. Temperatures of 5 and 25 °C resulted in similar curves for chromatin dispersion although chromatin integrities differed with storage for periods longer than 4 h. At 37 °C, results using both chromatin evaluations indicated there was damage after 2 h of incubation. Oxidative stress at 5 and 25 °C was similar when there was 24 h of storage. Intriguingly, there were no interaction between temperature and storage duration for peroxidized sperm membrane and total peroxidation status. These findings indicated that with a prolonged storage at 5 °C there were not marked changes in chicken spermatozoa, whereas at 25 °C there did not appear to be sperm damage occurring as a result of short-term storage.

Genomic methods reveal independent demographic histories despite strong morphological conservatism in fish species.

Human overexploitation of natural resources has placed conservation and management as one of the most pressing challenges in modern societies, especially in regards to highly vulnerable marine ecosystems. In this context, cryptic species are particularly challenging to conserve because they are hard to distinguish based on morphology alone, and thus it is often unclear how many species coexist in sympatry, what are their phylogenetic relationships and their demographic history. We answer these questions using morphologically similar species of the genus Mugil that are sympatric in the largest coastal Marine Protected Area in the Tropical Southwestern Atlantic marine province. Using a sub-representation of the genome, we show that individuals are assigned to five highly differentiated genetic clusters that are coincident with five mitochondrial lineages, but discordant with morphological information, supporting the existence of five species with conserved morphology in this region. A lack of admixed individuals is consistent with strong genetic isolation between sympatric species, but the most likely species tree suggests that in one case speciation has occurred in the presence of interspecific gene flow. Patterns of genetic diversity within species suggest that effective population sizes differ up to two-fold, probably reflecting differences in the magnitude of population expansions since species formation. Together, our results show that strong morphologic conservatism in marine environments can lead to species that are difficult to distinguish morphologically but that are characterized by an independent evolutionary history, and thus that deserve species-specific management strategies.

Fecal sex steroids and reproductive behaviors in harpy eagles (Harpia harpyja).

Aiming to improve our reproductive knowledge of large birds of prey, behavioral data and fecal steroids were assessed in captive pairs of Harpy eagles, a keystone species that is monogamous and resides in the Neotropics year-round. Adult individuals exhibited different reproductive outcomes and a breeding season extending beyond summer solstice (5-9 months) suggests that harpy eagles may not be absolutely photorefractory. Comparisons among breeding stages in males revealed that mean androgen levels in courtship were higher than in copulation and incubation, but no differences were detected in fecal progestagens or estrogens. Females had higher mean estrogen concentrations in courtship and copulation, whereas mean progestagen levels peaked during egg laying. Mean androgen concentrations were not significantly different among breeding stages in females. Assessment of six egg-lay cycles from three females demonstrated that fecal estrogens peaked predominantly between 31 and 18 days before oviposition (-31 to -18 days), and then remained low until 45 days after laying the first egg (+45 days). In contrast, fecal progestagens raised mostly between -20 and +1 day, lowering to baseline concentrations by +3 days. To our knowledge, this is the first study to describe in detail endocrine and behavioral data regarding reproduction in tropical eagles, which may serve in the future as a reference to developing breeding programs.

Immunolocalisation and expression of oxytocin receptors and sex hormone-binding globulin in the testis and epididymis of dogs: correlation with sperm function.

The aim of this study was to confirm gene and protein expression of oxytocin receptor (OTR) and sex hormone-binding globulin (SHBG) in the testis and epididymis of dogs, correlating these data with sperm quality and production and testosterone concentrations. Positive correlations were found between OTR and SHBG expression in both the testis and epididymis. Testicular OTR expression was positively associated with plasma membrane and acrosome integrity in canine spermatozoa, whereas SHBG expression in the testis was positively correlated with various sperm characteristics, such as sperm concentration, total and progressive motility, plasma membrane integrity and acrosome integrity. Testicular expression of both OTR and SHBG was negatively correlated with low sperm mitochondrial activity. In the epididymis, SHBG expression was only positively correlated with plasma membrane integrity. Analysis of protein expression revealed that testicular OTR was positively correlated with testosterone concentrations and negatively correlated with the absence of sperm mitochondrial activity. In addition, SHBG expression in the testes was associated with epididymis SHBG expression and morphologically normal cells. Immunohistochemical (IHC) analysis revealed the presence of both OTR and SHBG in testicular smooth muscles and Leydig cells. However, in the epididymis, OTR was only located in smooth muscle cells, whereas neither IHC nor western blotting detected SHBG. Together, the results of this study suggest that OTR and SHBG play key roles in spermatogenesis and sperm maturation, being essential for male reproductive success.

Effect of simple and low-cost enrichment items on behavioral, clinical, and productive variables of caged laying hens.

Housing layers in battery cages is a practice still used by many countries but it has been criticized because of its influence on behavioral repertoire of birds. We investigated whether simple and affordable enrichment devices alone impact behavior, foot condition and performance of laying hens housed in conventional cages. Hens were divided into plain cages (CON), cages with perches (PER), and cages with tassels and scratch-pads (ENR), and parameters were evaluated before and after enrichment placement. After perch placement inactivity, drinking and competition for space reduced 35.6%, 40.8% and 70.3%, respectively, whereas social interaction increased 19.3%. Both modifications decreased locomotion (75.0% and 42.4% for PER and ENR respectively) and abnormal behaviors (62.5% and 43.9.4% for PER and ENR respectively). None of the performance variables were affected by ENR or PER. Thermography was more efficient than visual inspection in detecting subclinical bumblefoot, and it confirmed that PER reduced subclinical and clinical cases. Our findings indicate that perches increased welfare-related behaviors and foot health of hens, supporting the use of these inexpensive and highly adaptable alternatives for the enrichment of battery cages.

Semen collection by electro-stimulation in a variety of bird orders.

It is unquestinable that artificial insemination (AI) offers many benefits to avian conservation programs, but a serious impediment towards implementing AI for wild species is the development of effective techniques to consistently collect good quality ejaculates. Thus, we aimed to examine the success rate of electro-stimulation (ES) in collecting semen from 49 unconditioned males from orders Piciformes, Strigiformes, Accipitriformes, Cathartiformes, Galiformes, Anseriformes and Psittaciformes at different times of the year. Sixty out of 299 ES attempts provided ejaculates with sperm, but collection success rates varied widely (0-50%) depending on the species. Except for swans whose greater results were registered during spring-summer, males from most orders responded better to ES during winter-spring, suggesting seasonal variations on semen collection success rates. Overall, ES enabled successful semen collection from males of unproven and proven fertility under mixed pairing conditions. However, the highest success rate occurred in paired males with fertile clutches (40.6%) followed by unpaired males (22.1%), paired males without clutches (13.9%), and paired males with infertile clutches (6.8%). Behavioral responses of male birds to electrical impulses were also recorded to assess any discomfort during semen collection. Furthermore, macroscopic and microscopic analysis provided ejaculate parameters from several species, even from orders that hitherto have never been assessed for semen collection, which may serve as a starting point in the future. Altogether, these findings demonstrate the feasibility of ES in collecting semen from unpaired, unconditioned and non-imprinted males from a variety of bird orders. In the medium to long term, the use of this technique in both captive and free-ranging populations offers new perspectives to ensure genetic diversity in avian conservation programs.

Avanços aplicados à reprodução de aves domésticas e silvestres / Progress towards reproduction of domestic and wild birds

Técnicas reprodutivas em aves de produção e silvestres possuem objetivos e desafios distintos, visto que um nicho visa resultados econômicos enquanto o outro almeja a conservação de espécies raras e ameaçadas. Por essa razão, as biotecnologias desenvolvidas e empregadas na avicultura industrial buscam melhorar a performance reprodutiva de linhas puras, bisavós, avós e matrizes (p.e. fertilidade, produção de ovos, eclodibilidade, etc.), além de métodos operáveis em larga escala para avaliação de reprodutores (ambos machos e fêmeas) e incubação de ovos. Em contraste, a principal meta daqueles que trabalham com aves selvagens é a formação e utilização de bancos de germoplasma, dado que aproximadamente 12% de todas as espécies aviárias do mundo encontram-se atualmente ameaçadas de extinção. Considerando ambos cenários, o objetivo deste trabalho foi descrever alguns dos principais desafios e avanços relacionados a reprodução de aves domésticas e selvagens, de aspectos pretensamente mais conhecidos como a coleta e análise seminal ou manejo de ovos em incubatórios industriais, até métodos mais inovadores como transplantes interespecíficos de células germinativas e transplantes de tecido ovariano.

Reproductive techniques applied to poultry and wild birds have very different goals and challenges as one niche aims economical benefits while the other seeks to conserve rare and endangered species. Therefore, the development and application of biotechnologies in poultry industry strive to improve reproductive performance of pure lines, great grandparents, grandparents and parents (e.g. fertility, egg production, hatchability, etc.), in addition to feasible methods for large-scale operations with regard to breeders’ assessment (both males and females) and egg incubation. Conversely, the ultimate goal of those who work with wild birds is the development and use of germplasm banks since about 12% of the bird species worldwide are considered endangered. Taking both scenarios into consideration, we attempted to outline some challenges and advances in breeding domestic and wild birds, from supposedly well-known aspects such as semen collection and analysis or egg incubation, to more innovative techniques such germ cell and tissue transplantation.

Avanços aplicados à reprodução de aves domésticas e silvestres / Progress towards reproduction of domestic and wild birds

Técnicas reprodutivas em aves de produção e silvestres possuem objetivos e desafios distintos, visto que um nicho visa resultados econômicos enquanto o outro almeja a conservação de espécies raras e ameaçadas. Por essa razão, as biotecnologias desenvolvidas e empregadas na avicultura industrial buscam melhorar a performance reprodutiva de linhas puras, bisavós, avós e matrizes (p.e. fertilidade, produção de ovos, eclodibilidade, etc.), além de métodos operáveis em larga escala para avaliação de reprodutores (ambos machos e fêmeas) e incubação de ovos. Em contraste, a principal meta daqueles que trabalham com aves selvagens é a formação e utilização de bancos de germoplasma, dado que aproximadamente 12% de todas as espécies aviárias do mundo encontram-se atualmente ameaçadas de extinção. Considerando ambos cenários, o objetivo deste trabalho foi descrever alguns dos principais desafios e avanços relacionados a reprodução de aves domésticas e selvagens, de aspectos pretensamente mais conhecidos como a coleta e análise seminal ou manejo de ovos em incubatórios industriais, até métodos mais inovadores como transplantes interespecíficos de células germinativas e transplantes de tecido ovariano.(AU)

Reproductive techniques applied to poultry and wild birds have very different goals and challenges as one niche aims economical benefits while the other seeks to conserve rare and endangered species. Therefore, the development and application of biotechnologies in poultry industry strive to improve reproductive performance of pure lines, great grandparents, grandparents and parents (e.g. fertility, egg production, hatchability, etc.), in addition to feasible methods for large-scale operations with regard to breeders assessment (both males and females) and egg incubation. Conversely, the ultimate goal of those who work with wild birds is the development and use of germplasm banks since about 12% of the bird species worldwide are considered endangered. Taking both scenarios into consideration, we attempted to outline some challenges and advances in breeding domestic and wild birds, from supposedly well-known aspects such as semen collection and analysis or egg incubation, to more innovative techniques such germ cell and tissue transplantation.(AU)

Validation of simple and cost-effective stains to assess acrosomal status, DNA damage and mitochondrial activity in rooster spermatozoa.

Several methods have been developed to evaluate spermatozoa function in birds but many of these are sometimes complicated, costly and not applicable to field studies (i.e., performed within poultry breeding facilities). The objective was, therefore, to validate efficient, practical and inexpensive procedures to determine DNA fragmentation, acrosomal integrity, and mitochondrial activity in poultry spermatozoa. Initially, ejaculates were individually diluted and divided into control (4°C, 4h) and UV-irradiated aliquots (room temperature, 4h), and then samples containing different percentages of DNA-damaged spermatozoa (0%, 25%, 50%, 75% and 100%) were subjected to Toluidine Blue (TB) and Sperm Chromatin Dispersion assessments (SCD). Fast Green-Rose Bengal (FG-RB) and FITC-PSA staining protocols were subsequently used to assess acrosome status in aliquots comprising assorted amounts of acrosome-reacted spermatozoa. Furthermore, to validate 3,3'-diaminobenzidine (DAB) assay, ejaculates containing different gradients of spermatozoa with great amounts of mitochondrial activity were concurrently evaluated using DAB and JC-1 stains. The proportion of spermatozoa with abnormal DNA integrity when evaluated using the TB assessment correlated significantly with the expected percentages of UV-irradiated spermatozoa and with SCD results. A significant linear regression coefficient was also observed between expected amounts of acrosome-intact spermatozoa and FG-RB readings, and there was a significant correlation of the data when FG-RB and FITC-PSA were used. Likewise, the use of the DAB assay enabled for accurately ascertaining percentages of rooster spermatozoa with greater and lesser mitochondrial function, and results were highly correlated to results with staining with JC-1. Altogether, findings of the present study indicate acrosomal status, DNA integrity and mitochondrial activity in rooster spermatozoa can be easily and reliably determined using FG-RB, TB and DAB stains.

Adaptation to a latitudinal thermal gradient within a widespread copepod species: the contributions of genetic divergence and phenotypic plasticity.

Understanding how populations adapt to heterogeneous thermal regimes is essential for comprehending how latitudinal gradients in species diversification are formed, and how taxa will respond to ongoing climate change. Adaptation can occur by innate genetic factors, by phenotypic plasticity, or by a combination of both mechanisms. Yet, the relative contribution of such mechanisms to large-scale latitudinal gradients of thermal tolerance across conspecific populations remains unclear. We examine thermal performance in 11 populations of the intertidal copepod Tigriopus californicus, ranging from Baja California Sur (Mexico) to British Columbia (Canada). Common garden experiments show that survivorship to acute heat-stress differs between populations (by up to 3.8°C in LD50 values), reflecting a strong genetic thermal adaptation. Using a split-brood experiment with two rearing temperatures, we also show that developmental phenotypic plasticity is beneficial to thermal tolerance (by up to 1.3°C), and that this effect differs across populations. Although genetic divergence in heat tolerance strongly correlates with latitude and temperature, differences in the plastic response do not. In the context of climate warming, our results confirm the general prediction that low-latitude populations are most susceptible to local extinction because genetic adaptation has placed physiological limits closer to current environmental maxima, but our results also contradict the prediction that phenotypic plasticity is constrained at lower latitudes.

Impact of induced levels of specific free radicals and malondialdehyde on chicken semen quality and fertility.

Over the past decades, scientists endeavored to comprehend oxidative stress in poultry spermatozoa and its relationship with fertilizing ability, lipid peroxidation (LPO), free-radical scavenging systems, and antioxidant therapy. Although considerable progress has been made, further improvement is needed in understanding how specific reactive oxygen species (ROS) and malondialdehyde (MDA, a toxic byproduct of LPO) disrupt organelles in avian spermatozoon. Hence, this study examined functional changes in chicken spermatozoa after incubation with different ROS, and their implications for the fertility. First, semen samples from 14 roosters were individually diluted and aliquoted into five equal parts: control, superoxide anion, hydrogen peroxide (H2O2), hydroxyl radicals, and MDA. After incubation with these molecules, aliquots were analyzed for motility, plasma membrane and acrosome integrity, mitochondrial activity, and LPO and DNA damage. Hydrogen peroxide was more detrimental for sperm motility than hydroxyl radicals, whereas the superoxide anion and MDA exhibited no differences compared with controls. In turn, plasma membrane and acrosome integrity, mitochondrial activity, LPO and DNA integrity rates were only affected by hydroxyl radicals. Thereafter, semen aliquots were incubated under the same conditions and used for artificial insemination. In accordance to our in vitro observations, H2O2 and hydroxyl radicals sharply reduced egg fertility, whereas superoxide anion and MDA only induced slight declines. Thus, chicken sperm function was severely impaired by H2O2 and hydroxyl radicals, but their mechanisms of action seemingly comprise different pathways. Further analysis regarding susceptibility of spermatozoon organelles to specific radicals in other poultry will help us to understand the development of interspecific differences in scavenging systems and to outline more oriented antioxidant approaches.

Population Structure of the Rockpool Blenny Entomacrodus vomerinus Shows Source-Sink Dynamics among Ecoregions in the Tropical Southwestern Atlantic.

The Tropical Southwestern Atlantic is characterized by prominent ecosystems with large-scale oceanographic complexity. Yet, the evolutionary processes underlying genetic differentiation and connectivity in this region remain largely unknown. Entomacrodus vomerinus (Valenciennes, 1836) is a demersal fish with planktonic larvae endemic to this marine province, inhabiting shallow tidal pools in continental and oceanic reef environments. We evaluated the population structure, genetic diversity and gene flow of E. vomerinus using mitochondrial data (CYTB and COI) and nuclear (rhodopsin, RHO) DNA sequences. We sampled a total of 85 individuals, comprising 46 from three oceanic archipelagos with varying distance from the coast (São Pedro and São Paulo-SS, Fernando de Noronha-FE and Rocas Atoll-RA) and 39 from two localities in northeastern Brazilian coast (Rio Grande do Norte-RN and Bahia-BA). Multilocus analysis revealed the presence of three Evolutionarily Significant Units-ESUs (SS, FE+RA, and RN+BA), which are in accordance with distinct marine ecoregions. Coalescent analyses showed that the central ESU has a larger effective population size than the other two, suggesting strong asymmetries in the genetic diversity across the species range. Moreover, they showed that gene flow is highly asymmetric, suggesting a source-sink dynamics from the central ESU into the remaining ones, in agreement with oceanic currents. Together, these results provide insights in the evolutionary mechanisms facilitating diversification in this marine province.

Conservation of avian germplasm by xenogeneic transplantation of spermatogonia from sexually mature donors.

Approximately 12.5% of all 9,920 extant bird species in the world are threatened with extinction, and yet conservation efforts through natural breeding of captive species continue to encounter difficulties. However, sperm cryopreservation and artificial insemination offer potential benefits over natural breeding, but their applicability is still limited in nondomestic species. In this study, we aimed to exploit the potential of germ cell xenotransplantation as an alternative tool for preserving germplasm of endangered birds. The study was designed to investigate whether transfer of either spermatogonia-enriched cell fraction (SEF) or crude testicular cell fraction (CTF) from adult Japanese quails (as a model for wild species) would result in recolonization of gamma-irradiated gonads of adult recipient chickens. One month after transplantation, 75% of recipients injected with SEF and 25% of recipients injected with CTF resumed spermatogenesis. However, it took more than 3 months for 33% of the negative controls to resume marginal production of sperm. Some SEF recipients produced more spermatozoa bearing head morphology compared with donor controls. DNA analysis using quail-specific primers did not detect donor's DNA in these recipients' semen. However, 6 months after xenotransplantation, presence of quail germ cells was demonstrated by polymerase chain reaction and by immunohistochemistry in 1 rooster injected with SEF. These findings indicate that spermatogonia from adult quails were capable of colonizing immunocompetent testis of adult chickens but failed to produce sufficient sperm. Despite this limitation, the present approach represents a potential conservation tool that may be used to rescue germ cells of endangered adult male birds.

Ring distributions leading to species formation: a global topographic analysis of geographic barriers associated with ring species.

BACKGROUND: In the mid 20th century, Ernst Mayr and Theodosius Dobzhansky championed the significance of circular overlaps or ring species as the perfect demonstration of speciation, yet in the over 50 years since, only a handful of such taxa are known. We developed a topographic model to evaluate whether the geographic barriers that favor processes leading to ring species are common or rare, and to predict where other candidate ring barriers might be found. RESULTS: Of the 952,147 geographic barriers identified on the planet, only about 1% are topographically similar to barriers associated with known ring taxa, with most of the likely candidates occurring in under-studied parts of the world (for example, marine environments, tropical latitudes). Predicted barriers separate into two distinct categories: (i) single cohesive barriers (< 50,000 km2), associated with taxa that differentiate at smaller spatial scales (salamander: Ensatina eschscholtzii; tree: Acacia karroo); and (ii) composite barriers - formed by groups of barriers (each 184,000 to 1.7 million km2) in close geographic proximity (totaling 1.9 to 2.3 million km2) - associated with taxa that differentiate at larger spatial scales (birds: Phylloscopus trochiloides and Larus (sp. argentatus and fuscus)). When evaluated globally, we find a large number of cohesive barriers that are topographically similar to those associated with known ring taxa. Yet, compared to cohesive barriers, an order of magnitude fewer composite barriers are similar to those that favor ring divergence in species with higher dispersal. CONCLUSIONS: While these findings confirm that the topographic conditions that favor evolutionary processes leading to ring speciation are, in fact, rare, they also suggest that many understudied natural systems could provide valuable demonstrations of continuous divergence towards the formation of new species. Distinct advantages of the model are that it (i) requires no a priori information on the relative importance of features that define barriers, (ii) can be replicated using any kind of continuously distributed environmental variable, and (iii) generates spatially explicit hypotheses of geographic species formation. The methods developed here - combined with study of the geographical ecology and genetics of taxa in their environments - should enable recognition of ring species phenomena throughout the world.