Honey bees and bumble bees occupying the same landscape have distinct gut microbiomes and amplicon sequence variant-level responses to infections.

The gut microbiome of bees is vital for the health of their hosts. Given the ecosystem functions performed by bees, and the declines faced by many species, it is important to improve our understanding of the amount of natural variation in the gut microbiome, the level of sharing of bacteria among co-occurring species (including between native and non-native species), and how gut communities respond to infections. We conducted 16S rRNA metabarcoding to discern the level of microbiome similarity between honey bees (Apis mellifera, N = 49) and bumble bees (Bombus spp., N = 66) in a suburban-rural landscape. We identified a total of 233 amplicon sequence variants (ASVs) and found simple gut microbiomes dominated by bacterial taxa belonging to Gilliamella, Snodgrassella, and Lactobacillus. The average number of ASVs per species ranged from 4.00-15.00 (8.79 ± 3.84, mean ± SD). Amplicon sequence variant of one bacterial species, G. apicola (ASV 1), was widely shared across honey bees and bumble bees. However, we detected another ASV of G. apicola that was either exclusive to honey bees, or represented an intra-genomic 16S rRNA haplotype variant in honey bees. Other than ASV 1, honey bees and bumble bees rarely share gut bacteria, even ones likely derived from outside environments (e.g., Rhizobium spp., Fructobacillus spp.). Honey bee bacterial microbiomes exhibited higher alpha diversity but lower beta and gamma diversities than those of bumble bees, likely a result of the former possessing larger, perennial hives. Finally, we identified pathogenic or symbiotic bacteria (G. apicola, Acinetobacter sp. and Pluralibacter sp.) that associate with Trypanosome and/or Vairimorpha infections in bees. Such insights help to determine bees' susceptibility to infections should gut microbiomes become disrupted by chemical pollutants and contribute to our understanding of what constitutes a state of dysbiosis.

Minimal sharing of nosematid and trypanosomatid parasites between honey bees and other bees, but extensive sharing of Crithidia between bumble and mason bees.

We document gut parasites in co-occurring Apis, Bombus, and Osmia spp. in the Northern Virginia region, USA. Trypanosomatidea occurred in sixty percent of specimens and 13% carried Nosematidae. We found strong host partitioning: Lotmaria passim and Vairimorpha (Nosema) ceranae predominated in Apis, and Crithidia bombi and V. bombi in Bombus. We did not detect pathogen spread from Apis to Bombus but did detect sharing of C. bombi between Bombus and Osmia, higher parasite levels in Apis at sites with apiaries, and clustering of Vairimopha infection. Given the presence of C. bombi in Osmia, we suggest disease sharing across taxa be monitored.

Maintenance of local adaptation despite gene flow in a coastal songbird.

Adaptation to local environments is common in widespread species and the basis of ecological speciation. The song sparrow (Melospiza melodia) is a widespread, polytypic passerine that occurs in shrubland habitats throughout North America. We examined the population structure of two parapatric subspecies that inhabit different environments: the Atlantic song sparrow (M. m. atlantica), a coastal specialist, and the eastern song sparrow (M. m. melodia), a shrubland generalist. These populations lacked clear mitochondrial population structure, yet coastal birds formed a distinct nuclear genetic cluster. We found weak overall genomic differentiation between these subspecies, suggesting either recent divergence, extensive gene flow, or a combination thereof. There was a steep genetic cline at the transition to coastal habitats, consistent with isolation by environment, not isolation by distance. A phenotype under divergent selection, bill size, varied with the amount of coastal ancestry in transitional areas, but larger bill size was maintained in coastal habitats regardless of ancestry, further supporting a role for selection in the maintenance of these subspecies. Demographic modeling suggested a divergence history of limited gene flow followed by secondary contact, which has emerged as a common theme in adaptive divergence across taxa.

Clinical health issues, reproductive hormones, and metabolic hormones associated with gut microbiome structure in African and Asian elephants.

BACKGROUND: The gut microbiome is important to immune health, metabolism, and hormone regulation. Understanding host-microbiome relationships in captive animals may lead to mediating long term health issues common in captive animals. For instance, zoo managed African elephants (Loxodonta africana) and Asian elephants (Elephas maximus) experience low reproductive rates, high body condition, and gastrointestinal (GI) issues. We leveraged an extensive collection of fecal samples and health records from the Elephant Welfare Study conducted across North American zoos in 2012 to examine the link between gut microbiota and clinical health issues, reproductive hormones, and metabolic hormones in captive elephants. We quantified gut microbiomes of 69 African and 48 Asian elephants from across 50 zoos using Illumina sequencing of the 16S rRNA bacterial gene. RESULTS: Elephant species differed in microbiome structure, with African elephants having lower bacterial richness and dissimilar bacterial composition from Asian elephants. In both species, bacterial composition was strongly influenced by zoo facility. Bacterial richness was lower in African elephants with recent GI issues, and richness was positively correlated with metabolic hormone total triiodothyronine (total T3) in Asian elephants. We found species-specific associations between gut microbiome composition and hormones: Asian elephant gut microbiome composition was linked to total T3 and free thyroxine (free T4), while fecal glucocorticoid metabolites (FGM) were linked to African elephant gut microbiome composition. We identified many relationships between bacterial relative abundances and hormone concentrations, including Prevotella spp., Treponema spp., and Akkermansia spp. CONCLUSIONS: We present a comprehensive assessment of relationships between the gut microbiome, host species, environment, clinical health issues, and the endocrine system in captive elephants. Our results highlight the combined significance of host species-specific regulation and environmental effects on the gut microbiome between two elephant species and across 50 zoo facilities. We provide evidence of clinical health issues, reproductive hormones, and metabolic hormones associated with the gut microbiome structure of captive elephants. Our findings establish the groundwork for future studies to investigate bacterial function or develop tools (e.g., prebiotics, probiotics, dietary manipulations) suitable for conservation and zoo management.

Sexual Selection and Introgression in Avian Hybrid Zones: Spotlight on Manacus.

Hybrid zones offer a window into the processes and outcomes of evolution, from species formation or fusion to genomic underpinnings of specific traits and isolating mechanisms. Sexual selection is believed to be an important factor in speciation processes, and hybrid zones present special opportunities to probe its impact. The manakins (Aves, Pipridae) are a promising group in which to study the interplay of sexual selection and natural hybridization: they show substantial variation across the family in the strength of sexual selection they experience, they readily hybridize within and between genera, and they appear to have formed hybrid species, a rare event in birds. A hybrid zone between two manakins in the genus Manacus is unusual in that plumage and behavioral traits of one species have introgressed asymmetrically into populations of the second species through positive sexual selection, then apparently stalled at a river barrier. This is one of a handful of documented examples of asymmetric sexual trait introgression with a known selective mechanism. It offers opportunities to examine reproductive isolation, introgression, plumage color evolution, and natural factors enhancing or constraining the effects of sexual selection in real time. Here, we review previous work in this system, propose new hypotheses for observed patterns, and recommend approaches to test them.

Gut Microbiomes Differ Among Dietary Types and Stool Consistency in the Captive Red Wolf (Canis rufus).

Captive management of many wildlife species can be challenging, with individuals displaying health disorders that are not generally described in the wild population. Retrospective studies have identified gastrointestinal (GI) diseases, in particular inflammatory bowel disease (IBD), as the second leading cause of captive adult red wolf (Canis rufus) mortality. Recent molecular studies show that imbalanced gut microbial composition is tightly linked to IBD in the domestic dog. The goal of the present study was to address two main questions: (1) how do red wolf gut microbiomes differ between animals with loose stool consistency, indicative of GI issues, and those with normal stool consistency and (2) how does dietary type relate to stool consistency and red wolf gut microbiomes? Fresh fecal samples were collected from 48 captive wolves housed in eight facilities in the United States and from two wild wolves living in Alligator River National Wildlife Refuge, NC, United States. For each individual, the stool consistency was categorized as loose or normal using a standardized protocol and their diet was categorized as either wild, whole meat, a mix of whole meat and kibble or kibble. We characterized gut microbiome structure using 16S rRNA gene amplicon sequencing. We found that red wolves with a loose stool consistency differed in composition than wolves with normal stool consistency, suggesting a link between GI health and microbiome composition. Diet was not related to stool consistency but did significantly impact gut microbiome composition; gut microbiome composition of wolves fed a kibble diet were significantly different than the gut microbiome composition of wolves fed a mixed, whole meat and wild diet. Findings from this study increase the understanding of the interplay between diet and GI health in the red wolf, a critical piece of information needed to maintain a healthy red wolf population ex situ.

Little genetic structure in a Bornean endemic small mammal across a steep ecological gradient.

Janzen's influential "mountain passes are higher in the tropics" hypothesis predicts restricted gene flow and genetic isolation among populations spanning elevational gradients in the tropics. Few studies have tested this prediction, and studies that focus on population genetic structure in Southeast Asia are particularly underrepresented in the literature. Here, we test the hypothesis that mountain treeshrews (Tupaia montana) exhibit limited dispersal across their broad elevational range which spans ~2,300 m on two peaks in Kinabalu National Park (KNP) in Borneo: Mt Tambuyukon (MT) and Mt Kinabalu (MK). We sampled 83 individuals across elevations on both peaks and performed population genomics analyses on mitogenomes and single nucleotide polymorphisms from 4,106 ultraconserved element loci. We detected weak genetic structure and infer gene flow both across elevations and between peaks. We found higher genetic differentiation on MT than MK despite its lower elevation and associated environmental variation. This implies that, contrary to our hypothesis, genetic structure in this system is not primarily shaped by elevation. We propose that this pattern may instead be the result of historical processes and limited upslope gene flow on MT. Importantly, our results serve as a foundational estimate of genetic diversity and population structure from which to track potential future effects of climate change on mountain treeshrews in KNP, an important conservation stronghold for the mountain treeshrew and other montane species.

Opening the door to greater phylogeographic inference in Southeast Asia: Comparative genomic study of five codistributed rainforest bird species using target capture and historical DNA.

Indochina and Sundaland are biologically diverse, interconnected regions of Southeast Asia with complex geographic histories. Few studies have examined phylogeography of bird species that span the two regions because of inadequate population sampling. To determine how geographic barriers/events and disparate dispersal potential have influenced the population structure, gene flow, and demographics of species that occupy the entire area, we studied five largely codistributed rainforest bird species: Arachnothera longirostra, Irena puella, Brachypodius atriceps, Niltava grandis, and Stachyris nigriceps. We accomplished relatively thorough sampling and data collection by sequencing ultraconserved elements (UCEs) using DNA extracted from modern and older (historical) specimens. We obtained a genome-wide set of 753-4,501 variable loci and 3,919-18,472 single nucleotide polymorphisms. The formation of major within-species lineages occurred within a similar span of time (0.5-1.5 mya). Major patterns in population genetic structure are largely consistent with the dispersal potential and habitat requirements of the study species. A population break across the Isthmus of Kra was shared only by the two hill/submontane insectivores (N. grandis and S. nigriceps). Across Sundaland, there is little structure in B. atriceps, which is a eurytopic and partially frugivorous species that often utilizes forest edges. Two other eurytopic species, A. longirostra and I. puella, possess highly divergent populations in peripheral Sunda Islands (Java and/or Palawan) and India. These species probably possess intermediate dispersal abilities that allowed them to colonize new areas, and then remained largely isolated subsequently. We also observed an east-west break in Indochina that was shared by B. atriceps and S. nigriceps, species with very different habitat requirements and dispersal potential. By analyzing high-throughput DNA data, our study provides an unprecedented comparative perspective on the process of avian population divergence across Southeast Asia, a process that is determined by geography, species characteristics, and the stochastic nature of dispersal and vicariance events.

Genomics-based re-examination of the taxonomy and phylogeny of human and simian Mastadenoviruses: an evolving whole genomes approach, revealing putative zoonosis, anthroponosis, and amphizoonosis.

With the advent of high-resolution and cost-effective genomics and bioinformatics tools and methods contributing to a large database of both human (HAdV) and simian (SAdV) adenoviruses, a genomics-based re-evaluation of their taxonomy is warranted. Interest in these particular adenoviruses is growing in part due to the applications of both in gene transfer protocols, including gene therapy and vaccines, as well in oncolytic protocols. In particular, the re-evaluation of SAdVs as appropriate vectors in humans is important as zoonosis precludes the assumption that human immune system may be naïve to these vectors. Additionally, as important pathogens, adenoviruses are a model organism system for understanding viral pathogen emergence through zoonosis and anthroponosis, particularly among the primate species, along with recombination, host adaptation, and selection, as evidenced by one long-standing human respiratory pathogen HAdV-4 and a recent re-evaluation of another, HAdV-76. The latter reflects the insights on amphizoonosis, defined as infections in both directions among host species including "other than human", that are possible with the growing database of nonhuman adenovirus genomes. HAdV-76 is a recombinant that has been isolated from human, chimpanzee, and bonobo hosts. On-going and potential impacts of adenoviruses on public health and translational medicine drive this evaluation of 174 whole genome sequences from HAdVs and SAdVs archived in GenBank. The conclusion is that rather than separate HAdV and SAdV phylogenetic lineages, a single, intertwined tree is observed with all HAdVs and SAdVs forming mixed clades. Therefore, a single designation of "primate adenovirus" (PrAdV) superseding either HAdV and SAdV is proposed, or alternatively, keeping HAdV for human adenovirus but expanding the SAdV nomenclature officially to include host species identification as in ChAdV for chimpanzee adenovirus, GoAdV for gorilla adenovirus, BoAdV for bonobo adenovirus, and ad libitum.

Publisher Correction: Spatial Organization of the Gastrointestinal Microbiota in Urban Canada Geese.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Spatial Organization of the Gastrointestinal Microbiota in Urban Canada Geese.

Recent reviews identified the reliance on fecal or cloacal samples as a significant limitation hindering our understanding of the avian gastrointestinal (gut) microbiota and its function. We investigated the microbiota of the esophagus, duodenum, cecum, and colon of a wild urban population of Canada goose (Branta canadensis). From a population sample of 30 individuals, we sequenced the V4 region of the 16S SSU rRNA on an Illumina MiSeq and obtained 8,628,751 sequences with a median of 76,529 per sample. These sequences were assigned to 420 bacterial OTUs and a single archaeon. Firmicutes, Proteobacteria, and Bacteroidetes accounted for 90% of all sequences. Microbiotas from the four gut regions differed significantly in their richness, composition, and variability among individuals. Microbial communities of the esophagus were the most distinctive whereas those of the colon were the least distinctive, reflecting the physical downstream mixing of regional microbiotas. The downstream mixing of regional microbiotas was also responsible for the majority of observed co-occurrence patterns among microbial families. Our results indicate that fecal and cloacal samples inadequately represent the complex patterns of richness, composition, and variability of the gut microbiota and obscure patterns of co-occurrence of microbial lineages.

High-throughput SNP genotyping of historical and modern samples of five bird species via sequence capture of ultraconserved elements.

Sample availability limits population genetics research on many species, especially taxa from regions with high diversity. However, many such species are well represented in museum collections assembled before the molecular era. Development of techniques to recover genetic data from these invaluable specimens will benefit biodiversity science. Using a mixture of freshly preserved and historical tissue samples, and a sequence capture probe set targeting >5000 loci, we produced high-confidence genotype calls on thousands of single nucleotide polymorphisms (SNPs) in each of five South-East Asian bird species and their close relatives (N = 27-43). On average, 66.2% of the reads mapped to the pseudo-reference genome of each species. Of these mapped reads, an average of 52.7% was identified as PCR or optical duplicates. We achieved deeper effective sequencing for historical samples (122.7×) compared to modern samples (23.5×). The number of nucleotide sites with at least 8× sequencing depth was high, with averages ranging from 0.89 × 10(6)  bp (Arachnothera, modern samples) to 1.98 × 10(6)  bp (Stachyris, modern samples). Linear regression revealed that the amount of sequence data obtained from each historical sample (represented by per cent of the pseudo-reference genome recovered with ≥8× sequencing depth) was positively and significantly (P ≤ 0.013) related to how recently the sample was collected. We observed characteristic post-mortem damage in the DNA of historical samples. However, we were able to reduce the error rate significantly by truncating ends of reads during read mapping (local alignment) and conducting stringent SNP and genotype filtering.

Test of the invasive pathogen hypothesis of bumble bee decline in North America.

Emergent fungal diseases are critical factors in global biodiversity declines. The fungal pathogenNosema bombiwas recently found to be widespread in declining species of North American bumble bees (Bombus), with circumstantial evidence suggesting an exotic introduction from Europe. This interpretation has been hampered by a lack of knowledge of global genetic variation, geographic origin, and changing prevalence patterns ofN. bombiin declining North American populations. Thus, the temporal and spatial emergence ofN. bombiand its potential role in bumble bee decline remain speculative. We analyzeNosemaprevalence and genetic variation in the United States and Europe from 1980, before an alleged introduction in the early 1990s, to 2011, extractingNosemaDNA fromBombusnatural history collection specimens from across this time period.Nosema bombiprevalence increased significantly from low detectable frequency in the 1980s to significantly higher frequency in the mid- to late-1990s, corresponding to a period of reported massive infectious outbreak ofN. bombiin commercial bumble bee rearing stocks in North America. Despite the increased frequency, we find no conclusive evidence of an exoticN. bombiorigin based on genetic analysis of globalNosemapopulations; the widespreadNosemastrain found currently in declining United States bumble bees was present in the United States before commercial colony trade. Notably, the USN. bombiis not detectably different from that found predominantly throughout Western Europe, with both regions characterized by low genetic diversity compared with high levels of diversity found in Asia, where commercial bee breeding activities are low or nonexistent.

Deep sequencing and ecological characterization of gut microbial communities of diverse bumble bee species.

Gut bacterial communities of bumble bees are correlated with defense against pathogens. Further understanding this host-microbe association is vitally important as bumble bees are currently experiencing global population declines, potentially due in part to emergent diseases. In this study, we used pyrosequencing and community fingerprinting (ARISA) to characterize the gut microbial communities of nine bumble species from across the Bombus phylogeny. Overall, we delimited 74 bacterial taxa (operational taxonomic units or OTUs) belonging to Betaproteobacteria, Gammaproteobacteria, Bacilli, Actinobacteria, Flavobacteria and Alphaproteobacteria. Each bacterial community was taxonomically simple, containing an average of 1.9 common (relative abundance per sample > 5%) bacterial OTUs. The most abundant and prevalent (occurring in 92% of the samples) bacterial OTU, based on 16S rRNA sequences, closely matched that of the previously described Betaproteobacteria species Snodgrassella alvi. Bacteria that were first described in bee-related external environments dominated a number of gut bacterial communities, suggesting that they are not strictly dependent on the internal gut environment. The ARISA data showed a correlation between bacterial community structures and the geographic locations where the bees were sampled, suggesting that at least a subset of the bacterial species may be transmitted environmentally. Using light and fluorescent microscopy, we demonstrated that the gut bacteria form a biofilm on the internal epithelial surface of the ileum, corroborating results obtained from Apis mellifera.

Molecular phylogeny and insular biogeography of the lowland tailorbirds of Southeast Asia (Cisticolidae: Orthotomus).

The lowland tailorbirds of Southeast Asia (Orthotomus) offer an excellent opportunity for comparative biogeography because of their diversity in the Greater Sunda and Philippine islands. We reconstructed the phylogeny of all species in the genus using maximum likelihood, Bayesian, and coalescent methods on DNA sequences of three gene segments: an autosomal intron (TGF), a Z-linked intron (MUSK), and a mitochondrial coding gene (ND2). Although resolution is low in parts of the phylogeny, several well defined clades emerge. When considered in light of distribution, these clades indicate that the Greater Sunda and Philippine islands were occupied early in Orthotomus history by the ancestors of O. sericeus in the Greater Sundas and O. frontalis in the Philippines. Subsequently, tailorbirds diversified further in each island group: O. atrogularis, O. ruficeps, and O. sepium arose in the Greater Sundas, and O. castaneiceps castaneiceps, O. c. chloronotus, O. derbianus, O. samarensis, O. nigriceps, and O. cinereiceps in the Philippines. Among the continental taxa (including Sundaic birds), the older lineages (O. sutorius and O. sericeus) are habitat generalists and the recently evolved taxa are more specialized. In the Philippines, several taxa once considered conspecific with O. atrogularis turn out to be highly divergent species (>9% in ND2). Indeed, all Philippine allospecies are well diverged from one another. This finding supports the recent assertion of higher-than-appreciated bird endemicity in the Philippines.

Multilocus analysis of the evolutionary dynamics of rainforest bird populations in Southeast Asia.

Sundaland has a dynamic geographic history because its landmasses were periodically interconnected when sea levels fell during glacial periods. Superimposed on this geographic dynamism were environmental changes related to climatic oscillations. To investigate how tropical taxa responded to such changes, we studied the divergence and demographic history of two co-distributed rainforest passerine species, Arachnothera longirostra and Malacocincla malaccensis. We sampled birds primarily from Borneo and the Malay Peninsula, which straddle the now-submerged Sunda shelf, and analysed multilocus DNA data with a variety of coalescent and gene genealogy methods. Cross-shelf divergence in both species occurred well before the last glacial maximum, i.e., before the most recent land connection. However, post-divergence gene flow occurred, and it was more pronounced in A. longirostra (a highly vagile nectarivore/insectivore) than in M. malaccensis (an understory insectivore). Despite current habitat continuity on Borneo, the population of M. malaccensis in northeastern Borneo is substantially divergent from that on the rest of the island. The NE population experienced dramatic demographic fluctuations, probably because of competition with the other population, which expanded from western Borneo after the mid-Pleistocene. In contrast, the Borneo population of A. longirostra has little structure and appears to have experienced demographic expansion 16 kya, long after it had diverged from the Malay Peninsula population (630-690 kya). Malay Peninsula populations of both species have remained relatively stable. Overall, the most recent glacial period was not the chief determinant of the evolutionary dynamics of our study species, and in this respect, they are different from temperate species.

Revisiting Wallace's haunt: coalescent simulations and comparative niche modeling reveal historical mechanisms that promoted avian population divergence in the Malay Archipelago.

Sundaland, a biogeographic region of Southeast Asia, is a major biodiversity hotspot. However, little is known about the relative importance of Pleistocene habitat barriers and rivers in structuring populations and promoting diversification here. We sampled 16 lowland rainforest bird species primarily from peninsular Malaysia and Borneo to test the long-standing hypothesis that animals on different Sundaic landmasses intermixed extensively when lower sea-levels during the Last Glacial Maximum (LGM) exposed land-bridges. This hypothesis was rejected in all but five species through coalescent simulations. Furthermore, we detected a range of phylogeographic patterns; Bornean populations are often genetically distinct from each other, despite their current habitat connectivity. Environmental niche modeling showed that the presence of unsuitable habitats between western and eastern Sundaland during the LGM coincided with deeper interpopulation genetic divergences. The location of this habitat barrier had been hypothesized previously based on other evidence. Paleo-riverine barriers are unlikely to have produced such a pattern, but we cannot rule out that they acted with habitat changes to impede population exchanges across the Sunda shelf. The distinctiveness of northeastern Borneo populations may be underlied by a combination of factors such as rivers, LGM expansion of montane forests and other aspects of regional physiography.

Phylogeny and biogeography of the Asian trogons (Aves: Trogoniformes) inferred from nuclear and mitochondrial DNA sequences.

We present the first species-level molecular phylogenetic hypothesis for the Asian trogons, using DNA sequences of multiple mitochondrial and nuclear loci, and Bayesian and maximum likelihood tree reconstruction methods. The two genera of Asian trogons, Harpactes and Apalharpactes, are distantly related to each other. Within the widespread Southeast Asian genus Harpactes, we recovered three species groups: (1) H. oreskios; (2) H. orrhophaeus and H. duvaucelii; and (3) a clade of the seven large-bodied species. Short internal branch lengths link species in the large-bodied group, suggesting rapid diversification. Apalharpactes, which is currently restricted to the montane forests of Sumatra and Java, appears to be a relictual lineage distantly related to all other trogons. Bayesian and maximum likelihood analyses indicate Apalharpactes is sister to the African genus Apaloderma, although this result was not strongly supported. Overall, the extant Asian trogon species appear to have diversified prior to the Pleistocene, based on large pair-wise mitochondrial divergences between taxa.

Molecular phylogenetic analysis of the Grey-cheeked Fulvetta (Alcippe morrisonia) of China and Indochina: a case of remarkable genetic divergence in a "species".

The Grey-cheeked Fulvetta, Alcippe morrisonia, is a polytypic species of Quaker babbler (Timaliidae) occurring mainly in highlands from Burma across southern China to Taiwan. To examine gene flow among populations, we sequenced the mitochondrial ND2 gene of 39 individuals of six of the seven subspecies, plus multiple individuals of three outgroup Alcippe species. A lack of shared haplotypes and high FST values suggested no gene flow among populations. The nucleotide divergence between geographically juxtaposed subspecies ranged from 0.8% between Guangdong and Hainan to 9.4% between Yunnan and Vietnam. Phylogenetic analysis of the populations yielded a well resolved tree with two major clades. One clade consisted of the geographically central subspecies schaefferi and davidi, which are located largely in the "Mid-central" zoogeographic region of China's "Oriental" realm. The other clade, the geographically peripheral group, consisted of all other A. morrisonia subspecies, as well as an erstwhile outgroup, the Mountain Fulvetta (Alcippe peracensis annamensis) from central Vietnam. This peripheral group was further divided into two clades, one consisting of taxa occurring in China's "Southwest" zoogeographic region (fratercula and A. p. annamensis), and one occurring in China's "Southern" region (morrisonia, rufescentior, and hueti). These three geographic and phylogenetic groups represent at least four different species based on plumage differences and genetic differentiation. The phylogeny provides the first avian molecular evidence of area relationships among China's zoogeographic zones. It also highlights a remarkable and unexpected amount of genetic divergence and structure in a Sino-Indian "species". If such diversity occurs in other groups of birds with similar distribution, the ramifications are important for conservation planning.