Neurons in the monkey frontopolar cortex encode learning stage and goal during a fast learning task.

The frontopolar cortex (FPC) is, to date, one of the least understood regions of the prefrontal cortex. The current understanding of its function suggests that it plays a role in the control of exploratory behaviors by coordinating the activities of other prefrontal cortex areas involved in decision-making and exploiting actions based on their outcomes. Based on this hypothesis, FPC would drive fast-learning processes through a valuation of the different alternatives. In our study, we used a modified version of a well-known paradigm, the object-in-place (OIP) task, to test this hypothesis in electrophysiology. This paradigm is designed to maximize learning, enabling monkeys to learn in one trial, which is an ability specifically impaired after a lesion of the FPC. We showed that FPC neurons presented an extremely specific pattern of activity by representing the learning stage, exploration versus exploitation, and the goal of the action. However, our results do not support the hypothesis that neurons in the frontal pole compute an evaluation of different alternatives. Indeed, the position of the chosen target was strongly encoded at its acquisition, but the position of the unchosen target was not. Once learned, this representation was also found at the problem presentation, suggesting a monitoring activity of the synthetic goal preceding its acquisition. Our results highlight important features of FPC neurons in fast-learning processes without confirming their role in the disengagement of cognitive control from the current goals.

Hippocampal lesions impair non-navigational spatial memory in macaques.

Decades of studies robustly support a critical role for the hippocampus in spatial memory across a wide range of species. Hippocampal damage produces clear and consistent deficits in allocentric spatial memory that requires navigating through space in rodents, non-human primates, and humans. By contrast, damage to the hippocampus spares performance in most non-navigational spatial memory tasks-which can typically be resolved using egocentric cues. We previously found that transient inactivation of the hippocampus impairs performance in the Hamilton Search Task (HST), a self-ordered non-navigational spatial search task. A key question, however, still needs to be addressed. Acute, reversible inactivation of the hippocampus may have resulted in an impairment in the HST because this approach does not allow for neuroplastic compensation, may prevent the development of an alternative learning strategy, and/or may produce network-based effects that disrupt performance. We compared learning and performance on the HST in male rhesus macaques (six unoperated control animals and six animals that underwent excitotoxic lesions of the hippocampus). We found a significant impairment in animals with hippocampal lesions. While control animals improved in performance over the course of 45 days of training, performance in animals with hippocampal lesions remained at chance levels. The HST thus represents a sensitive assay for probing the integrity of the hippocampus in non-human primates. These data provide evidence demonstrating that the hippocampus is critical for this type of non-navigational spatial memory, and help to reconcile the many null findings previously reported.

Chemogenetic Disconnection between the Orbitofrontal Cortex and the Rostromedial Caudate Nucleus Disrupts Motivational Control of Goal-Directed Action.

The orbitofrontal cortex (OFC) and its major downstream target within the basal ganglia-the rostromedial caudate nucleus (rmCD)-are involved in reward-value processing and goal-directed behavior. However, a causal contribution of the pathway linking these two structures to goal-directed behavior has not been established. Using the chemogenetic technology of designer receptors exclusively activated by designer drugs with a crossed inactivation design, we functionally and reversibly disrupted interactions between the OFC and rmCD in two male macaque monkeys. We injected an adeno-associated virus vector expressing an inhibitory designer receptor, hM4Di, into the OFC and contralateral rmCD, the expression of which was visualized in vivo by positron emission tomography and confirmed by postmortem immunohistochemistry. Functional disconnection of the OFC and rmCD resulted in a significant and reproducible loss of sensitivity to the cued reward value for goal-directed action. This decreased sensitivity was most prominent when monkeys had accumulated a certain amount of reward. These results provide causal evidence that the interaction between the OFC and the rmCD is needed for motivational control of action on the basis of the relative reward value and internal drive. This finding extends the current understanding of the physiological basis of psychiatric disorders in which goal-directed behavior is affected, such as obsessive-compulsive disorder.SIGNIFICANCE STATEMENT In daily life, we routinely adjust the speed and accuracy of our actions on the basis of the value of expected reward. Abnormalities in these kinds of motivational adjustments might be related to behaviors seen in psychiatric disorders such as obsessive-compulsive disorder. In the current study, we show that the connection from the orbitofrontal cortex to the rostromedial caudate nucleus is essential for motivational control of action in monkeys. This finding expands our knowledge about how the primate brain controls motivation and behavior and provides a particular insight into disorders like obsessive-compulsive disorder in which altered connectivity between the orbitofrontal cortex and the striatum has been implicated.

Molecular ecology of highest priority critically important antibiotic resistant Escherichia coli from mammals housed at an urban zoo.

OBJECTIVES: Zoos are environments where species of highly valued animals are kept largely separated from others and the wider world. We report the molecular ecology of critically important antibiotic resistant (ABR) Escherichia coli carried by 28 mammalian species housed in a zoo located in an urban residential district. METHODS: Over 3 months we collected 167 faecal samples from captive mammals and processed for E. coli resistant to third-generation cephalosporins (3GC-R) and fluoroquinolones (FQ-R). Isolates were sequenced using Illumina. RESULTS: We identified high rates of faecal sample-level positivity, with 50%, 57% and 36% of mammalian species excreting 3GC-R, FQ-R or dual 3GC-R/FQ-R E. coli, respectively. Isolates represented multiple ST and ABR mechanisms; CTX-M-15 and CMY-2 dominated for 3GC-R, and target-site mutation caused 75% of FQ-R. We identified multiple examples of ABR E. coli transmission between mammalian species in separate enclosures, and a variant of the epidemic plasmid pCT within the zoo. There was no evidence for ABR E. coli leaving the zoo, based on comparative analysis with E. coli from humans, cattle and dogs isolated from the 50 × 50 km region in which the zoo is located. Amoxicillin/clavulanate was the most widely used antibiotic in the zoo, and we identified four widely disseminated amoxicillin/clavulanate resistance mechanisms, including a previously unreported inhibitor-resistant TEM, and the carbapenemase OXA-181. CONCLUSIONS: We conclude that the zoo studied here is a 'melting pot' for the selection and circulation of 3GC-R and FQ-R E. coli, but these circulating E. coli appear captive within the zoo.

Contributions of the Monkey Inferior Temporal Areas TE and TEO to Visual Categorization.

The ability to categorize images is thought to depend on neural processing within the ventral visual stream. Recently, we reported that after removal of architectonic area TE, the terminal region of the ventral stream, monkeys were still able to categorize images as cats or dogs moderately well. Here, we investigate the contribution of TEO, the architectonically defined region located one step earlier than area TE in the ventral stream. Bilateral removal of TEO caused only a mild impairment in categorization. However, combined TE + TEO removal was followed by a severe, long-lasting impairment in categorization. All of the monkeys tested, including those with combined TE + TEO removals, had normal low-level visual functions, such as visual acuity. These results support the conclusion that categorization based on visual similarity is processed in parallel in TE and TEO.

Information-Limiting Correlations in Large Neural Populations.

Understanding the neural code requires understanding how populations of neurons code information. Theoretical models predict that information may be limited by correlated noise in large neural populations. Nevertheless, analyses based on tens of neurons have failed to find evidence of saturation. Moreover, some studies have shown that noise correlations can be very small, and therefore may not affect information coding. To determine whether information-limiting correlations exist, we implanted eight Utah arrays in prefrontal cortex (PFC; area 46) of two male macaque monkeys, recording >500 neurons simultaneously. We estimated information in PFC about saccades as a function of ensemble size. Noise correlations were, on average, small (∼10-3). However, information scaled strongly sublinearly with ensemble size. After shuffling trials, destroying noise correlations, information was a linear function of ensemble size. Thus, we provide evidence for the existence of information-limiting noise correlations in large populations of PFC neurons.SIGNIFICANCE STATEMENT Recent theoretical work has shown that even small correlations can limit information if they are "differential correlations," which are difficult to measure directly. However, they can be detected through decoding analyses on recordings from a large number of neurons over a large number of trials. We have achieved both by collecting neural activity in dorsal-lateral prefrontal cortex of macaques using eight microelectrode arrays (768 electrodes), from which we were able to compute accurate information estimates. We show, for the first time, strong evidence for information-limiting correlations. Despite pairwise correlations being small (on the order of 10-3), they affect information coding in populations on the order of 100 s of neurons.

MRI monitoring of macaque monkeys in neuroscience: Case studies, resource and normative data comparisons.

Information from Magnetic Resonance Imaging (MRI) is useful for diagnosis and treatment management of human neurological patients. MRI monitoring might also prove useful for non-human animals involved in neuroscience research provided that MRI is available and feasible and that there are no MRI contra-indications precluding scanning. However, MRI monitoring is not established in macaques and a resource is urgently needed that could grow with scientific community contributions. Here we show the utility and potential benefits of MRI-based monitoring in a few diverse cases with macaque monkeys. We also establish a PRIMatE MRI Monitoring (PRIME-MRM) resource within the PRIMatE Data Exchange (PRIME-DE) and quantitatively compare the cases to normative information drawn from MRI data from typical macaques in PRIME-DE. In the cases, the monkeys presented with no or mild/moderate clinical signs, were well otherwise and MRI scanning did not present a significant increase in welfare impact. Therefore, they were identified as suitable candidates for clinical investigation, MRI-based monitoring and treatment. For each case, we show MRI quantification of internal controls in relation to treatment steps and comparisons with normative data in typical monkeys drawn from PRIME-DE. We found that MRI assists in precise and early diagnosis of cerebral events and can be useful for visualising, treating and quantifying treatment response. The scientific community could now grow the PRIME-MRM resource with other cases and larger samples to further assess and increase the evidence base on the benefits of MRI monitoring of primates, complementing the animals' clinical monitoring and treatment regime.

Dimensionality, information and learning in prefrontal cortex.

Learning leads to changes in population patterns of neural activity. In this study we wanted to examine how these changes in patterns of activity affect the dimensionality of neural responses and information about choices. We addressed these questions by carrying out high channel count recordings in dorsal-lateral prefrontal cortex (dlPFC; 768 electrodes) while monkeys performed a two-armed bandit reinforcement learning task. The high channel count recordings allowed us to study population coding while monkeys learned choices between actions or objects. We found that the dimensionality of neural population activity was higher across blocks in which animals learned the values of novel pairs of objects, than across blocks in which they learned the values of actions. The increase in dimensionality with learning in object blocks was related to less shared information across blocks, and therefore patterns of neural activity that were less similar, when compared to learning in action blocks. Furthermore, these differences emerged with learning, and were not a simple function of the choice of a visual image or action. Therefore, learning the values of novel objects increases the dimensionality of neural representations in dlPFC.

EARLY-ONSET LEUKOENCEPHALOMYELOPATHY AND POLYNEUROPATHY IN EASTERN QUOLLS (DASYURUS VIVERRINUS) IN THE EUROPEAN CAPTIVE POPULATION.

Leukoencephalomyelopathy (LEM) is suggested to be an age-related degenerative condition in geriatric Eastern quolls (Dasyurus viverrinus), manifesting in animals greater than 3.5 yr of age. This case series describes four LEM cases from two zoologic collections; three in nongeriatric animals, with one only 1 yr of age, and details advanced diagnostic investigation, including magnetic resonance imaging, cerebrospinal fluid analysis, and electrodiagnostic studies, not previously reported in Eastern quolls. Animals presented clinically with forelimb proprioceptive deficits and hindlimb and lumbar muscle hypotrophy, which were not noted in previous reports, in addition to hindlimb ataxia. Blindness and emaciation, which have been reported previously, were not seen. Disease progression was variable, and time from first clinical signs to euthanasia ranged from 46 days to over 2 yr. Histopathologic findings in the central nervous system were typical of those in previous LEM cases; concomitant polyneuropathy was observed in two quolls. Our findings suggest that age-related degeneration may not be the only cause of LEM in Eastern quolls. Because all quolls were related, a familial component cannot be excluded. LEM should be further investigated for its potential impact on future captive breeding programs, and our findings suggest that daily quality-of-life assessment should guide euthanasia of affected animals.

Molecular phylogenetics and floral evolution of the Cirrhopetalum alliance (Bulbophyllum, Orchidaceae): Evolutionary transitions and phylogenetic signal variation.

The Cirrhopetalum alliance is a loosely circumscribed species-rich group within the mega-diverse genus Bulbophyllum (Orchidaceae). The monophyletic status of the alliance has been challenged by previous studies, although established sectional classifications have yet to be tested in a phylogenetic context. We used maximum likelihood and Bayesian analyses of DNA sequence data (cpDNA: matK and psbA-trnH; nrDNA: ITS and Xdh; 3509 aligned characters; 117 taxa), including all sections putatively associated with the Cirrhopetalum alliance, to reconstruct the phylogeny. We mapped 11 selected categorical floral characters onto the phylogeny to identify synapomorphies and assess potential evolutionary transitions across major clades. Our results unequivocally support the recognition of an amended Cirrhopetalum alliance as a well-supported monophyletic group characterized by clear synapomorphies, following the inclusion of sect. Desmosanthes and the exclusion of five putative Cirrhopetalum-allied sections. Most sections within the Cirrhopetalum alliance are demonstrated to be polyphyletic or paraphyletic, necessitating a new sectional classification. The inclusion of sect. Desmosanthes revolutionizes our understanding of the alliance, with significant evolutionary transitions in floral characters detected. We further investigated six continuously variable characters of the sepals and labellum, and detect phylogenetic conservatism in labellum width and the evolutionary lability of lateral sepal length, which can partly be explained by the different functional roles they play in pollination and pollinator trapping.

A symbiotic balancing act: arbuscular mycorrhizal specificity and specialist fungus gnat pollination in the mycoheterotrophic genus Thismia (Thismiaceae).

BACKGROUND AND AIMS: Mycorrhizal associations in mycoheterotrophic plants are generally more specialized than in autotrophs. Mycoheterotrophs typically bear small, inconspicuous flowers that often self-pollinate to maximize seed set, although some have structurally complex flowers indicative of xenogamy. A trade-off has previously been proposed between specialization in these above- and below-ground symbioses, although empirical data are lacking. METHODS: We used next-generation DNA sequencing to compare the mycorrhizal communities from the roots of a mycoheterotrophic species, Thismia tentaculata (Thismiaceae), and its neighbouring autotrophs. We furthermore conducted detailed assessments of floral phenology and pollination ecology, and performed artificial pollination experiments to determine the breeding system. KEY RESULTS: Thismia tentaculata maintains a symbiotic association with a single arbuscular mycorrhizal Rhizophagus species. The flowers are pollinated by a single species of fungus gnats (Corynoptera, Sciaridae), which are attracted by the yellow pigments and are temporarily restrained within the perianth chamber before departing via apertures between the anthers. The plants are self-compatible but predominantly xenogamous. CONCLUSIONS: Our findings demonstrate that T. tentaculata maintains highly specialized associations with pollinators and mycorrhizal fungi, both of which are widely distributed. We suggest that specialization in multiple symbiotic interactions is possible in mycoheterotrophs if redundant selective pressures are not exerted to further restrict an already constrained suite of life-history traits.

Accommodative esotropia: the state of the art.

PURPOSE: To review the state of the art of Accommodative Esotropia (AE) through careful study of what has been reported up to the point in literature. METHODS: A literature search was done on PubMed using key words including "Accommodative esotropia", "Infantile esotropia", "Strabismus" and "Accommodation". We systematically reviewed and critically appraised what has been written about AE and we tried to analyze that according to the current management of AE. RESULTS: Accommodative Esotropia (AE) is a form of strabismus characterized by convergent misalignment of the visual axes that can be associated with hyperopia and abnormal fusional divergence. Also abnormal accommodative convergence/accommodation ratio could be found. In lots of cases, AE initially presents as an intermittent esodeviation at age 1.5 to 4 years. The prevalence of AE has been estimated near 1-2% in the United States. The only treatment with an optical correction usually is successful in re-establishing alignment, but surgical correction is necessary in approximately 30% of cases.

Gene tree discordance and coalescent methods support ancient intergeneric hybridisation between Dasymaschalon and Friesodielsia (Annonaceae).

Although hybridisation between closely related species is common and known to be important in plant evolution, hybridisation at the generic level or above is comparatively rare. We address ancient intergeneric hybridisation in the early-divergent angiosperm family Annonaceae by phylogenetic reconstruction, divergence time estimation and coalescent simulation of the genus Dasymaschalon using a multi-locus approach based on molecular data from five chloroplast (matK, psbA-trnH, ndhF, rbcL, and trnL-F) and five nuclear (ITS, ETS, AP3, PhyA, and PhyC) DNA markers. We demonstrate incongruence among different gene trees: Dasymaschalon is retrieved as monophyletic in the nuclear ribosomal tree (based on ITS and ETS), but is non-monophyletic in the chloroplast and Phy-gene trees (with poor resolution in the AP3 tree), with the majority of species assigned to a strongly supported clade but three species (D. filipes, D. longiflorum and D. tibetense) more closely related to the sister genus Friesodielsia. Three contrasting approaches-a coalescent method based on molecular dating, incongruence pattern comparison, and a multi-accession phylogenetic reconstruction-are used to assess the patterns of this gene tree incongruence and test hypotheses of ancient hybridisation and incomplete lineage sorting. Our results support a late Miocene intergeneric hybridisation between members of the Dasymaschalon and Friesodielsia lineages in continental Asia-west Malesia.

Determination of vertebral heart score in three species of Spider monkey (Ateles fusciceps, A. hybridus and A. paniscus).

BACKGROUND: The vertebral heart score (VHS) is a method of evaluation of cardiac size well documented in domestic mammals and in other primate species, and the aim of this study was to determine the VHS in three species of Spider monkey. METHODS: In this retrospective study, right lateral radiographs of thirty clinically well animals were reviewed and VHS determined. The species included were Ateles fusciceps (n=17), Ateles hybridus (n=8) and Ateles paniscus (n=5). RESULTS: The VHS was found to vary between species and was 9.73±0.81 for A. fusciceps, 10.53±0.37 for A. hybridus and 10.45±0.27 for A. paniscus. CONCLUSIONS: The observed values appear consistent with values determined for other primate species. There was statistically significant variation noted between species, and so VHS should be considered species-specific in this genus. The values determined may be of benefit in objectively evaluating cardiac size in the species investigated.

Intrinsic Connections of the Core Auditory Cortical Regions and Rostral Supratemporal Plane in the Macaque Monkey.

In the ventral stream of the primate auditory cortex, cortico-cortical projections emanate from the primary auditory cortex (AI) along 2 principal axes: one mediolateral, the other caudorostral. Connections in the mediolateral direction from core, to belt, to parabelt, have been well described, but less is known about the flow of information along the supratemporal plane (STP) in the caudorostral dimension. Neuroanatomical tracers were injected throughout the caudorostral extent of the auditory core and rostral STP by direct visualization of the cortical surface. Auditory cortical areas were distinguished by SMI-32 immunostaining for neurofilament, in addition to established cytoarchitectonic criteria. The results describe a pathway comprising step-wise projections from AI through the rostral and rostrotemporal fields of the core (R and RT), continuing to the recently identified rostrotemporal polar field (RTp) and the dorsal temporal pole. Each area was strongly and reciprocally connected with the areas immediately caudal and rostral to it, though deviations from strictly serial connectivity were observed. In RTp, inputs converged from core, belt, parabelt, and the auditory thalamus, as well as higher order cortical regions. The results support a rostrally directed flow of auditory information with complex and recurrent connections, similar to the ventral stream of macaque visual cortex.

Superficial white matter fiber systems impede detection of long-range cortical connections in diffusion MR tractography.

In vivo tractography based on diffusion magnetic resonance imaging (dMRI) has opened new doors to study structure-function relationships in the human brain. Initially developed to map the trajectory of major white matter tracts, dMRI is used increasingly to infer long-range anatomical connections of the cortex. Because axonal projections originate and terminate in the gray matter but travel mainly through the deep white matter, the success of tractography hinges on the capacity to follow fibers across this transition. Here we demonstrate that the complex arrangement of white matter fibers residing just under the cortical sheet poses severe challenges for long-range tractography over roughly half of the brain. We investigate this issue by comparing dMRI from very-high-resolution ex vivo macaque brain specimens with histological analysis of the same tissue. Using probabilistic tracking from pure gray and white matter seeds, we found that ∼50% of the cortical surface was effectively inaccessible for long-range diffusion tracking because of dense white matter zones just beneath the infragranular layers of the cortex. Analysis of the corresponding myelin-stained sections revealed that these zones colocalized with dense and uniform sheets of axons running mostly parallel to the cortical surface, most often in sulcal regions but also in many gyral crowns. Tracer injection into the sulcal cortex demonstrated that at least some axonal fibers pass directly through these fiber systems. Current and future high-resolution dMRI studies of the human brain will need to develop methods to overcome the challenges posed by superficial white matter systems to determine long-range anatomical connections accurately.

Weight management in adults with intellectual and developmental disabilities: A randomized controlled trial of two dietary approaches.

BACKGROUND: The prevalence of obesity among individuals with intellectual and developmental disabilities (IDD) is equal to or greater than the general population. METHODS: Overweight/obese adults (BMI ≥25 kg/m2 ) with mild-to-moderate intellectual and developmental disabilities were randomized to an enhanced stop light diet (eSLD = SLD + portion-controlled meals, n = 78) or a conventional diet (CD, n = 72) for an 18 months trial (6 months weight loss, 12 months maintenance). Participants were asked to increase physical activity (150 min/week), self-monitor diet and physical activity and attend counselling/educational sessions during monthly home visits. RESULTS: Weight loss (6 months) was significantly greater in the eSLD (-7.0% ± 5.0%) compared with the CD group (-3.8% ± 5.1%, p < .001). However, at 18 months, weight loss between groups did not differ significantly (eSLD = -6.7% ± 8.3%; CD = 6.4% ± 8.6%; p = .82). CONCLUSION: The eSLD and CD provided clinically meaningful weight loss over 18 months in adults with intellectual and developmental disabilities.

Mild Perceptual Categorization Deficits Follow Bilateral Removal of Anterior Inferior Temporal Cortex in Rhesus Monkeys.

In primates, visual recognition of complex objects depends on the inferior temporal lobe. By extension, categorizing visual stimuli based on similarity ought to depend on the integrity of the same area. We tested three monkeys before and after bilateral anterior inferior temporal cortex (area TE) removal. Although mildly impaired after the removals, they retained the ability to assign stimuli to previously learned categories, e.g., cats versus dogs, and human versus monkey faces, even with trial-unique exemplars. After the TE removals, they learned in one session to classify members from a new pair of categories, cars versus trucks, as quickly as they had learned the cats versus dogs before the removals. As with the dogs and cats, they generalized across trial-unique exemplars of cars and trucks. However, as seen in earlier studies, these monkeys with TE removals had difficulty learning to discriminate between two simple black and white stimuli. These results raise the possibility that TE is needed for memory of simple conjunctions of basic features, but that it plays only a small role in generalizing overall configural similarity across a large set of stimuli, such as would be needed for perceptual categorical assignment. SIGNIFICANCE STATEMENT: The process of seeing and recognizing objects is attributed to a set of sequentially connected brain regions stretching forward from the primary visual cortex through the temporal lobe to the anterior inferior temporal cortex, a region designated area TE. Area TE is considered the final stage for recognizing complex visual objects, e.g., faces. It has been assumed, but not tested directly, that this area would be critical for visual generalization, i.e., the ability to place objects such as cats and dogs into their correct categories. Here, we demonstrate that monkeys rapidly and seemingly effortlessly categorize large sets of complex images (cats vs dogs, cars vs trucks), surprisingly, even after removal of area TE, leaving a puzzle about how this generalization is done.

Historical biogeography and ecological niche modelling of the Asimina-Disepalum clade (Annonaceae): role of ecological differentiation in Neotropical-Asian disjunctions and diversification in Asia.

BACKGROUND: The Asimina-Disepalum clade (Annonaceae subfam. Annonoideae tribe Annoneae) includes a major Neotropical-Asian biogeographical disjunction. We evaluate whether this disjunction can be explained by the Eocene boreotropics hypothesis, which relies on the existence of extensive boreotropical forests during the Late Palaeocene-Early Eocene thermal maximum (52-50 Ma), followed by disruption of boreotropical vegetation during post-Eocene cooling. Molecular dating using an uncorrelated relaxed molecular clock (UCLD) model with two fossil calibrations, ancestral range estimation, and ecological niche modelling across evolutionary time were performed. Our focus was the geographical origin of Disepalum and general biogeographic patterns within this genus. Comparison of ecological tolerance among extant species and niche reconstructions at ancestral nodes within the clade enabled insights in likely migration routes of lineages, as well as evaluating the role of bioclimatic ecological differentiation in the diversification of Disepalum within Southeast Asia. RESULTS: The inferred vicariance event associated with the Asimina-Disepalum disjunction is estimated to have originated ca. 40 Mya [95% highest posterior density (HPD): 44.3-35.5 Mya]. The Disepalum crown lineage is estimated to have originated ca. 9 Mya (95% HPD: 10.6-7.6), either in western Malesia and continental Southeast Asia, or exclusively in western Malesia. Ecological niche modelling shows that seasonality of temperature and precipitation are major contributors determining the geographical range of species. Ancestral niche modelling furthermore indicates that the ancestor of the Asimina-Disepalum clade likely had bioclimatic preferences close to conditions found in current tropical and subtropical climates across Asia, whereas the ancestors of the Asimina and Disepalum crown groups are projected onto the more subtropical and tropical regions, respectively. CONCLUSIONS: The vicariance event associated with the Neotropical-Asian disjunction within the Asimina-Disepalum clade likely coincided with climatic deterioration at the Eocene-Oligocene boundary. Although detrended component analyses (DCA) indicate that altitude and seasonality of temperature and precipitation have the greatest influence in determining the geographical range of species, isolation due to palaeogeographic and palaeoclimatic events appears to be of greater significance than climate niche differentiation in driving diversification in Disepalum.

Preponderance of clonality triggers loss of sex in Bulbophyllum bicolor, an obligately outcrossing epiphytic orchid.

Vegetative propagation (clonal growth) conveys several evolutionary advantages that positively affect life history fitness and is a widespread phenomenon among angiosperms that also reproduce sexually. However, a bias towards clonality can interfere with sexual reproduction and lead to sexual extinction, although a dearth of effective genetic tools and mathematical models for clonal plants has hampered assessment of these impacts. Using the endangered tropical epiphytic or lithophytic orchid Bulbophyllum bicolor as a model, we integrated an examination of breeding system with 12 microsatellite loci and models valid for clonal species to test for the "loss of sex" and infer likely consequences for long-term reproductive dynamics. Bagging experiments and field observations revealed B. bicolor to be self-incompatible and pollinator-dependent, with an absence of fruit-set over 4 years. Challenging the assumptions that clonal populations can be as genotypically diverse as sexually reproducing ones and that clonality does not greatly influence genetic structure, just 22 multilocus genotypes were confirmed among all 15 extant natural populations, 12 of the populations were found to be monoclonal, and all three multiclonal ones exhibited a distinct phalanx clonal architecture. Our results suggest that all B. bicolor populations depend overwhelmingly on clonal growth for persistence, with a concomitant loss of sex due to an absence of pollinators and a lack of mating opportunities at virtually all sites, both of which are further entrenched by habitat fragmentation. Such cryptic life history impacts, potentially contributing to extinction debt, could be widespread among similarly fragmented, outcrossing tropical epiphytes, demanding urgent conservation attention.