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RESEARCH ARTICLE
The critically endangered forest owlet
Heteroglaux blewitti is nested within the
currently recognized Athene clade: A century-
old debate addressed
Pankaj Koparde
1,2,3
*, Prachi Mehta
4
, Sushma Reddy
5
, Uma Ramakrishnan
6
,
Shomita Mukherjee
1☯
, V. V. Robin
3☯
1Division of Conservation Biology, Sa
´lim Ali Centre for Ornithology & Natural History, Coimbatore, Tamil
Nadu, India, 2Manipal Academy of Higher Education, Madhav Nagar, Manipal, Karnataka, India, 3Indian
Institute of Science Education and Research, Tirupati, Andhra Pradesh, India, 4Wildlife Research and
Conservation Society, Pune, Maharashtra, India, 5Loyola University, Chicago, Illinois, United States of
America, 6National Centre for Biological Science, TIFR, Bangalore, Karnataka, India
☯These authors contributed equally to this work.
*pankajkoparde@gmail.com
Abstract
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 mor-
phology place the small owls of Asia into three genera, namely, Athene,Glaucidium, and
Heteroglaux. Based on morphological and anatomical data, H.blewitti has been alterna-
tively hypothesized to belong within Athene,Glaucidium, or its own monotypic genus Het-
eroglaux. 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 com-
bined 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.
PLOS ONE | https://doi.org/10.1371/journal.pone.0192359 February 5, 2018 1 / 15
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OPEN ACCESS
Citation: Koparde P, Mehta P, Reddy S,
Ramakrishnan U, Mukherjee S, Robin VV (2018)
The critically endangered forest owlet Heteroglaux
blewitti is nested within the currently recognized
Athene clade: A century-old debate addressed.
PLoS ONE 13(2): e0192359. https://doi.org/
10.1371/journal.pone.0192359
Editor: Matthias Sto¨ck, Leibniz-Institute of
Freshwater Ecology and Inland Fisheries,
GERMANY
Received: June 15, 2017
Accepted: January 21, 2018
Published: February 5, 2018
Copyright: ©2018 Koparde et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: Department of Biotechnology,
Government of India (http://www.dbtindia.nic.in/)
provided a research grant (file no. BT/PR4812/BCE/
8/898/2012) to SM; US National Science
Foundation (https://www.nsf.gov/) (DEB-1457624)
provided a grant to SR for partial museum work. In
Introduction
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 [1–3]. 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 [5–7], 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 [8–10].
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 catego-
rized 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.ble-
witti by ornithologists over the centuries. In the past, the species has alternatively been placed
in either the genus Heteroglaux [12–16] or Athene [17–22]. 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 place-
ment 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 Eur-
asian Athene (with global distribution encompassing India) and A.superciliaris and A.cunicu-
laria 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, Ko¨nig 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 mono-
typic genus Heteroglaux claimed by Rasmussen & Collar (2013) is based solely on an assess-
ment 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 dis-
puted [28–31], 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
The critically endangered forest owlet Heteroglaux blewitti is nested within the Athene clade
PLOS ONE | https://doi.org/10.1371/journal.pone.0192359 February 5, 2018 2 / 15
both cases, the funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
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].
We test three proposed phylogenetic relationships as competing hypotheses—Rasmussen &
Collar (2013) [16], Ko¨nig 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 [8–10], 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 & Rip-
ley (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
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.
https://doi.org/10.1371/journal.pone.0192359.g001
The critically endangered forest owlet Heteroglaux blewitti is nested within the Athene clade
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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 han-
dling 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 col-
lected 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 iden-
tified 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 cri-
teria mentioned above to ensure species identity. In case of A.brama and G.radiatum, we col-
lected 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 Chhattis-
garh). 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.superci-
liaris 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 mini-
mize 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 optimiza-
tions (S2 Table) of reaction conditions. We sequenced the purified PCR products in both for-
ward 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 [34–42]. 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 trans-
lated 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
The critically endangered forest owlet Heteroglaux blewitti is nested within the Athene clade
PLOS ONE | https://doi.org/10.1371/journal.pone.0192359 February 5, 2018 4 / 15
v4.0 [44] for counting the proportion of variable sites, parsimony informative sites, and single-
tons. We tested for positive selection, to avoid introducing possible error in phylogenetic infer-
ence 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) [53–56].
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 con-
ducted 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 fre-
quency 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 posi-
tions 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 anal-
ysis for 1.5 billion runs. We viewed and edited the trees in FigTree v1.4.2. We also used Densi-
Tree 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 seri-
ally removed individual genes from the concatenated dataset to see which gene/s may influ-
ence 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 con-
sistency 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 cali-
brations. We used different combinations of data (concatenated, mitochondrial, and nuclear,
partitioned alignments), to check for consistency in results, to obtain molecular dates after per-
forming tests for a molecular clock [65] in MEGA v4.0 and using both the strict and the uncor-
related relaxed lognormal clocks. Substitution models, clock models and trees option were set
The critically endangered forest owlet Heteroglaux blewitti is nested within the Athene clade
PLOS ONE | https://doi.org/10.1371/journal.pone.0192359 February 5, 2018 5 / 15
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 (www.cipres.org) 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 Mlı
´kovsky
´(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 [53–56]. We used Effective Sample Size (ESS)
values as one of the criteria to compare among analyses.
Results
1. Phylogenetic analysis
In the concatenated tree analysis, we recovered H.blewitti as nested within the Athene, and sis-
ter to the other Athene from Madagascar and Americas (Fig 2 and S3 Fig). We observed that
in all gene trees (S4–S9 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 congru-
ence (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.
2. Molecular dating analysis
The molecular dating analyses resulted in varied estimates of divergence (Table 1). Our mito-
chondrial 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.
Discussion
1. Heteroglaux as a synonym of Athene
Our analysis using mitochondrial, nuclear and concatenated datasets (Figs 2and 3, and S4–
S11 Figs) show that H.blewitti is nested within the Athene clade, rejecting the Ko¨nig et al.
(1999) hypothesis that the species is nested within Glaucidium [23], and the Rasmussen & Col-
lar (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 tarso-
metatarsi as compared to A.noctua to occupy arboreal niche [16]. Therefore, our results sup-
port 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
The critically endangered forest owlet Heteroglaux blewitti is nested within the Athene clade
PLOS ONE | https://doi.org/10.1371/journal.pone.0192359 February 5, 2018 6 / 15
(concatenated and nuclear datasets, Figs 2and 3,S3–S9 and S11 Figs) or Eurasian Athene
(mitochondrial dataset, Fig 3 and S10 Fig), making the exact phylogenetic position of the spe-
cies 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 pro-
vide a better resolution. Based on our results, we propose that Heteroglaux is treated as a syno-
nym 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.
Fig 2. A species tree reconstruction using BEAST on concatenated (mitochondrial + nuclear) dataset indicatethat 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.
https://doi.org/10.1371/journal.pone.0192359.g002
The critically endangered forest owlet Heteroglaux blewitti is nested within the Athene clade
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Pavia et al. (2014) argue that the genus Athene originated in Africa and had a much wider dis-
tribution than previously thought [64]. Given the rich island endemic Athene fossil records,
from Late Pliocene of Palearctic [67], Nearctic [68–71], 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].
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.
https://doi.org/10.1371/journal.pone.0192359.g003
Table 1. Summary of molecular dating analysis using (uncorrelated) relaxed lognormal clock.
Attribute Analysis 1 Analysis 2 Analysis 3
Dataset Mitochondrial Nuclear Concatenated
Substitution model GTR+I+G HKY+I+G HKY+I+G
MCMC runs (X10^7) 150 150 200
Overall ESS <200 >>200 <200
Posterior -13117.02 -6161.38 -16360.55
Prior 69.56 -334.72 -11.17
Likelihood -13186.6 -5826.66 -16349.37
tmrca (AB/AN) 4.89 ±0.63 4.82 ±0.95 1.24 ±0.06
tmrca (AC/AS) 4.21 ±0.66 3.05 ±0.86 0.97 ±0.07
tmrca (Athene) 7.22 ±0.6 5.28 ±0.44 1.91 ±0.05
tmrca (HB/AC/AS) 7.21 ±0.61 4.94 ±0.65 1.78 ±0.05
tmrca (HB/AN/AB) 6.63 ±0.65 5.26 ±0.47 1.91 ±0.06
tmrca (all) (Tytonidae / Strigidae) 19.33 ±2.17 45.1 ±2.6 4.13 ±0.14
tmrca (Asioninae) 11.52 ±1.01 16.36 ±1.24 2.22 ±0.05
tmrca (GR/GC) 2.34 ±0.47 0.98 ±0.85 0.46 ±0.07
AB:A.brama,AN:A.noctua,AC:A.cunicularia,AS:A.superciliaris,HB:H.blewitti,GR:G.radiatum,GC:G.cuculoides.
https://doi.org/10.1371/journal.pone.0192359.t001
The critically endangered forest owlet Heteroglaux blewitti is nested within the Athene clade
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Perhaps the Plio-Pleistocene climatic fluctuations and the subsequent retraction of ever-
green 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.leuco-
tis 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 [54–56].
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 phyloge-
netic status of A. [H.] blewitti does not directly impact the IUCN status of the species, its rank-
ing 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 can-
not 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 dem-
onstrate 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 data-
set that needs to be followed up with further spatially explicit sampling that can be used for
conservation prioritization.
The critically endangered forest owlet Heteroglaux blewitti is nested within the Athene clade
PLOS ONE | https://doi.org/10.1371/journal.pone.0192359 February 5, 2018 9 / 15
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.
(TIF)
S2 Fig. Map of sampling locations.
(TIF)
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.
(TIF)
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.superci-
liaris;ATHNB:A.brama;ANOCT:A.noctua;ACUN:A.cunicularia;GLRAD:G.radiatum
and GCUCU:G.cuculoides.
(TIF)
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.
(TIF)
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.superci-
liaris;ATHNB:A.brama;ANOCT:A.noctua;ACUN:A.cunicularia;GLRAD:G.radiatum
and GCUCU:G.cuculoides.
(TIF)
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.superci-
liaris;ATHNB:A.brama;ANOCT:A.noctua;ACUN:A.cunicularia;GLRAD:G.radiatum
and GCUCU:G.cuculoides.
(TIF)
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.superci-
liaris;ATHNB:A.brama;ANOCT:A.noctua;ACUN:A.cunicularia;GLRAD:G.radiatum
and GCUCU:G.cuculoides.
(TIF)
The critically endangered forest owlet Heteroglaux blewitti is nested within the Athene clade
PLOS ONE | https://doi.org/10.1371/journal.pone.0192359 February 5, 2018 10 / 15
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.superci-
liaris;ATHNB:A.brama;ACUN:A.cunicularia;GLRAD:G.radiatum and GCUCU:G.cucu-
loides.
(TIF)
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.cunicu-
laria;GLRAD:G.radiatum and GCUCU:G.cuculoides.
(TIF)
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.cunicu-
laria;GLRAD:G.radiatum and GCUCU:G.cuculoides.
(TIF)
S1 Table. Location data of the samples used. BNHS: Bombay Natural History Society
museum; JCT: Jivdaya Charitable Trust (rescued bird); CKV: C.K. Vishnudas.
(DOCX)
S2 Table. List of primers used. Tm: Optimal annealing temperature.
(DOCX)
S3 Table. Provisional GenBank accession numbers of the sequences generated during the
study.
(DOCX)
S4 Table. GenBank accession numbers of the sequences used in the current study. NA: Not
available/Not used.
(DOCX)
S5 Table. Best-fit partitioning scheme for genes used in the study.
(DOCX)
Acknowledgments
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.
Author Contributions
Conceptualization: Shomita Mukherjee, V. V. Robin.
Data curation: Pankaj Koparde, V. V. Robin.
Formal analysis: Pankaj Koparde.
The critically endangered forest owlet Heteroglaux blewitti is nested within the Athene clade
PLOS ONE | https://doi.org/10.1371/journal.pone.0192359 February 5, 2018 11 / 15
Funding acquisition: Uma Ramakrishnan, Shomita Mukherjee, V. V. Robin.
Investigation: Pankaj Koparde, Sushma Reddy, Shomita Mukherjee, V. V. Robin.
Methodology: Pankaj Koparde, Prachi Mehta, Sushma Reddy.
Project administration: Pankaj Koparde, Prachi Mehta, Uma Ramakrishnan, Shomita
Mukherjee, V. V. Robin.
Resources: Prachi Mehta, Uma Ramakrishnan, Shomita Mukherjee, V. V. Robin.
Software: Uma Ramakrishnan, V. V. Robin.
Supervision: Sushma Reddy, Shomita Mukherjee, V. V. Robin.
Validation: Pankaj Koparde, Shomita Mukherjee, V. V. Robin.
Visualization: Pankaj Koparde, Shomita Mukherjee, V. V. Robin.
Writing – original draft: Pankaj Koparde.
Writing – review & editing: Pankaj Koparde, Shomita Mukherjee, V. V. Robin.
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