On the road to losing connectivity: Faecal samples provide genome-wide insights into anthropogenic impacts on two large herbivore species in central India.

Humans have impacted most of the planet, and the ensuing fragmentation results in small, isolated habitat patches posing a risk of genetic diversity loss, inbreeding, and genetic load. Understanding how natural and anthropogenic landscape features affect gene flow among habitat patches is critical for maintaining connectivity. Genome-wide data are required to comprehend the impacts of recent fragmentation, which can be challenging when only non-invasive samples are available. Here, we build upon advancements in conservation genomics to address connectivity of two large herbivores, gaur (Bos gaurus) and sambar (Rusa unicolor) in central India. Given their habitat associations, we expected these species to respond similarly to habitat fragmentation. We used faecal-DNA and methylation-based host-DNA enrichment with modified ddRAD protocol to generate genome-wide single-nucleotide polymorphism (SNP) data for 124 gaur and 99 sambar individuals. Our findings reveal that gaur populations in central India are fragmented, displaying high genetic differentiation, with drift significantly affecting small populations like Umred Karhandla Wildlife Sanctuary. Although sambar shows low genetic structure, another small population, Bor Tiger Reserve is genetically differentiated. Our results suggest that although land cover change and roads restrict animal movement, the extent of this impact varies across the two species. We show that different species respond differently to landscape features, even with similar habitat associations. We highlight small and isolated populations requiring urgent conservation intervention. Such multi-species approaches enhance our understanding of cross-species connectivity patterns. We suggest shifting from single-species to multi-species holistic conservation approach in rapidly developing landscapes to better manage co-occurring endangered species.

Genomic evidence for inbreeding depression and purging of deleterious genetic variation in Indian tigers.

Increasing habitat fragmentation leads to wild populations becoming small, isolated, and threatened by inbreeding depression. However, small populations may be able to purge recessive deleterious alleles as they become expressed in homozygotes, thus reducing inbreeding depression and increasing population viability. We used whole-genome sequences from 57 tigers to estimate individual inbreeding and mutation load in a small-isolated and two large-connected populations in India. As expected, the small-isolated population had substantially higher average genomic inbreeding (FROH = 0.57) than the large-connected (FROH = 0.35 and FROH = 0.46) populations. The small-isolated population had the lowest loss-of-function mutation load, likely due to purging of highly deleterious recessive mutations. The large populations had lower missense mutation loads than the small-isolated population, but were not identical, possibly due to different demographic histories. While the number of the loss-of-function alleles in the small-isolated population was lower, these alleles were at higher frequencies and homozygosity than in the large populations. Together, our data and analyses provide evidence of 1) high mutation load, 2) purging, and 3) the highest predicted inbreeding depression, despite purging, in the small-isolated population. Frequency distributions of damaging and neutral alleles uncover genomic evidence that purifying selection has removed part of the mutation load across Indian tiger populations. These results provide genomic evidence for purifying selection in both small and large populations, but also suggest that the remaining deleterious alleles may have inbreeding-associated fitness costs. We suggest that genetic rescue from sources selected based on genome-wide differentiation could offset any possible impacts of inbreeding depression.

High frequency of an otherwise rare phenotype in a small and isolated tiger population.

Most endangered species exist today in small populations, many of which are isolated. Evolution in such populations is largely governed by genetic drift. Empirical evidence for drift affecting striking phenotypes based on substantial genetic data are rare. Approximately 37% of tigers (Panthera tigris) in the Similipal Tiger Reserve (in eastern India) are pseudomelanistic, characterized by wide, merged stripes. Camera trap data across the tiger range revealed the presence of pseudomelanistic tigers only in Similipal. We investigated the genetic basis for pseudomelanism and examined the role of drift in driving this phenotype's frequency. Whole-genome data and pedigree-based association analyses from captive tigers revealed that pseudomelanism cosegregates with a conserved and functionally important coding alteration in Transmembrane Aminopeptidase Q (Taqpep), a gene responsible for similar traits in other felid species. Noninvasive sampling of tigers revealed a high frequency of the Taqpep p.H454Y mutation in Similipal (12 individuals, allele frequency = 0.58) and absence from all other tiger populations (395 individuals). Population genetic analyses confirmed few (minimal number) tigers in Similipal, and its genetic isolation, with poor geneflow. Pairwise FST (0.33) at the mutation site was high but not an outlier. Similipal tigers had low diversity at 81 single nucleotide polymorphisms (mean heterozygosity = 0.28, SD = 0.27). Simulations were consistent with founding events and drift as possible drivers for the observed stark difference of allele frequency. Our results highlight the role of stochastic processes in the evolution of rare phenotypes. We highlight an unusual evolutionary trajectory in a small and isolated population of an endangered species.

Recent Evolutionary History of Tigers Highlights Contrasting Roles of Genetic Drift and Selection.

Species conservation can be improved by knowledge of evolutionary and genetic history. Tigers are among the most charismatic of endangered species and garner significant conservation attention. However, their evolutionary history and genomic variation remain poorly known, especially for Indian tigers. With 70% of the world's wild tigers living in India, such knowledge is critical. We re-sequenced 65 individual tiger genomes representing most extant subspecies with a specific focus on tigers from India. As suggested by earlier studies, we found strong genetic differentiation between the putative tiger subspecies. Despite high total genomic diversity in India, individual tigers host longer runs of homozygosity, potentially suggesting recent inbreeding or founding events, possibly due to small and fragmented protected areas. We suggest the impacts of ongoing connectivity loss on inbreeding and persistence of Indian tigers be closely monitored. Surprisingly, demographic models suggest recent divergence (within the last 20,000 years) between subspecies and strong population bottlenecks. Amur tiger genomes revealed the strongest signals of selection related to metabolic adaptation to cold, whereas Sumatran tigers show evidence of weak selection for genes involved in body size regulation. We recommend detailed investigation of local adaptation in Amur and Sumatran tigers prior to initiating genetic rescue.

The evolutionary history of an accidental model organism, the leopard gecko Eublepharis macularius (Squamata: Eublepharidae).

The leopard gecko, Eublepharis macularius, is a widely used model organism in laboratory and experimental studies. The high phenotypic diversity in the pet trade, the fact that the provenance of different breeding lines is unknown, and that distinct Eublepharis species are known to hybridize, implies that the continued use of E. macularius as a model requires clarity on the origin of the lineages in the pet trade. We combine multi-locus sequence data and the first range-wide sampling of the genus Eublepharis to reconstruct the evolutionary history of the Eublepharidae and Eublepharis, with an updated time-tree for the Eublepharidae. Our sampling includes five of the six recognized species and additional nominal taxa of uncertain status comprising 43 samples from 34 localities plus 48 pet-trade samples. The Eublepharidae began diversifying in the Cretaceous. Eublepharis split from its sister genera in Africa in the Palaeocene-Eocene, and began diversifying in the Oligocene-Miocene, with late Miocene-Pliocene cladogenesis giving rise to extant species. The current species diversity within this group is moderately underestimated. Our species delimitation suggests 10 species with four potentially unnamed divergent lineages in Iran, India and Pakistan. All 30 individuals of E. macularius that we sampled from the pet trade, which include diverse morphotypes, come from a few shallow E. macularius clades, confirming that lab and pet trade strains are part of a single taxon. One of the wild-caught haplotypes of E. macularius, from near Karachi, Pakistan, is identical to (10) pet-trade samples and all other captive populations are closely related to wild-caught animals from central/southern Pakistan (0.1-0.5 % minimum pairwise uncorrected ND2 sequence divergence).

Recapitulating whole genome based population genetic structure for Indian wild tigers through an ancestry informative marker panel.

Identification of genetic structure within wildlife populations have implications in their conservation and management. Accurately inferring population genetic structure requires whole-genome data across the geographical range of the species, which can be resource-intensive. A cheaper strategy is to employ a subset of markers that can efficiently recapitulate the population genetic structure inferred by the whole genome data. Such ancestry informative markers (AIMs), have rarely been developed for endangered species such as tigers utilizing single nucleotide polymorphisms (SNPs). Here, we first identify the population structure of the Indian tiger using whole-genome sequences and then develop an AIMs panel with a minimum number of SNPs that can recapitulate this structure. We identified four population clusters of Indian tigers with North-East, North-West, and South Indian tigers forming three separate groups, and Terai and Central Indian tigers forming a single cluster. To evaluate the robustness of our AIMs, we applied it to a separate dataset of tigers from across India. Out of 92 SNPs present in our AIMs panel, 49 were present in the new dataset. These 49 SNPs were sufficient to recapitulate the population genetic structure obtained from the whole genome data. To the best of our knowledge, this is the first-ever SNP-based AIMs panel for big cats, which can be used as a cost-effective alternative to whole-genome sequencing for detecting the biogeographical origin of Indian tigers. Our study can be used as a guideline for developing an AIMs panel for the management of other endangered species where obtaining whole genome sequences are difficult.

Synthesizing habitat connectivity analyses of a globally important human-dominated tiger-conservation landscape.

As ecological data and associated analyses become more widely available, synthesizing results for effective communication with stakeholders is essential. In the case of wildlife corridors, managers in human-dominated landscapes need to identify both the locations of corridors and multiple stakeholders for effective oversight. We synthesized 5 independent studies of tiger (Panthera tigris) connectivity in central India, a global priority landscape for tiger conservation, to quantify agreement on landscape permeability for tiger movement and potential movement pathways. We used the latter analysis to identify connectivity areas on which studies agreed and stakeholders associated with these areas to determine relevant participants in corridor management. Three or more of the 5 studies' resistance layers agreed in 63% of the study area. Areas in which all studies agree on resistance were of primarily low (66%, e.g., forest) and high (24%, e.g., urban) resistance. Agreement was lower in intermediate resistance areas (e.g., agriculture). Despite these differences, the studies largely agreed on areas with high levels of potential movement: >40% of high average (top 20%) current-flow pixels were also in the top 20% of current-flow agreement pixels (measured by low variation), indicating consensus connectivity areas (CCAs) as conservation priorities. Roughly 70% of the CCAs fell within village administrative boundaries, and 100% overlapped forest department management boundaries, suggesting that people live and use forests within these priority areas. Over 16% of total CCAs' area was within 1 km of linear infrastructure (437 road, 170 railway, 179 transmission line, and 339 canal crossings; 105 mines within 1 km of CCAs). In 2019, 78% of forest land diversions for infrastructure and mining in Madhya Pradesh (which comprises most of the study region) took place in districts with CCAs. Acute competition for land in this landscape with globally important wildlife corridors calls for an effective comanagement strategy involving local communities, forest departments, Appendix 1 and infrastructure planners. This article is protected by copyright. All rights reserved.

Global Commitments to Conserving and Monitoring Genetic Diversity Are Now Necessary and Feasible.

Global conservation policy and action have largely neglected protecting and monitoring genetic diversity-one of the three main pillars of biodiversity. Genetic diversity (diversity within species) underlies species' adaptation and survival, ecosystem resilience, and societal innovation. The low priority given to genetic diversity has largely been due to knowledge gaps in key areas, including the importance of genetic diversity and the trends in genetic diversity change; the perceived high expense and low availability and the scattered nature of genetic data; and complicated concepts and information that are inaccessible to policymakers. However, numerous recent advances in knowledge, technology, databases, practice, and capacity have now set the stage for better integration of genetic diversity in policy instruments and conservation efforts. We review these developments and explore how they can support improved consideration of genetic diversity in global conservation policy commitments and enable countries to monitor, report on, and take action to maintain or restore genetic diversity.

Coalescent Theory of Migration Network Motifs.

Natural populations display a variety of spatial arrangements, each potentially with a distinctive impact on genetic diversity and genetic differentiation among subpopulations. Although the spatial arrangement of populations can lead to intricate migration networks, theoretical developments have focused mainly on a small subset of such networks, emphasizing the island-migration and stepping-stone models. In this study, we investigate all small network motifs: the set of all possible migration networks among populations subdivided into at most four subpopulations. For each motif, we use coalescent theory to derive expectations for three quantities that describe genetic variation: nucleotide diversity, FST, and half-time to equilibrium diversity. We describe the impact of network properties on these quantities, finding that motifs with a high mean node degree have the largest nucleotide diversity and the longest time to equilibrium, whereas motifs with low density have the largest FST. In addition, we show that the motifs whose pattern of variation is most strongly influenced by loss of a connection or a subpopulation are those that can be split easily into disconnected components. We illustrate our results using two example data sets-sky island birds of genus Sholicola and Indian tigers-identifying disturbance scenarios that produce the greatest reduction in genetic diversity; for tigers, we also compare the benefits of two assisted gene flow scenarios. Our results have consequences for understanding the effect of geography on genetic diversity, and they can assist in designing strategies to alter population migration networks toward maximizing genetic variation in the context of conservation of endangered species.

Transcriptome analysis reveals plasticity in gene regulation due to environmental cues in Primula sikkimensis, a high altitude plant species.

BACKGROUND: Studying plasticity in gene expression in natural systems is crucial, for predicting and managing the effects of climate change on plant species. To understand the contribution of gene expression level variations to abiotic stress compensation in a Himalaya plant (Primula sikkimensis), we carried out a transplant experiment within (Ambient), and beyond (Below Ambient and Above Ambient) the altitudinal range limit of species. We sequenced nine transcriptomes (three each from each altitudinal range condition) using Illumina sequencing technology. We compared the fitness variation of transplants among three transplant conditions. RESULTS: A large number of significantly differentially expressed genes (DEGs) between below ambient versus ambient (109) and above ambient versus ambient (85) were identified. Transcripts involved in plant growth and development were mostly up-regulated in below ambient conditions. Transcripts involved in signalling, defence, and membrane transport were mostly up-regulated in above ambient condition. Pathway analysis revealed that most of the genes involved in metabolic processes, secondary metabolism, and flavonoid biosynthesis were differentially expressed in below ambient conditions, whereas most of the genes involved in photosynthesis and plant hormone signalling were differentially expressed in above ambient conditions. In addition, we observed higher reproductive fitness in transplant individuals at below ambient condition compared to above ambient conditions; contrary to what we expect from the cold adaptive P. sikkimensis plants. CONCLUSIONS: We reveal P. sikkimensis's capacity for rapid adaptation to climate change through transcriptome variation, which may facilitate the phenotypic plasticity observed in morphological and life history traits. The genes and pathways identified provide a genetic resource for understanding the temperature stress (both the hot and cold stress) tolerance mechanism of P. sikkimensis in their natural environment.

Geographical and host species barriers differentially affect generalist and specialist parasite community structure in a tropical sky-island archipelago.

Understanding why some parasites emerge in novel host communities while others do not has broad implications for human and wildlife health. In the case of haemosporidian blood parasites, epidemic wild bird mortalities on oceanic islands have been linked to Plasmodium spp., but not genera like Haemoproteus. Indeed, Haemoproteus is absent from many oceanic islands. By contrast, birds on continental islands share long coevolutionary histories with both Plasmodium and Haemoproteus, and are thus ideal model systems to elucidate eco-evolutionary endpoints associated with these parasites in oceanic islands. Here, we examine eco-evolutionary dynamics of avian haemosporidian in the Shola sky-island archipelago of the Western Ghats, India. Our analyses reveal that compared to Plasmodium, Haemoproteus lineages were highly host-specific and diversified via co-speciation with their hosts. We show that community structure of host-generalist Plasmodium was primarily driven by geographical factors (e.g. biogeographic barriers), while that of host-specialist Haemoproteus was driven by host species barriers (e.g. phylogenetic distance). Consequently, a few host species can harbour a high diversity of Plasmodium lineages which, in turn, are capable of infecting multiple host species. These two mechanisms can act in concert to increase the risk of introduction, establishment, and emergence of novel Plasmodium lineages in island systems.

Two new genera of songbirds represent endemic radiations from the Shola Sky Islands of the Western Ghats, India.

BACKGROUND: A long-standing view of Indian biodiversity is that while rich in species, there are few endemics or in-situ radiations within the subcontinent. One exception is the Western Ghats biodiversity hotspot, an isolated mountain range with many endemic species. Understanding the origins of the montane-restricted species is crucial to illuminate both taxonomic and environmental history. RESULTS: With evidence from genetic, morphometric, song, and plumage data, we show that two songbird lineages endemic to the Western Ghats montane forest each have diversified into multiple distinct species. Historically labeled as single species of widespread Asian genera, these two lineages are highly divergent and do not group with the taxa in which they were previously classified but rather are distinct early divergences in larger Asian clades of flycatchers and babblers. Here we designated two new genera, the Western Ghats shortwings as Sholicola and the laughingthrushes as Montecincla, and evaluated species-limits to reflect distinct units by revising six previously named taxa and describing one novel species. Divergence dating showed that both these montane groups split from their Himalayan relatives during the Miocene, which is coincident with a shift towards arid conditions that fragmented the previously contiguous humid forest across peninsular India and isolated these lineages in the Western Ghats. Furthermore, these two genera showed congruent patterns of diversification across the Western Ghats Sky Islands, coincident with other climatic changes. CONCLUSION: Our study reveals the existence of two independent endemic radiations in the high montane Western Ghats or Shola Sky Islands with coincident divergence times, highlighting the role of climate in the diversification of these ancient lineages. The endemic and highly divergent nature of these previously unrecognized species underscores the dearth of knowledge about the biogeography of the Asian tropics, even for comparatively well-known groups such as birds. The substantial increase in the diversity of this region underscores the need for more rigorous systematic analysis to inform biodiversity studies and conservation efforts.

Genetics, morphology and ecology reveal a cryptic pika lineage in the Sikkim Himalaya.

Asian pika species are morphologically ∼similar and have overlapping ranges. This leads to uncertainty and species misidentification in the field. Phylogenetic analyses of such misidentified samples leads to taxonomic ambiguity. The ecology of many pika species remains understudied, particularly in the Himalaya, where sympatric species could be separated by elevation and/or substrate. We sampled, measured, and acquired genetic data from pikas in the Sikkim Himalaya. Our analyses revealed a cryptic lineage, Ochotona sikimaria, previously reported as a subspecies of O. thibetana. The results support the elevation of this lineage to the species level, as it is genetically divergent from O. thibetana, as well as sister species, O. cansus (endemic to central China) and O. curzoniae (endemic to the Tibetan plateau). The Sikkim lineage diverged from its sister species' about 1.7-0.8myrago, coincident with uplift events in the Himalaya. Our results add to the recent spate of cryptic diversity identified from the eastern Himalaya and highlight the need for further study within the Ochotonidae.

Genetic Structure and Phylogeography of the Leopard Cat (Prionailurus bengalensis) Inferred from Mitochondrial Genomes.

The Leopard cat Prionailurus bengalensis is a habitat generalist that is widely distributed across Southeast Asia. Based on morphological traits, this species has been subdivided into 12 subspecies. Thus far, there have been few molecular studies investigating intraspecific variation, and those had been limited in geographic scope. For this reason, we aimed to study the genetic structure and evolutionary history of this species across its very large distribution range in Asia. We employed both PCR-based (short mtDNA fragments, 94 samples) and high throughput sequencing based methods (whole mitochondrial genomes, 52 samples) on archival, noninvasively collected and fresh samples to investigate the distribution of intraspecific genetic variation. Our comprehensive sampling coupled with the improved resolution of a mitochondrial genome analyses provided strong support for a deep split between Mainland and Sundaic Leopard cats. Although we identified multiple haplogroups within the species' distribution, we found no matrilineal evidence for the distinction of 12 subspecies. In the context of Leopard cat biogeography, we cautiously recommend a revision of the Prionailurus bengalensis subspecific taxonomy: namely, a reduction to 4 subspecies (2 mainland and 2 Sundaic forms).

Genome-wide data reveal cryptic diversity and genetic introgression in an Oriental cynopterine fruit bat radiation.

BACKGROUND: The Oriental fruit bat genus Cynopterus, with several geographically overlapping species, presents an interesting case study to evaluate the evolutionary significance of coexistence versus isolation. We examined the morphological and genetic variability of congeneric fruit bats Cynopterus sphinx and C. brachyotis using 405 samples from two natural contact zones and 17 allopatric locations in the Indian subcontinent; and investigated the population differentiation patterns, evolutionary history, and the possibility of cryptic diversity in this species pair. RESULTS: Analysis of microsatellites, cytochrome b gene sequences, and restriction digestion based genome-wide data revealed that C. sphinx and C. brachyotis do not hybridize in contact zones. However, cytochrome b gene sequences and genome-wide SNP data helped uncover a cryptic, hitherto unrecognized cynopterine lineage in northeastern India coexisting with C. sphinx. Further analyses of shared variation of SNPs using Patterson's D statistics suggest introgression between this lineage and C. sphinx. Multivariate analyses of morphology using genetically classified grouping confirmed substantial morphological overlap between C. sphinx and C. brachyotis, specifically in the high elevation contact zones in southern India. CONCLUSION: Our results uncover novel diversity and detect a pattern of genetic introgression in a cryptic radiation of bats, demonstrating the complicated nature of lineage diversification in this poorly understood taxonomic group. Our results highlight the importance of genome-wide data to study evolutionary processes of morphologically similar species pairs. Our approach represents a significant step forward in evolutionary research on young radiations of non-model species that may retain the ability of interspecific gene flow.

The Mycobacterium tuberculosis protein pair PE9 (Rv1088)-PE10 (Rv1089) forms heterodimers and induces macrophage apoptosis through Toll-like receptor 4.

Toll-like receptor (TLR)-mediated interactions of Mycobacterium tuberculosis (M. tb) with macrophages are major determinant in the outcome of innate immune defence and subsequent adaptive immune responses. Here we report a novel interaction of the M. tb protein pair PE9 (Rv1088)-PE10 (Rv1089) with the macrophage TLR4 leading to apoptosis and modulation of cytokine levels. We demonstrate that the two proteins physically interact, and that PE9 is required for the cell wall localization of PE10 in Mycobacterium smegmatis. Interaction of the PE9-PE10 complex with TLR4 in THP-1 macrophages was associated with increased levels of phospho-IRF-3, which correlated with an increase in transcript levels of its target gene interferon-ß. THP-1 macrophages treated with PE9-PE10 complex showed multiple hallmarks of apoptosis and modulation of interleukin (IL)-1b and IL-10 levels. All of these effects were abrogated when cells were treated either with an antibody to PE10 or an anti-TLR4 antibody, indicating that the complex specifically interacts with TLR4 through PE10, establishing this protein pair as a TLR4 ligand. This novel observation of two proline-glutamate (PE) proteins forming functional heterodimers represents a considerable expansion of the PE_PPE repertoire in the context of receptor engagement and the concomitant modulation of host responses by this unique class of proteins.

Higher speciation and lower extinction rates influence mammal diversity gradients in Asia.

BACKGROUND: Little is known about the patterns and correlates of mammal diversity gradients in Asia. In this study, we examine patterns of species distributions and phylogenetic diversity in Asia and investigate if the observed diversity patterns are associated with differences in diversification rates between the tropical and non-tropical regions. We used species distribution maps and phylogenetic trees to generate species and phylogenetic diversity measures for 1° × 1° cells across mainland Asia. We constructed lineage-through-time plots and estimated diversification shift-times to examine the temporal patterns of diversifications across orders. Finally, we tested if the observed gradients in Asia could be associated with geographical differences in diversification rates across the tropical and non-tropical biomes. We estimated speciation, extinction and dispersal rates across these two regions for mammals, both globally and for Asian mammals. RESULTS: Our results demonstrate strong latitudinal and longitudinal gradients of species and phylogenetic diversity with Southeast Asia and the Himalayas showing highest diversity. Importantly, our results demonstrate that differences in diversification (speciation, extinction and dispersal) rates between the tropical and the non-tropical biomes influence the observed diversity gradients globally and in Asia. For the first time, we demonstrate that Asian tropics act as both cradles and museums of mammalian diversity. CONCLUSIONS: Temporal and spatial variation in diversification rates across different lineages of mammals is an important correlate of species diversity gradients observed in Asia.

Deep and wide valleys drive nested phylogeographic patterns across a montane bird community.

Montane species distributions interrupted by valleys can lead to range fragmentation, differentiation and ultimately speciation. Paleoclimatic fluctuations may accentuate or reduce such diversification by temporally altering the extent of montane habitat and may affect species differentially. We examined how an entire montane bird community of the Western Ghats--a linear, coastal tropical mountain range--responds to topographic valleys that host different habitats. Using genetic data from 23 species (356 individuals) collected across nine locations, we examined if different species in the community reveal spatial concordance in population differentiation, and whether the timing of these divergences correlate with climatic events. Our results reveal a nested effect of valleys, with several species (10 of 23) demonstrating the oldest divergences associated with the widest and deepest valley in the mountain range, the Palghat Gap. Further, a subset of these 10 species revealed younger divergences across shallower, narrower valleys. We recovered discordant divergence times for all valley-affected montane birds, mostly in the Pleistocene, supporting the Pliestocene-pump hypotheses and highlighting the role of climatic fluctuations during this period in driving species evolution. A majority of species remain unaffected by valleys, perhaps owing to geneflow or extinction-recolonization dynamics. Studying almost the entire community allowed us to uncover a range of species' responses, including some generalizable and other unpredicted patterns.

Islands within islands: two montane palaeo-endemic birds impacted by recent anthropogenic fragmentation.

Anthropogenic habitat fragmentation of species that live in naturally patchy metapopulations such as mountaintops or sky islands experiences two levels of patchiness. Effects of such multilevel patchiness on species have rarely been examined. Metapopulation theory suggests that patchy habitats could have varied impacts on persistence, dependent on differential migration. It is not known whether montane endemic species, evolutionarily adapted to natural patchiness, are able to disperse between anthropogenic fragments at similar spatial scales as natural patches. We investigated historic and contemporary gene flow between natural and anthropogenic patches across the distribution range of a Western Ghats sky-island-endemic bird species complex. Data from 14 microsatellites for 218 individuals detected major genetic structuring by deep valleys, including one hitherto undescribed barrier. As expected, we found strong effects of historic genetic differentiation across natural patches, but not across anthropogenic fragments. Contrastingly, contemporary differentiation (D(PS)) was higher relative to historic differentiation (F(ST)) in anthropogenic fragments, despite the species' ability to historically traverse shallow valleys. Simulations of recent isolation resulted in high D(PS)/F(ST) values, confirming recent isolation in Western Ghats anthropogenic fragments and also suggesting that this ratio can be used to identifying recent fragmentation in the context of historic connectedness. We suggest that in this landscape, in addition to natural patchiness affecting population connectivity, anthropogenic fragmentation additionally impacts connectivity, making anthropogenic fragments akin to islands within natural islands of montane habitat, a pattern that may be recovered in other sky-island systems.