Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

The critically endangered forest owlet Heteroglaux blewitti is nested within the currently recognized Athene clade: A century-old debate addressed

  • Pankaj Koparde ,

    Roles Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliations Division of Conservation Biology, Sálim Ali Centre for Ornithology & Natural History, Coimbatore, Tamil Nadu, India, Manipal Academy of Higher Education, Madhav Nagar, Manipal, Karnataka, India, Indian Institute of Science Education and Research, Tirupati, Andhra Pradesh, India

  • Prachi Mehta,

    Roles Methodology, Project administration, Resources

    Affiliation Wildlife Research and Conservation Society, Pune, Maharashtra, India

  • Sushma Reddy,

    Roles Investigation, Methodology, Supervision

    Affiliation Loyola University, Chicago, Illinois, United States of America

  • Uma Ramakrishnan,

    Roles Funding acquisition, Project administration, Resources, Software

    Affiliation National Centre for Biological Science, TIFR, Bangalore, Karnataka, India

  • Shomita Mukherjee ,

    Contributed equally to this work with: Shomita Mukherjee, V. V. Robin

    Roles Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing

    Affiliation Division of Conservation Biology, Sálim Ali Centre for Ornithology & Natural History, Coimbatore, Tamil Nadu, India

  • V. V. Robin

    Contributed equally to this work with: Shomita Mukherjee, V. V. Robin

    Roles Conceptualization, Data curation, Funding acquisition, Investigation, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing

    Affiliation Indian Institute of Science Education and Research, Tirupati, Andhra Pradesh, India

The critically endangered forest owlet Heteroglaux blewitti is nested within the currently recognized Athene clade: A century-old debate addressed

  • Pankaj Koparde, 
  • Prachi Mehta, 
  • Sushma Reddy, 
  • Uma Ramakrishnan, 
  • Shomita Mukherjee, 
  • V. V. Robin


Range-restricted species generally have specific niche requirements and may often have unique evolutionary histories. Unfortunately, many of these species severely lack basic research, resulting in poor conservation strategies. The phylogenetic relationship of the Critically Endangered Forest Owlet Heteroglaux blewitti has been the subject of a century-old debate. The current classifications based on non-phylogenetic comparisons of morphology place the small owls of Asia into three genera, namely, Athene, Glaucidium, and Heteroglaux. Based on morphological and anatomical data, H. blewitti has been alternatively hypothesized to belong within Athene, Glaucidium, or its own monotypic genus Heteroglaux. To test these competing hypotheses, we sequenced six loci (~4300 bp data) and performed phylogenetic analyses of owlets. Mitochondrial and nuclear trees were not congruent in their placement of H. blewitti. However, both mitochondrial and nuclear combined datasets showed strong statistical support with high maximum likelihood bootstrap (>/ = 90) and Bayesian posterior probability values (>/ = 0.98) for H. blewitti being nested in the currently recognized Athene group, but not sister to Indian A. brama. The divergence of H. blewitti from its sister taxa was between 4.3 and 5.7 Ma coinciding with a period of drastic climatic changes in the Indian subcontinent. This study presented the first genetic analysis of H. blewitti, a Critically Endangered species, and addressed the long debate on the relationships of the Athene-Heteroglaux-Glaucidium complex. We recommend further studies with more data and complete taxon sampling to understand the biogeography of Indian Athene species.


Endemic and endangered species are often ecologically specialized and there is an urgent need to better understand the ecology and phylogenetic history of endangered species to uncover features that might be crucial for conservation. Such species could be viewed as model species to understand evolutionary processes in the landscape of their occurrence [13]. However, our knowledge of the evolutionary history of highly restricted, endemic species in the tropics is limited, due to their rarity and incomplete genetic sampling [4]. This could have far-reaching consequences on policy level decisions regarding species conservation.

Although the phylogeny of the higher order avian taxa has undergone several changes in the past three decades [57], relationships among clades still remain poorly known. Owls (Order: Strigiformes) is one such groups. Many rare or range-restricted species such as the Critically Endangered Forest Owlet Heteroglaux blewitti (Hume, 1873), Spotted Owlet Athene brama (Temmink, 1821) and Jungle Owlet Glaucidium radiatum (Tickell, 1833) were not included in the most recently published owl phylogenies [810].

H. blewitti, endemic to India, has been a taxonomic mystery since its discovery in 1872. Owing to its severely fragmented distribution and low population, H. blewitti has been categorized as a “Critically Endangered” species by the International Union for Conservation of Nature (IUCN) [11]. There have been many opinions on the phylogenetic affinities of H. blewitti by ornithologists over the centuries. In the past, the species has alternatively been placed in either the genus Heteroglaux [1216] or Athene [1722]. Additionally, some researchers have claimed Heteroglaux to be a subgenus of Glaucidium [23], related to the tail-flicking behavior typical in the genus Glaucidium. Nevertheless, none of these opinions were founded on formal phylogenetic analyses.

The genus Athene is represented by four species—Burrowing Owl A. cunicularia, Spotted Owlet A. brama, Little Owl A. noctua and White-browed Owl A. superciliaris [9, 15]. All the extant Athene members were classified in the genus Strix when first described. Following a revision in taxonomy [17], A. brama and A. noctua were placed in the genus Athene, a placement that remains unchanged to date. A. cunicularia was moved from Strix to Speotyto [24] based on DNA-DNA hybridization studies, and later to Athene [8], based on mitochondrial CYTB and nuclear RAG-1 gene data. Similarly, A. superciliaris was moved from Strix to Ninox [25], and then to Athene [9]. Throughout this article, we refer A. brama and A. noctua as Eurasian Athene (with global distribution encompassing India) and A. superciliaris and A. cunicularia as Athene from Madagascar and the Americas.

Since H. blewitti and A. brama are morphologically similar in appearance [17] and are co-distributed (Fig 1, S1 Fig), they would be expected to form a sister group. Although Wolters (1975) hypothesized that H. blewitti and A. brama together form a subgenus Heteroglaux, nested within Athene [26], he did not provide an explanation for this classification [16]. In contrast, König et al. (1999) argue that the tail flicking behavior, a characteristic of Glaucidium, shown by H. blewitti, suggests that the species is closely related to Glaucidium and could be nested within Athene or Glaucidium [23]. The current classification of H. blewitti in a monotypic genus Heteroglaux claimed by Rasmussen & Collar (2013) is based solely on an assessment of morphological [12, 16] and osteological characteristics [16], without phylogenetic data. This classification needs further scrutiny by incorporating phylogenetic information. Furthermore, a report on the hybridization between H. blewitti and A. brama [27], was disputed [2831], and this underscores the need to examine the taxonomic status of the species. Data available on acoustic [32], morphological, osteological and behavioral characters [16] of H. blewitti show that the species differs from other Athene species in osteological features such as multiple cranial characters, especially, wider, inflated anterior and posterior frontals, larger lacrimals and maxillopalatines, stouter tarsometatarsi, and behavioral features such as non-undulating flight and tail flicking. Rasmussen & Collar (2013) argue that this difference could well be at the genus level [16]. The authors further propose that the plumage similarities in A. brama and H. blewitti could be due to convergence but given the distinct osteological and behavioral features of H. blewitii, another possibility is that H. blewitti evolved from an ancient divergence event separating the genus Heteroglaux from Athene [16].

Fig 1. Co-distributed Indian owlets show plumage similarity, however can be identified based on size and markings on the chest and forehead.

Presence of white spots and brown bars in case of A. brama and G. radiatum respectively are identification keys. Photo credits: color banded H. blewitti individual by PM, A. brama and G. radiatum by PK.

We test three proposed phylogenetic relationships as competing hypotheses—Rasmussen & Collar (2013) [16], König et al. (1999) [23], and Wolters (1975) [26] using molecular data to infer phylogenetic relationships among H. blewitti, A. brama, and G. radiatum. Our study will also address the debate about Athene-Glaucidium-Heteroglaux relationships using genetic data. We also expect that this new phylogenetic information on an endemic and Critically Endangered species will help understand priorities in conservation strategies.

Materials and methods

1. Taxon sampling

Based on data from extant phylogenies [810], we generated data on the three Indian Owlets H. blewitti, A. brama, and G. radiatum as well as the Madagascan species A. superciliaris. We sampled, three out of five subspecies of A. brama namely A. b. brama (North India), A. b. indica (South India) and A. b. albida (Parts of Gujarat, Pakistan, and Iran), and two subspecies of G. radiatum namely G. r. radiatum (Peninsular India) and G. r. malabaricum (Southwest India). The assignment of subspecies was based on distributional limits described in Ali & Ripley (1983) [19]. For field-based sample collection from the three species of owls (H. blewitti, A. brama, and G. radiatum), we followed all legalities and obtained prior permissions from State Forest Departments (Madhya Pradesh, Maharashtra, Gujarat and Chhattisgarh). The Madhya Pradesh Forest Department granted one of the authors (PM) permits to capture and color tag H. blewitii individuals as part of an independent study on the species. The Chhattisgarh Forest Department permitted capture and blood collection, whereas our permits from Maharashtra and Gujarat were limited to visual surveying of the Forest Owlet (H. blewitti). We captured Forest Owlets using Bal-chatri traps, known to be the most effective trapping technique for capturing birds of prey without inflicting injury [33]. The capture and release protocol was reviewed by the Madhya Pradesh State Forest Department’s expert committee before granting the research permit to PM.

For this study, during the capture process, we handled captured owlets for a maximum of twelve minutes and released them immediately after banding and biometrics procedure. We carried out the banding procedure very close to where the owlet was captured so we could release it at the same spot. Once captured, we covered the head of the owlet with a cloth to minimize stress. We collected feathers that were shed during the process of capture and handling from each bird. We collected up to two feathers per individual. We stored the feathers in separate paper envelopes and placed these in airtight containers for transport. Color tagging of birds ensured that we could identify different individuals and sources for the samples. We used these feathers to create reference genetic data for the species. In two instances, we collected broken eggshells fallen below known nest-sites of H. blewitti. We compared genetic data collected from eggshells (S1 Table) with the reference genetic data to identify species. We identified the species using a criterion of ≥99% sequence identity with the reference data. After species assignment, we proceeded with further analysis. We also obtained a museum feather sample of H. blewitti from the Bombay Natural History Society (BNHS) and used the same criteria mentioned above to ensure species identity. In case of A. brama and G. radiatum, we collected fallen feathers below known nest-sites outside Protected Areas or shed feathers from injured bird rescued by NGOs. We trapped G. radiatum individuals in mist nets (in Chhattisgarh). We sampled spatially non-overlapping and distant nest-sites to avoid resampling the same individuals. For both the species, for ensuring species identity, we followed the same approach as for H. blewitti. We sequenced target genes from a vouchered tissue of A. superciliaris obtained from the Field Museum of Natural History (details of samples and sources are provided in S1 Table and S2 Fig).

2. Laboratory procedure

We extracted DNA using DNeasy blood and tissue kit (Qiagen, Hilden, Germany, Product no. 69504), following the manufacturer’s protocol with a few modifications. We added 20 μl of 1% Dithiothreitol (DTT) in the lysis reaction. To ensure a higher concentration of DNA and minimize loss, we eluted DNA twice in separate vials, each time in 100 μl of AE buffer, instead of the recommended one elute of 200 ul and used the first elute for further analysis. We amplified two mitochondrial genes (CYTB, COI), a nuclear exon (RAG-1), and three nuclear introns (TGFB2, LDH, MYO). These genes were chosen based on previously available data on other species of Athene and Glaucidium [8, 9]. We carried out all PCR amplifications after optimizations (S2 Table) of reaction conditions. We sequenced the purified PCR products in both forward and reverse directions with an ABI 3730 Genetic Analyzer and analyzed raw sequences with the ABI 3730 Genetic Analyzer software (Applied Biosystems, Foster City, USA). We designed two owlet specific primers for amplifying COI gene from A. brama and G. radiatum. We used primers for other genes available from published studies [3442]. We submitted all the sequences from the study to GenBank (see S3 Table for accession numbers).

3. Sequence analyses and phylogenetic reconstruction

We viewed and manually edited the sequences in Chromas Lite 2.1.1 (Technelysium, Brisbane, Australia) and aligned them using the software Geneious v7.0.6 (Biomatters, Auckland, New Zealand) [43]. We downloaded sequences of other owls from GenBank (S4 Table). We translated coding sequences in Geneious to check for the presence of stop codons and/or nuclear inserts of mitochondrial DNA (numts). We processed individual gene alignments in MEGA v4.0 [44] for counting the proportion of variable sites, parsimony informative sites, and singletons. We tested for positive selection, to avoid introducing possible error in phylogenetic inference as shown in [45, 46], in CYTB, COI, and RAG-1, using HyPhy [47] and Tajima’s test of neutrality [48] implemented in MEGA v4.0.

We conducted the analyses using three different sets of data—mitochondrial (CYTB + COI), nuclear (RAG1 + TGFB2 + MYO), and concatenated (CYTB + COI + RAG1 + TGFB2 + MYO). There was a missing in-group taxon (A. noctua) in the LDH dataset; therefore, we did not include the dataset in the final combined analysis. The concatenated dataset contained <9% missing data. We used codon-specific DNA substitution models (PartitionFinder v1.1.1, S5 Table) [49]. We tested the separate gene as well as concatenated datasets for best-fit DNA substitution models (Details in S5 Table).

We conducted phylogenetic analysis with maximum likelihood using RaxML v8.0 [50], Bayesian Inference using MrBayes v3.2.2 [51], and multi-species coalescent tree using BEAST [52]. We used members of the Tytonidae family (Tyto alba and Phodilus badius) as outgroup taxa since this is the family closest to the Strigidae family with an estimated known divergence time for the Strigidae / Tytonidae split (54–83 Ma) [5356].

We used PartitionFinder to first determine the best partitioning scheme of gene regions based on evolutionary rates. We used these partitions in RAxML and MrBayes. In RAxML, we used ML+rapid bootstrap function with 10000 bootstraps for all analyses. In MrBayes, we conducted two runs of five chains (one cold) for 30–70 million generations and sampling every 1000th generation. We set the temperatures of the heated chains to 0.25. We discarded the first 25% of samples (burnin) and continued the MCMC run till the standard deviation of split frequency dropped below 0.005.

We ran each ML and Bayesian analysis thrice, to ensure consistency in the results, for the concatenated dataset with the following options—partitioning of the dataset in all codon positions of coding sequences, only the third codon position of coding sequences, and all codon positions for the mitochondrial genes and only the third codon position for the nuclear exon.

We used the concatenated dataset, without LDH data, to build species phylogeny in Beast v1.8.1. The species tree analysis does not take into consideration columns with missing data; hence, we did not include LDH dataset for which A. noctua data was missing. We ran the analysis for 1.5 billion runs. We viewed and edited the trees in FigTree v1.4.2. We also used DensiTree v2.2.5 [57], based on the Bayesian output of BEAST, to plot sets of trees.

To test for congruency in mitochondrial and nuclear datasets, we performed a Shimodaira-Hasegawa test [58] in RaxML. We also conducted gene jack-knifing analysis in which we serially removed individual genes from the concatenated dataset to see which gene/s may influence the phylogenetic analysis [39].

4. Fossil calibrations and molecular dating

Owls have an adequate fossil and sub-fossil record, largely from Europe and North America [59]; however, classification of many of the sub-fossils remains ambiguous [60]. In addition, very few phylogenetic studies of Strigiformes have used molecular dating and there is no consistency in fossil calibrations used. Only fossil calibrations with sufficient support, as discussed in [61, 62], and those that have been used in multiple studies, were used in this study. We used A. otus / O. leucotis (23.7–16.4 Ma) [63] and the oldest Athene fossil (3.6–5.3 Ma) [64] for calibrations. We used different combinations of data (concatenated, mitochondrial, and nuclear, partitioned alignments), to check for consistency in results, to obtain molecular dates after performing tests for a molecular clock [65] in MEGA v4.0 and using both the strict and the uncorrelated relaxed lognormal clocks. Substitution models, clock models and trees option were set to unlinked for all the partitions. We used the lognormal distribution for fossil calibrations with the means of distributions set such that 95% of the distribution probability fell within expected ranges of time intervals. We ran BEAST on CIPRES portal ( for 1 to 2 billion MCMC runs. We set up the sampling frequency at 1000 and re-sampled data using Log Combiner v1.8.1. The BEAST output was viewed in Tracer v1.6 and trees were combined in TreeAnnotator v1.8.1. We compared our results with other studies to check for consistency of our molecular date estimates. We first compared our results with Fuchs et al. (2008) [66] who used Mlíkovský (1998) fossils of A. otus and O. leucotis [63], along with a geographical event dating which does not include our study area. Further, we compared our Strigidae / Tytonidae split dates with other relevant studies [5356]. We used Effective Sample Size (ESS) values as one of the criteria to compare among analyses.


1. Phylogenetic analysis

In the concatenated tree analysis, we recovered H. blewitti as nested within the Athene, and sister to the other Athene from Madagascar and Americas (Fig 2 and S3 Fig). We observed that in all gene trees (S4S9 Figs), A. brama and A. noctua were sisters to each other. Similarly, G. radiatum and G. cuculoides were sisters in all the analyses. We did not find significant congruence (P < 0.01) at the H. blewitti node in mitochondrial (S10 Fig) and nuclear (S11 Fig) trees when we performed the Shimodaira-Hasegawa test (Fig 3 and S3 Fig). In the mitochondrial tree, H. blewitti was sister to the Eurasian Athene clade, whereas in the nuclear tree it was sister to Athene from Madagascar and the Americas (Fig 3). We always recovered the mitochondrial tree topology when any one of the three nuclear genes (TGFB2, MYO and RAG-1) were removed from the concatenated dataset during gene jack-knifing.

Fig 2. A species tree reconstruction using BEAST on concatenated (mitochondrial + nuclear) dataset indicate that H. blewitti is nested within the Athene clade.

The brown text indicates the species sampled in the present study. The nodal values show Bayesian posterior probability (PP). All the nodes are highly supported (PP = 1) except for those where PP is mentioned as nodal value.

Fig 3. A Maximum Likelihood Phylogenetic tree of Athene-Heteroglaux-Glaucidium members.

3A: Tree constructed using mitochondrial (CYTB + COI) dataset; 3B: Tree constructed using nuclear (RAG-1 + TGFB2 + MYO) dataset. The red text indicates the species sampled in the present study. The nodal values indicate Bayesian posterior probability separated by maximum likelihood bootstrap support.

2. Molecular dating analysis

The molecular dating analyses resulted in varied estimates of divergence (Table 1). Our mitochondrial and concatenated datasets, however, failed to converge in dating runs. Based on the analyses for the nuclear dataset (Table 1, analysis 2), for which ESS values were the highest and the Tytonidae / Strigidae and A. otus / O. leucotis divergence estimates matched with other published estimates, we predicted diversification dates for H. blewitti between 4.3 and 5.7 Ma, A. brama / A. noctua split between 3.9 and 5.8 Ma, A. cunicularia / A. superciliaris split between 2.2 and 3.9 Ma and G. radiatum / G. cuculoides split between 0.1 and 1.8 Ma.

Table 1. Summary of molecular dating analysis using (uncorrelated) relaxed lognormal clock.


1. Heteroglaux as a synonym of Athene

Our analysis using mitochondrial, nuclear and concatenated datasets (Figs 2 and 3, and S4S11 Figs) show that H. blewitti is nested within the Athene clade, rejecting the König et al. (1999) hypothesis that the species is nested within Glaucidium [23], and the Rasmussen & Collar (2013) hypothesis that Heteroglaux is a monotypic genus sister to Athene [16]. Our results also refute the Wolters (1975) hypothesis [26] that H. blewitti is sister to A. brama. H. blewitti and A. brama show similarity in lengths of tibiotarsus and ulna, and relatively shorter tarsometatarsi as compared to A. noctua to occupy arboreal niche [16]. Therefore, our results support the Rasmussen & Collar (2013) interpretation of the morphological similarities in A. brama and H. blewitti being either convergence of traits or plesiomorphies, further supported by the observation that a strong arboreal nature is absent in A. noctua. We find two contrasting results–H. blewitti as a sister clade, either to Athene from Madagascar and the Americas (concatenated and nuclear datasets, Figs 2 and 3, S3S9 and S11 Figs) or Eurasian Athene (mitochondrial dataset, Fig 3 and S10 Fig), making the exact phylogenetic position of the species uncertain. Phylogenetic analyses with additional sampling of genetic markers, individuals per species, and distinct subspecies of A. brama, A. noctua, and A. cunicularia may help provide a better resolution. Based on our results, we propose that Heteroglaux is treated as a synonym of Athene, identifying Heteroglaux blewitti as Athene [Heteroglaux] blewitti.

2. Molecular dating and biogeography of A. [H.] blewitti

The overlapping dates of diversification of A. [H.] blewitti (4.3–5.7 Ma), A. brama / A. noctua split (3.9–5.8 Ma), and A. cunicularia / A. superciliaris split (2.2–3.9 Ma), based on the nuclear dataset (Table 1, analysis 2), indicate a rapid diversification of all three owlets in India, perhaps in response to Plio-Pleistocene climatic fluctuations. This diversification is from the same period as A. inexpectanta (3.6–5.3 Ma, Early Pliocene), the oldest fossil Athene owl from Africa. Pavia et al. (2014) argue that the genus Athene originated in Africa and had a much wider distribution than previously thought [64]. Given the rich island endemic Athene fossil records, from Late Pliocene of Palearctic [67], Nearctic [6871], and Early Pleistocene of Palearctic [60, 72, 73], we speculate that Athene species might have undergone multiple diversifications and extinction events, possibly as a response to Plio-Pleistocene climate change, as observed in other groups such as the Western Ghats montane birds [7, 74].

Perhaps the Plio-Pleistocene climatic fluctuations and the subsequent retraction of evergreen forests [75, 76], during the Upper Sivalik time (1.6–5.1 Ma) of India, facilitated A. brama expansion into Peninsular India, while A. noctua expanded northward to a cooler climate. Recent studies have shown that H. blewitti occupies moderately dense dry deciduous forests, with intermittent open spaces [77, 78]. This peculiar choice of habitat influenced by climate, along with prey preference and ecological interactions with other similar-sized competitors might have restricted the range of A. [H.] blewitti. The species’ diurnal, ambushing predatory nature might be an adaptation to maximally utilize the available niche, given the presence of other co-distributed crepuscular and nocturnal owlets such as A. brama and G. radiatum in the same area. Further information on the dietary preference of H. blewitti and its ecological interactions with other species would help understand its adaptations. Nevertheless, our study provides another line of evidence to the role of climatic fluctuations in the diversification of Indian birds.

Our divergence estimates, based on the nuclear dataset (Table 1, analysis 2), are overlapping but more recent (15.1–17.6 Ma) than those derived by Fuchs et al. (2008) for A. otus / O. leucotis split (16.7–19.3 Ma) [66]. For the Tytonidae / Strigidae split, our divergence estimate of 42.5–47.7 Ma (Table 1, analysis 2) overlaps with Ericsson et al. (2006) estimate of 40–60 Ma [53], however, it also presents an underestimate when compared with other studies [5456]. Our molecular dating analyses runs that included mitochondrial DNA (Table 1, analysis 1 and 3) did not converge despite 1.5 to 2 billion runs, perhaps due to the saturation of signal for these deep lineages.

3. Conservation implications

Genetic sampling of tropical birds is poor, especially in the Old World Tropics, thereby impacting, regional conservation needs [4, 79]. Although the new information on the phylogenetic status of A. [H.] blewitti does not directly impact the IUCN status of the species, its ranking in international conservation listings that use phylogenetic information may change. The Evolutionary Distinct and Globally Endangered (EDGE) listing will perhaps no longer carry the same evolutionary distinctness score for the species [79].

A. [H.] blewitti is a species of Central Indian old growth dry deciduous forests, occurring in protected as well as non-protected areas [11]. Across most of its range, it is also co-distributed with A. brama, a phylogenetically close relative based on this study. A. [H.] blewitti is under severe threat of habitat loss due to large-scale logging, timber harvesting, and land-use change [11, 77, 78]. A. brama, on the other hand, occurs in the vicinity of human habitation [19]. Although we detected no admixture between A. [H.] blewitti and A. brama (a mitochondrial genetic distance of 16 ± 1% indicative of low sharing of alleles) in this study, hybridization cannot be wholly ruled out. In the rapidly changing human-dominated landscape of the Central India, circumstances are similar to other owls such as Barred and Spotted Owls [80] and Northern and California Spotted Owls [81], where hybridization facilitated by anthropogenic activities, has led to numerous conservation challenges.

With this first molecular phylogenetic study of this Critically Endangered species, we demonstrate that crucial information can only be obtained through capture-based sampling that strengthens and supports ecological data collected through conventional methods. Capture-based genetic studies still do not find support from conservation managers in India [82, 83], but such studies are instrumental in providing vital information on taxonomy, evolutionary biogeography, and in identifying conservation units. Our study provides the first genetic dataset that needs to be followed up with further spatially explicit sampling that can be used for conservation prioritization.

The new information provided here will facilitate both the taxonomic revision of the Athene / Heteroglaux clade and highlight the need for studies predicting species responses to climate change.

Supporting information

S1 Fig. Distribution of few Palearctic and Oriental owlets as per Birdlife International (2015).

H. blewitti is the only range-restricted, rare owlet among Indian owlets.


S3 Fig. Densitree representation, based on the Bayesian output of BEAST analysis, of the concatenated phylogenetic tree.

Blue line: the consensus tree (primary hypothesis), magenta line: the next most popular tree after consensus (secondary hypothesis), green lines: tertiary hypotheses.


S4 Fig. A Bayesian phylogenetic tree constructed using CYTB data.

The species code used can be referred from S3 and S4 Tables. The nodal values show Bayesian posterior probability (PP) separated by Maximum Likelihood bootstrap support. HB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.


S5 Fig. A Bayesian phylogenetic tree constructed using COI data.

The species code used can be referred from S3 and S4 Tables. The nodal values show Bayesian posterior probability (PP) separated by Maximum Likelihood bootstrap support. HB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.


S6 Fig. A Bayesian phylogenetic tree constructed using RAG-1 data.

The species code used can be referred from S3 and S4 Tables. The nodal values show Bayesian posterior probability (PP) separated by Maximum Likelihood bootstrap support. HB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.


S7 Fig. A Bayesian phylogenetic tree constructed using TGFB2 data.

The species code used can be referred from S3 and S4 Tables. The nodal values show Bayesian posterior probability (PP) separated by Maximum Likelihood bootstrap support. HB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.


S8 Fig. A Bayesian phylogenetic tree constructed using MYO data.

The species code used can be referred from S3 and S4 Tables. The nodal values show Bayesian posterior probability (PP) separated by Maximum Likelihood bootstrap support. HB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.


S9 Fig. A Bayesian phylogenetic tree constructed using LDH data.

The species code used can be referred from S3 and S4 Tables. The nodal values show Bayesian posterior probability (PP) separated by Maximum Likelihood bootstrap support. HB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.


S10 Fig. A Bayesian phylogenetic tree constructed using mitochondrial dataset (CYTB + COI).

The species code used can be referred from the S4 Table. The nodal values show Bayesian posterior probability (PP) separated by Maximum Likelihood bootstrap support. HB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.


S11 Fig. A Bayesian phylogenetic tree constructed using nuclear dataset (RAG-1 + TGFB2 + MYO).

The species code used can be referred from the S4 Table. The nodal values show Bayesian posterior probability (PP) separated by Maximum Likelihood bootstrap support. HB: H. blewitti; ASUP: A. superciliaris; ATHNB: A. brama; ANOCT: A. noctua; ACUN: A. cunicularia; GLRAD: G. radiatum and GCUCU: G. cuculoides.


S1 Table. Location data of the samples used.

BNHS: Bombay Natural History Society museum; JCT: Jivdaya Charitable Trust (rescued bird); CKV: C.K. Vishnudas.


S2 Table. List of primers used.

Tm: Optimal annealing temperature.


S3 Table. Provisional GenBank accession numbers of the sequences generated during the study.


S4 Table. GenBank accession numbers of the sequences used in the current study.

NA: Not available/Not used.


S5 Table. Best-fit partitioning scheme for genes used in the study.



We would like to thank State Forest Departments (Maharashtra, Madhya Pradesh, Gujarat, and Chhattisgarh) for providing permits to work. We would like to thank Bombay Natural History Society (BNHS), Jivdaya Charitable Trust (JCT) and C. K. Vishnudas for helping with sample collection. We also thank Praveen Karanth, Rajah Jaypal, Ishan Agrawal and NCBS Lab-3 members for their valuable suggestions.


  1. 1. Ribeiro ÂM, Lloyd P, Bowie RC. A tight balance between natural selection and gene flow in a southern African arid‐zone endemic bird. Evol. 2011;65(12): 3499–3514.
  2. 2. Holmes IA, Mautz WJ, Rabosky AR. Historical Environment Is Reflected in Modern Population Genetics and Biogeography of an Island Endemic Lizard (Xantusia riversiana reticulata). PLoS ONE. 2016;11(11): e0163738. pmid:27828958
  3. 3. Kahindo CM, Bates JM, Bowie RC. Population genetic structure of Grauer’s Swamp Warbler Bradypterus graueri, an Albertine Rift endemic. Ibis. 2017;159(2): 415–429.
  4. 4. Reddy S. What’s missing from avian global diversification analyses? Mol Phylogenet Evol. 2014;77: 159–165. pmid:24780750
  5. 5. Edwards SV, Jennings WB, Shedlock AM. Phylogenetics of modern birds in the era of genomics. Proc R Soc B. 2005;272(1567): 979–92. pmid:16024355
  6. 6. Prum RO, Berv JS, Dornburg A, Field DJ, Townsend JP, Lemmon EM, et al. A comprehensive phylogeny of birds (Aves) using targeted next-generation DNA sequencing. Nature. 2015;526: 569–573. pmid:26444237
  7. 7. Robin VV, Vishnudas CK, Gupta P, Rheindt FE, Hooper DM, Ramakrishnan U, et al. Two new genera of songbirds represent endemic radiations from the Shola Sky Islands of the Western Ghats, India. BMC Evol Biol. 2017;17(1): 31–45. pmid:28114902
  8. 8. Wink M, El-Sayed AA, Sauer-Gürth H, Gonzalez J. Molecular phylogeny of owls (Strigiformes) inferred from DNA sequences of the mitochondrial cytochrome b and the nuclear RAG-1 gene. Ardea. 2009;97(4): 581–591.
  9. 9. Wink M. Molekulare Phylogenie der Eulen (Strigiformes). Vogelwarte. 2014;52(4): 325–326. German.
  10. 10. Wood JR, Mitchell KJ, Scofield RP, De Pietri VL, Rawlence NJ, Cooper A. Phylogenetic relationships and terrestrial adaptations of the extinct laughing owl, Sceloglaux albifacies (Aves: Strigidae). Zool. J. Linn. Soc. 2016;179(4): 907–18.
  11. 11. BirdLife International. Heteroglaux blewitti. The IUCN Red List of Threatened Species. Version 2015.2. Downloaded on 20 June 2015.
  12. 12. Hume AO. Heteroglaux blewitti, sp. nov. Stray Feathers. 1873;1: 468–469.
  13. 13. Dubois A. Synopsis avium. Nouveau manuel d’ornithologie. Deuxième Partie. Bruxelles: H. Lamertin; 1904. French.
  14. 14. Dickinson EC, Remsen JV jr, editors. The Howard & Moore Complete Checklist of the Birds of the World. Vol 1. 4th ed. Eastbourne: Aves Press; 2013.
  15. 15. Gill F, Donsker D, editors. IOC World Bird List (v 3.5). 2013. Downloaded on 12 December 2013.
  16. 16. Rasmussen PC, Collar NJ. Phenotypic evidence for the specific and generic validity of Heteroglaux blewitti. Forktail. 2013;29: 78–87.
  17. 17. Blanford WT. The Fauna of British India, including Ceylon and Burma. Birds—Vol. 3. 1st ed. London: Taylor and Francis; 1895.
  18. 18. Baker ECS. The Nidification of Birds of the Indian Empire. Vol. III–Ploceidae—Asionidae. 1st ed. London: Taylor and Francis; 1934.
  19. 19. Ali S, Ripley D. Handbook of the Birds of India and Pakistan. Volume 3: Stone Curlews to Owls. 2nd ed. New Delhi: Oxford University Press; 1983.
  20. 20. Monroe BL Jr., Sibley CG. A World Checklist of Birds. London: Yale University Press; 1993.
  21. 21. del Hoyo J, Elliot A, Sargatal J, editors. Handbook of The Birds of The World. Volume 5: Barn Owls to Hummingbirds. 1st ed. London: Lynx Edicions; 1999.
  22. 22. Clements JF, Schulenberg TS, Iliff MJ, Sullivan BL, Wood CL, Roberson D. The eBird/Clements checklist of birds of the world: Version 6.8. 2013. Downloaded on 20 June 2015.
  23. 23. König C, Weick F, Becking JH. Owls: A Guide to the Owls of the World. London: Pica Press; 1999.
  24. 24. Sibley CG, Monroe BL. Distribution and taxonomy of birds of the world. New York: Yale University Press; 1990.
  25. 25. König C, Weick F. Owls of the World. 1st ed. London: Christopher Helm; 2008.
  26. 26. Wolters HE. Die Vogelarten der Erde. Hamburg: Parey; 1975. German.
  27. 27. Pande S, Pawashe A, Kasambe R, Yosef R. Discovery of a possible hybrid of the Critically Endangered Forest Owlet Athene blewitti and Spotted Owlet Athene brama (Aves: Strigiformes) from northern Maharashtra, India. J Threat Taxa. 2011;3(4): 1727–1730.
  28. 28. Ishtiaq F. Response to “Discovery of a possible hybrid of the Critically Endangered Forest Owlet Athene blewitti and Spotted Owlet Athene brama (Aves: Strigiformes) from northern Maharashtra, India” by Pande et al. J Threat Taxa. 2011;3(5): 1798.
  29. 29. Jathar G, Patil D. A review of “Discovery of possible hybrid of the Critically Endangered Forest Owlet Athene blewitti and Spotted Owlet Athene brama from northern Maharashtra”. J Threat Taxa. 2011;3(5): 1800–1803.
  30. 30. Pande S, Pawashe A, Kasambe R, Yosef R. Reply to the Response to Pande et al. by Ishtiaq. J Threat Taxa. 2011;3(5): 1799.
  31. 31. Pande S, Pawashe A, Kasambe R, Yosef R. Reply to the Response to Pande et al. by Jathar & Patil. J Threat Taxa. 2011;3(5): 1804.
  32. 32. Rasmussen PC, Ishtiaq F. Vocalizations and behavior of the Forest Owlet Athene (Heteroglaux) blewitti. Forktail. 1999;15: 61–66.
  33. 33. Berger DD, Mueller HC. The bal-chatri: a trap for the birds of prey. Bird-banding. 1959;30(1): 18–26.
  34. 34. Lanyon SM. Polyphyly of the blackbird genus Agelaius and the importance of assumptions of monophyly in comparative studies. Evolution. 1994;48(3): 679–93. pmid:28568260
  35. 35. Groth JG, Barrowclough GF. Basal divergences in birds and the phylogenetic utility of the nuclear RAG-1gene. Mol Phylogenet Evol. 1999;12(2): 115–123. pmid:10381315
  36. 36. Bures S, Nadvornik P, Saetre GP. Hybridization and apparent hybridization between meadow pipit (Anthus pratensis) and water pipit (A. spinoletta). Hereditas. 2002;136(3): 254–256. pmid:12471675
  37. 37. Primmer CR, Borge T, Lindell J, Saetre GP. Single‐nucleotide polymorphism characterization in species with limited available sequence information: high nucleotide diversity revealed in the avian genome. Mol Ecol. 2002;11(3): 603–612. pmid:11918793
  38. 38. Hebert PD, Stoeckle MY, Zemlak TS, Francis CM. Identification of birds through DNA barcodes. PLoS Biol. 2004;2: 1657–1663.
  39. 39. Hackett SJ, Kimball RT, Reddy S, Bowie RC, Braun EL, Braun MJ, et al. A phylogenomic study of birds reveals their evolutionary history. Science. 2008;320(5884): 1763–1768. pmid:18583609
  40. 40. Reddy S. Systematics and biogeography of the shrike-babblers (Pteruthius): species limits, molecular phylogenetics, and diversification patterns across southern Asia. Mol Phylogenet Evol. 2008;47(1): 54–72. pmid:18313946
  41. 41. Fregin S, Haase M, Olsson U, Alstrom P. Multi-locus phylogeny of the family Acrocephalidae (Aves: Passeriformes)–The traditional taxonomy overthrown. Mol Phylogenet Evol. 2009;52: 866–878. pmid:19393746
  42. 42. Dong F, Li SH, Yang XJ. Molecular systematics and diversification of the Asian scimitar babblers (Timaliidae, Aves) based on mitochondrial and nuclear DNA sequences. Mol Phylogenet Evol. 2010;57(3): 1268–1275. pmid:20937399
  43. 43. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinfo. 2012;28(12): 1647–9.
  44. 44. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24(8): 1596–1599. pmid:17488738
  45. 45. Castoe TA, de Koning AJ, Kim HM, Gu W, Noonan BP, Naylor G, et al. Evidence for an ancient adaptive episode of convergent molecular evolution. Proc Nat Acad Sci. 2009;106(22): 8986–91. pmid:19416880
  46. 46. Roje DM. Evaluating the effects of non-neutral molecular markers on phylogeny inference. PLoS ONE. 2014;9(2): e87428. pmid:24558367
  47. 47. Sergei LKP, Frost SDW, Muse SVM. HyPhy: hypothesis testing using phylogenies. Bioinfo. 2005;21: 676–679.
  48. 48. Nei M, Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol. 1986;3(5): 418–26. pmid:3444411
  49. 49. Lanfear R, Calcott B, Ho SY, Guindon S. PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Mol Biol Evol. 2012;29(6): 1695–1701. pmid:22319168
  50. 50. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinfo. 2014;30(9): 1312–1313.
  51. 51. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinfo. 2003;19(12): 1572–1574.
  52. 52. Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol. 2012;29(8): 1969–73. pmid:22367748
  53. 53. Ericson PG, Anderson CL, Britton T, Elzanowski A, Johansson US, Källersjö M, et al. Diversification of Neoaves: integration of molecular sequence data and fossils. Biol Let. 2006;2(4): 543–7.
  54. 54. Brown JW, Rest JS, García-Moreno J, Sorenson MD, Mindell DP. Strong mitochondrial DNA support for a Cretaceous origin of modern avian lineages. BMC Biol. 2008;6(1): 6–24.
  55. 55. Pacheco MA, Battistuzzi FU, Lentino M, Aguilar RF, Kumar S, Escalante AA. Evolution of modern birds revealed by mitogenomics: timing the radiation and origin of major orders. Mol Biol Evol. 2011;28(6): 1927–42. pmid:21242529
  56. 56. Claramunt S, Cracraft J. A new time tree reveals Earth history’s imprint on the evolution of modern birds. Sci Adv. 2015;1(11): e1501005. pmid:26824065
  57. 57. Bouckaert RR. DensiTree: making sense of sets of phylogenetic trees. Bioinfo. 2010;26(10): 1372–1373.
  58. 58. Shimodaira H, Hasegawa M. Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol. 1999;16(8): 1114–1116.
  59. 59. Kurochkin EN, Dyke GJ. The first fossil owls (Aves: Strigiformes) from the Paleogene of Asia and a review of the fossil record of Strigiformes. Paleontol J. 2011;45(4): 445–58.
  60. 60. Mlíkovský J. Cenozoic Birds of the World. Part 1: Europe. Amsterdam: Ninox Press; 2002.
  61. 61. Ho SY, Phillips MJ. Accounting for calibration uncertainty in phylogenetic estimation of evolutionary divergence times. Syst Biol. 2009;58(3): 367–380. pmid:20525591
  62. 62. Parham JF, Donoghue PC, Bell CJ, Calway TD, Head JJ, Holroyd PA, et al. Best practices for justifying fossil calibrations. Syst Biol. 2012;61(2): 346–359. pmid:22105867
  63. 63. Mlíkovský J. Two new owls (Aves: Strigidae) from the early Miocene of the Czech Republic, with comments on the fossil history of the subfamily Striginae. Buteo. 1998;10: 5–22.
  64. 64. Pavia M, Manegold A, Haarhoff P. New early Pliocene owls from Langebaanweg, South Africa, with the first evidence of Athene south of the Sahara and a new species of Tyto. Acta Palaeontol Polonica. 2014;60(4): 815–28.
  65. 65. Tajima F. Simple methods for testing molecular clock hypothesis. Genet. 1993;135: 599–607.
  66. 66. Fuchs J, Pons JM, Goodman SM, Bretagnolle V, Melo M, Bowie RC, et al. Tracing the colonization history of the Indian Ocean scops-owls (Strigiformes: Otus) with further insight into the spatio-temporal origin of the Malagasy avifauna. BMC Evol Biol. 2008;8(1): 197–212.
  67. 67. Boev ZN. Neogene avifauna of Bulgaria. In: Zhou Z, Zhang F, editors. Proceedings of the 5th Symposium of the Society of Avian Paleontology and Evolution. Pekin: Science Press, 2002. pp. 29–40.
  68. 68. Ford NL. Fossil owls from the Rexroad fauna of the Upper Pliocene of Kansas. Condor. 1966;68(5): 472–5.
  69. 69. Ford NL, Murray BG. Fossil owls from the Hagerman local fauna (Upper Pliocene) of Idaho. Auk. 1967;84(1): 115–7.
  70. 70. Feduccia JA. The avifauna of the Sand Draw local fauna (Aftonian) of Brown County, Nebraska. Wilson Bull. 1970;82(3): 332–4.
  71. 71. Bell CJ, Lundelius EL Jr, Barnosky AD, Graham RW, Lindsay EH, Ruez DR Jr, et al. The Blancan, Irvingtonian, and Rancholabrean mammal ages. In: Woodburne MO, editor. Late Cretaceous and Cenozoic mammals of North America: biostratigraphy and geochronology. New York: Columbia University Press; 2004. pp. 232–314.
  72. 72. Guerra C, Bover P, Alcover JA. A new species of extinct little owl from the Pleistocene of Mallorca (Balearic Islands). J Ornithol. 2012;153(2): 347–54.
  73. 73. Bedetti C, Pavia M. Early Pleistocene birds from Pirro Nord (Puglia, southern Italy). Palaeontographica Abteilung A-Palaozoologie-Stratigraphie. 2013;298: 31–53.
  74. 74. Robin VV, Vishnudas CK, Gupta P, Ramakrishnan U. Deep and wide valleys drive nested phylogeographic patterns across a montane bird community. Proc R Soc B. 2015; 282(1810): 20150861. pmid:26085588
  75. 75. Meher-Homji VM. On the Indo-Malaysian and Indo-African elements in India. Feddes Repertorium. 1983;94: 407–424.
  76. 76. Karanth KP. Evolution of disjunct distributions among Wet-zone species of the Indian subcontinent: Testing various hypotheses using a phylogenetic approach. Curr Sci. 2003;85(9): 1276–1283.
  77. 77. Mehta P, Kulkarni J, Patil D. A survey of the critically endangered Forest Owlet Heteroglaux blewitti in Central India BirdingASIA. 2008;10: 77–87.
  78. 78. Jathar G, Patil D, Kalra M, de Silva T, Peterson AT, Irfan-Ullah M, et al. Mapping the Potential Distribution of the Critically Endangered Forest Owlet Heteroglaux blewitti in India. J Bom Nat Hist Soc. 2015;112(2): 55–64.
  79. 79. Jetz W, Thomas GH, Joy JB, Redding DW, Hartmann K, Mooers AO. Global distribution and conservation of evolutionary distinctness in birds. Curr Biol. 2014;24(9): 919–930. pmid:24726155
  80. 80. Hamer TE, Forsman ED, Fuchs AD, Walters ML. Hybridization between barred and spotted owls. Auk. 1994;111(2): 487–492.
  81. 81. Barrowclough GF, Groth JG, Mertz LA, Gutiérrez RJ. Genetic structure, introgression, and a narrow hybrid zone between northern and California spotted owls (Strix occidentalis). Mol Ecol. 2005;14(4): 1109–1120. pmid:15773939
  82. 82. Bawa KS. Hurdles for conservation science in India. Curr Sci. 2006;91(8): 1005.
  83. 83. Madhusudan MD, Shanker K, Kumar A, Mishra C, Sinha A, Arthur R, et al. Science in the wilderness: the predicament of scientific research in India’s wildlife reserves. Curr Sci. 2006;91(8): 1015–1019.