Taxonomic implications of geographical variation in Rhinolophus affinis (Chiroptera: Rhinolophidae) in mainland Southeast Asia

Sample collection and study sites

A total of 170 specimens were examined from mainland Southeast Asia. Samples examined
were from existing museum collections and those arising from recent surveys. Specimens
were examined from collections held at Princess Maha Chakri Sirindhorn Natural History
Museum, Prince of Songkla University, Thailand [PSU collection]; Zoological Collection,
Centre for Biodiversity Conservation, Cambodia (CBC); Harrison Institute, UK (HZM);
Institute of Ecology and Biological Resources (IEBR), Vietnam; Natural Science and
Research Laboratory at Museum of Texas Tech University; and Zoological Museum at University
Malaysia Sarawak and Kim Hy Nature Reserve Collection (NF), Vietnam.

Specimens were collected by Saveng Ith and team (Small Mammals and Birds Research
Unit Team, PSU, Thailand) between November 2010 and March 2012 from survey sites in
Thailand. Animals were captured in the field using a combination of harp traps, mist
net, and hand nets.

Many specimens from Cambodia, Thailand, and Vietnam were previously included in Kingsada
et al. (2011]). All specimens and surveyed localities and habitats for the current study are listed
below and in Appendix 1 (Figure 1).

Figure 1. Collection localities ofRhinolophus affinisfrom mainland Southeast Asia. C = Cambodia, M = Myanmar, Ma = Malaysia, T = Thailand, and V = Vietnam. Black circles
are localities where materials were examined (based on sample collection and study sites in methodology and Appendix 1). The arrows indicate the type localities of the subspecies
in the research area.

Cambodia

Siem Reap Province: [C1] Phnom Kbal Spean, Banteay Srei District, and Phnom Kulen
National Park (14° 21? N 107° 22? E). Six males (five adult males and one sub adult)
and one nulliparous female were collected by Ben Hayes, Sarith Pen, and Sophany Pauk
between January and July 2010 on the mountain of evergreen forest. [C2] Ka Kek, Preah
Vihear Protected Forest (14° 04? N 105° 17? E). One nulliparous female was captured
by Gabor Csorba, Neil Furey, and Saveng Ith on 17 February 2011 in semi evergreen
forest.

Peninsular Malaysia

Kedah State: [Ma1] Langkawi Island (approximately 6° 23.204? N, 99° 47.831? E). An
adult male was collected by Mohd Isham Mohd Azhar; Penang State [Ma2] (05° 15? 795
N, 100° 29? 076 E). A nulliparous female was collected by Faisal Ali Anwarali Khan
on 15 June 2011; Kelantan State: [Ma3] Gua Madu, Gua Musang Division (approximately
5° 10.462? N, 101° 54.191? E). A parous female was captured by Faisal Ali Anwarali
Khan on 24 June 2011; Pahang State: [Ma4] Nature Study of Kuala Atok, Taman Negara
National Park (04° 16? 281 N, 102° 22? 316 E). One adult male and one nulliparous
female were collected by Faisal Ali Anwarali Khan on 19 to 22 May 2008.

Myanmar

Shan State: [M1] Mant Hai Village, Muse Twonship (23° 54? 962 N, 97° 49? 000 E); [M2]
Holin Village, Keng Taung (21° 27? 483 N, 99° 32? 000 E); [M3] Taung Pauk Village,
Inle Lake (20° 21? 175 N, 96° 53? 189 E). Three adult males and one female were collected
by Paul Bates and Iain Mackie between March 2002 and December 2003. All areas were
on the Shan plateau in areas of limestone karst, comprising limestone outcrops, deforested
agricultural land and small patches of deciduous forest. Taninthary Division: [M4]
Katalu Village, (12° 28? 436 N, 98° 24? 191 E); [M5] Kyi Village (12° 30? 113 N, 98°
24? 333 E), Kadan ID; specimens were collected in mist nets over a stream in open
heavily degraded forest and agricultural land and from a roost in granite boulders
in secondary forest; [M6] Hnedchey Khan Cave, Kyauk Taun Village (12° 11? 400 N, 99°
00? 600 E). The cave is in a limestone outcrop and surrounded by patches of degraded
evergreen forest and agricultural land. One adult male and four females were collected
by Paul Bates and Iain Mackie between June and November 2003.

Thailand

Chiang Mai Province: [T1] Khun Mae Ngai Ranger Station, Chiang Dao Wildlife Sanctuary
(approximately 19° 30.556? N, 98° 49.956? E). A sub-adult female was collected in
a harp trap on 28 June 2011 in hilly evergreen forest (approximately 19° 31? 55??
N, 98° 50? 26?? E; 864 m a.s.l); two adult males, one parous female, and one nulliparous
female were captured by Pipat Soisook between August 2005 and October 2006. Bats were
captured from the limestone cave surrounded by orchards, mixed deciduous, and bamboo
forest. Petchaboon Province: [T2] Nhong Mae Na, Thung Sa Lang Luang National Park
(16.34? 17?? N, 100.52? 35?? E). One adult male was captured by Charles Francis and
Sara Bumrungsri on 16 May 2006 in semi evergreen forest. Loei Province: [T3] Na Haeo
District, Phu Suan Sai National Park (17° 30? 19?? N, 100° 56? 18?? E, 620 m, 975
m a.s.l). Two adult males were captured by Sara Bumrungsri and Charles Francis on
18 to 20 May 2006. Bats were captured using harp traps set across the trails within
evergreen forest mixed with bamboo; [T4] Phu Ruea District, Phuluang Wildlife Sanctuary
(17° 25? 742 N, 101° 38? 006 E). Three adult males and one nulliparous female were
collected by Sara Bumrungsri and team on 17 to 18 March 1993. The habitat is unknown.
Chaiyapum Province: [T5] Thung Kamang, Khon San District, Phukieo Wildlife Sanctuary
(16° 18? N, 101° 52? E). One nulliparous female was captured by Pipat Soisook on 08
April 2006 in hilly semi evergreen forest. Tak Province: [T6] Kavackee, East Thung
Yai Naresuan Wildlife Sanctuary (15° 42? 26?? N, 98° 59? 28?? E). One adult male was
captured by Sara Bumrungsri on 11 March 2003 in semi evergreen forest. Surin Province:
[T7] Ta Muen Thom, Huai Thap Than-Huay Sumran Wildlife Sanctuary (14° 21? 08?? N,
103° 15? 54?? E). One adult male was captured by Sara Bumrungsri on 28 January 2000
in dry semi evergreen forest. Ratchaburi Province: [T8] Mae Nam Pha Chi Wildlife Sanctuary
(13° 18? 142 N, 99° 25? 009 E). A male adult was captured by a harp trap set over
a seasonal stream in dry evergreen forest by Pipat Soisook on 20 January 2008. Petchaburi
Province: [T9] Kaeng Kra Chan National Park (approximately 12° 47? 965 N, 99° 27?
812 E). Two adult males and one nulliparous female were collected by Saveng Ith and
team in August 2011. Three harp traps and two nets were set in bamboo forest, across
a stream and a trail in evergreen forest. Prachuap Kiri Khan Province: [T10] Pa La-ou
Ranger Station, Kaeng Kra Chan National Park (approx. 12° 32? 228 N, 99° 27? 812 E).
Two adult males and one nulliparous female were collected by Saveng Ith in August
2011. Three harp traps were set on forest trails in evergreen forest. Ranong Province:
[T11] Klong Sai On Waterfall, Krom Luang Chumpon Wildlife Sanctuary (10° 22? 21 N,
99° 04? 27 E). Three adult males and one nulliparous female were collected by Saveng
Ith in August 2011. Three harp traps were set on forest trails of evergreen forest
and surrounded rubber plantation and fruit orchards. Chumphon Province: [T12] Khao
Kram cave, Patiew District (10° 55? 08?? N, 99° 22? 26?? E, 67 m a.s.l). Three adult
males and three nulliparous females were captured by Sara Bumrungsri and team on 10
October 2006. The harp trap was set across the entrance of the cave surrounded by
rubber plantation; [T13] Huay Wang Cave, Tumbon Khao Talu, Sawi District (10° 10?
00?? N, 98° 55? 11?? E, 55 m, a.s.l). One adult male was captured by Sara Bumrungsri
and team on 10 January 2007. The harp trap was set across the entrance of a limestone
cave surrounded by deciduous forest and rubber plantation; [14] Klao Plu Cave, Lamae
District (09° 43? 36?? N, 99° 06? 30?? E). One adult male was captured by Sara Bumrungsri
and team on 09 January 2007. Harp traps were set across the trails in rubber plantation
and fruit orchards. Pang Nga Province: [T15] North Surin Island (approximately 8°
46? 200 N, 98° 18? 600 E). Two adult males were collected by Sara Bumrungsri on 02
February 2006. The specimens were captured in a harp trap set over the trail on hill
side surrounded by evergreen forest and close to the beach. Surat Thani Province:
[T16] Rajjaprabha Dam (close to people settlements and farms) and Khlong Saeng Wildlife
Sanctuary (approximately 7 km north of the reservoir) (approximately 8° 58? 885 N,
97° 47? 706 E). One adult male was collected by Saveng Ith on 31 August 2011 and one
adult male was collected by Sara Bumrungsri on 17 January 2012. The harp traps and
mist nets were set on small trails and streams surrounded by disturbed evergreen forest,
rubber plantations, and a mixed fruit orchard. Nakhon Si Thammarat Province: [T17]
Khao Phlu Cave, Khao Ro Commune, Ron Piboon District (8° 32? 250 N, 99° 43? 396 E).
One adult male and nulliparous female were collected by Sara Bumrungsri from the cave
on 15 October 2011. The cave is located in a limestone outcrop surrounded by rubber
and oil palm plantations. Krabi Province: [T18] Khao Pra Bang Kram Wildlife Sanctuary
(7° 55? 31 N, 99° 15? 47 E). One adult male was collected by Pipat Soisook on 04 May
2012. A harp trap was set across forest trail surrounded by lowland evergreen forest.
Pattalung Province: [T19] Khao Ban Tad Wildlife Sanctuary (approximately 7° 23? 48
N, 99° 58? 40 E). Two adult males, one parous female and one nulliparous female were
collected by Pipat Soisook in March 2012 using harp traps and mist nets set in evergreen
forest across a stream and forest trail. Trang Province: [T20] Sai Rung Waterfall,
Khao Ban Tad Wildlife Sanctuary (7° 18? 080 N, 99° 41? 988 E). One adult male and
two nulliparous females were collected by Pipat Soisook on 09 January 2011. Three
harp traps and a mist net were set on forest trails and across a stream. Songkhla
Province: [T21] Khuan Khao Wang Forest Park, Rattaphum District (7° 00? 776 N, 100°
01? 259 E). Four adult males and two nulliparous females were captured by Saveng Ith
in August 2011 and February 2012. Mist nets and harps were set on the forest trails
and across the small streams in evergreen forest surrounded by rubber plantation and
fruit orchards; [T22-25] Ton Nga Chang Wildlife Sanctuary (approximately 6° 55? 783
N, 100° 16? 299 E) including Boripatr Waterfall, Pha Dam Ranger Station, Makling Waterfall,
and Hin Sam Kon Waterfall. Fifteen adult males and four nulliparous females were collected
using harp traps and mist nets by Saveng Ith in February 2012 and Sara Bumrungsri
between October 2006 and January 2007. The traps and nets were set on small trails
and streams surrounded by evergreen forests and a rubber plantation; [T26] Khao Namkhang
National Park (6° 33? 108 N, 100° 16? 299 E). Two adult males were captured by hoop
net in a man-made tunnel by Saveng Ith on 16 May 2012. Narathiwat Province: [T27]
Hala Bala Wildlife Sanctuary (05° 47? 54?? N, 101° 49? 30?? E). Six adult males and
two nulliparous females were collected by Saveng Ith in January 2012. Harp traps were
set on forest trails in evergreen forest nearby the Wildlife Sanctuary Station.

Vietnam

Bac Kan Province: [V1] Kim Hy Nature Reserve (22° 11? 320? N, 106° 03? 530 E). One
immature male and seven parous females were captured by Neil Furey between June 2006
and February 2007. Bats were captured using mist net set in primary forest ridge.
Vinh Phuc Province: [V2] Tam Dao National Park (21° 30? 448 N, 105° 36? 4,924 E).
Five adult males were collected by Vu Dinh Thong on 24 November 2009. Son La Province:
[V3] Tin To Area, Sop Cop Nature Reserve (20° 49? 758 N, 103° 29? 519 E). Three adult
males were collected in November 2004 by Pham Duc Tien. Nghe An Province: four sites
were surveyed including [V4] Que Phong District, Pu Hoat Nature Reserve (approximately
19° 54? 221 N, 104° 50? 243 E); [V5] Ban Khom Cave, Que Phong District, Pu Hoat Nature
Reserve (approximately 19° 54? 221 N, 104° 50? 243 E); [V6] Phu Nong Mount, Pu Mat
National Park (19° 01? 340 N, 104° 44? 726 E); [V7] a cave at Khe Mat ridge, Pu Mat
National Park (approximately 19° 01? 340 N, 104° 44? 726 E). Twenty adult males, 13
females were collected between August 1998 and October 2008 by Pham Duc Tien, Vu Dinh
Thong, Thomas Howard, and Ben Hayes. Quang Binh Province: [V8] Hoa Son Village, Ke
Bang, Phong Nha National Park (17° 28? 200 N, 105° 31? 200 E). One adult male was
collected on 18 August 1998 by Ditte Hendrichsen. Thua Thien Hue Province: [V9] Bach
Ma National Park (16° 10? 989 N, 107° 52? 496 E). Three adult males were collected
between June and October 2001 by Pham Duc Tien and Vu Dinh Thong. Kon Tum Province:
[V10] Chu Mom Ray National Park (14° 29? 021 N, 107° 38? 139 E). Two parous females
and seven nulliparous females were collected by Vu Dinh Thong between May and August
2005. Gia Lai Province: two sites were surveyed including [V11] Kon Cha Rang Nature
Reserve (14° 17? 400 N, 108° 21? 600 E) and [V12] Kon Ka Kinh Nature Reserve (14°
11? 400 N, 108° 15? 000 E). Two males and two females were collected in March 1999
by Ben Hayes.

Morphological measurements

Multiple external and craniodental characters of each specimen were measured following
Bates and Harrison (1997]), Csorba et al. (2003]), Furey et al. (2009]), and Thomas (1997]). Wet specimens were measured using a pair of dial calipers to the nearest 0.1 mm,
whereas craniodental characters were measured to the nearest 0.01 mm using a digital
caliper under stereo microscope. Bacular morphology was also observed using a stereo
microscope.

External characters measured included the following: forearm length (FA) – from the
extremity of the elbow to the extremity of the carpus with the wings folded; ear length
(EL) – from the lower border of the external auditory meatus to the tip of the pinna;
tail length (TL) – from the tip of the tail to its base adjacent to the anus; hind
foot length (HF) – from the extremity of the heel behind the os calcis to the extremity
of the longest digit, not including the hairs or claws; tibia length (TIB) – from
the knee joint to the extremity of the heel behind the os calcis; length of metacarpals
(2MT, 3MT, 4MT, 5MT) – taken from the extremity of the carpus to the distal extremity
of the second, third, fourth and fifth metacarpals, respectively; length of the first
and second phalanges of the third, fourth, and fifth digits (1P3D, 2P3D, 1P4D, 2P4D,
1P5D, 2P5D), respectively – taken from the proximal to the distal end of the phalanx;
greatest width of nose leaf (GWN) – greatest diameter across the horseshoe; greatest
height of nose leaf (GHN) – from the base of the horseshoe to the tip of the lancet,
not including the hairs.

Craniodental characters measured included the following: skull length (SL) – the greatest
length from the occiput to the front of the canine; condyle-canine length (CCL) –
from the exoccipital condyle to the anterior alveolus of the canine; the greatest
width across the anterior lateral compartments of the rostrum (ALSW); anterior median
swellings width (AMSW) – the greatest width across the median swellings in dorsal
view; zygomatic width (ZYW) – the greatest width of the skull across the zygomata;
the braincase width (BW) – width of the braincase at the posterior roots of the zygomatic
arches; braincase width (BW1) – the greatest width across the braincase; mastoid width
(MAW) – greatest width of the braincase taken across the mastoid region; interorbital
width (IOW) – the narrowest width of the interorbital constriction; palatal bridge
(PB) – length of bony palate excluding the posterior spike; posterior palatal width
(M³M³W) – taken across the widest part of the outer borders of the third upper molar; anterior
palatal width (C¹C¹W) – taken across the widest part of the outer border of the upper canine; upper tooth
row length (CM³L) – from the front of the upper canine to the back of the crown of the third upper
molar; lower tooth row length (CM₃L) – from the front of the lower canine to the back of the crown of the third lower
molar; mandible length (ML) – from the most posterior part of the condyle to the most
anterior part of the mandible, including the lower incisors; least height of the coronoid
process (CPH) – from the tip of the coronoid process to the apex of the indentation
on the inferior surface of the ramus adjacent to the angular process.

Morphometric analysis

Statistical analyses were carried out using SPSS 16.0 (SPSS Inc., Chicago, IL, USA)
and PC-ORD 5.10 (MjM Software, Gleneden Beach, OR, USA) for Windows. Descriptive statistics
(minimum, maximum, mean, and standard deviation) were calculated for external and
craniodental measurements. Normality of data and homogeneity of variances were explored
prior to parametric t-tests to determine sexual dimorphism within the taxa. Non-parametric tests (Mann–Whitney
U-test) were used for characters that did not show normality of data (HF, p 0.05) and/or homogenous variances (ALSW, p 0.05). Multiple comparisons of characters between populations and colonies were
calculated using a multivariate analysis of variance (MANOVA). Linear regression was
used to examine the correlation between morphology and echolocation call frequencies.
Principal component analysis (PCA) on the correlation matrix was used to discriminate
between individuals. A series of t-test was run for morphological characters comparison between forms prior to PCA.
Characters which are significant different in size (p 0.05) were retained for PCA.

Echolocation call recording and measurement

Values for the frequency of maximum energy (FMAXE) for R. affinis in this study were largely obtained from survey work, with some additional data published
by Kingsada et al. (2011]) and Furey et al. (2009]). Echolocation calls were recorded using a Pettersson D-240X bat detector (Pettersson
Elektronik AB, Uppsala, Sweden) set in 10× time-expansion mode, and call data were
stored on a digital iRiver iHP-120 Multi Codec Jukebox recorder (iriver House, Seoul,
Korea). When available, a Pettersson D1000X was also used, and calls stored on a built
in compact flash card (type I). The detector was set to manual recording mode with
the maximum sampling rate at 768 kHz. A time expansion factor of 10 was used. Sound
files were recorded and saved in ‘wav’ format then transferred to a laptop computer
for analysis. Echolocation calls from Vietnam were recorded using the PCTape system,
which was custom-made by the University of Tuebingen, Germany. Call components were
displayed using spectrograms and oscillograms in BatSound Pro 3.31 (Pettersson Elektronik
AB, Sweden) in which sampling frequency was 44.10 kHz; spectrograms were set as 1,024
sampling size in fast Fourier transforms with Hanning windows. The constant frequency
portion of the call was selected for measuring FMAXE (kHz) from the power spectrum
feature in BatSound Pro 3.31. Multiple calls were measured for individuals where these
data were available.

Molecular systematics

Tissue collection and DNA extraction and analysis

Tissue (liver, tongue, and wing membrane) was collected from voucher specimens and
preserved in 95% concentration ethanol. Two mitochondrial DNA (mtDNA) gene fragments
were selected for analysis. A 657 base pair segment of cytochrome oxidase subunit
I (COI) was sequenced at the Canadian Center for DNA Barcoding (CCDB) using standardized
barcoding protocols (Ivanova et al. 2012]), and a 517 base pair segment of control region (D-loop) was analyzed at the Department
of Biotechnology and Molecular Biology, Prince of Songkla University, Thailand. For
comparison, sequences from GenBank were also accessed (Table 1).

Table 1. D-loop and cytochrome C oxidase subunit I (COI) genes accessed from GenBank

Genomic DNA was extracted using DNeasy Tissue Kits (Qiagen, Venlo, Limburg). The tRNA-proline
end of mitochondrial DNA control region containing the hypervariable domain (HVI)
was amplified (Chen et al. 2006]) by polymerase chain reaction (PCR) using the primers DL-H 16750 (5?-CCTGAAGTAGGAA-CCAGATG-3?)
(Wilkison and Chapman 1991]) and Thr-L 16272 (5?-CCCGGTCTTGTAAAC C-3?) (Stanley et al. 1996]). PCRs were carried out in 25 ?l volumes. Each reaction contained 7.5 ?l of water,
2 ?l of each primers (10 ?m), 12.5 ?l of Top Taq Master Mix Kit (Qiagen), and 1 to
2 ?l of DNA template (50 ng/?l). The amplification was run under the thermal conditions
of an initial denaturation at 95°C for 5 min followed by 34 cycles of 94°C for 30
s, 55°C for 30 s, 72°C for 40 s, and a final extension cycle at 72°C for 10 min. Possible
contamination was checked by gel electrophoresis of 6 ?l of PCR reaction including
a negative control (containing all reagents, but no DNA template). DNA present in
a 1.5% agarose gel was stained with ethidium bromide and visualized under UV using
gel analysis equipment (UVITEC, Cambridge, UK). PCR product was purified using QIA
quick Gel Extraction Kit (Qiagen) before sequencing. The ABI PRISM^TM Big Dye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Framingham, MA, USA)
was used to prepare the DNA samples for sequence analysis. Sequencing was performed
on an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems, Framingham, MA, USA).
The chromatograms were edited using Geneious Pro 5.6 trial version and BIOEDIT 7.0.0
(Hall 1999]) and aligned using CLUSTAL_X 1.83 (Thompson et al. 1997]) and MEGA 5.2.2 (Tamura et al. 2011]).

Phylogenetic relationships among sequences were reconstructed for each gene separately
using maximum-likelihood in the program MEGA 5.2.2 (Tamura et al.2011]). The most appropriate substitution model was determined using BIC as implemented
in jModel Test 2.14 (Darriba et al. 2012]). Among the 88 models in the 100% confidence interval, the Hasegawa-Kishino-Yano
substitution model (HKY) with proportion of invariant sites (I) was the best-fit model
selected for D-loop and Kimura 2-parameter (K80) was the best-fit model for COI. We
also performed Bayesian Analysis for each gene separately using Mr Bayes 3.2.2 (Huelsenbeck
and Ronquist 2001]). In Bayesian Analysis, convergence stationarity was searched by two independent
metropolis-coupled Markov chain Monte Carlo (MCMC), each comprising three incrementally
heated chains and one cold chain, run for six million generations, with parameters
sampled every 1,000 generations. Convergence stationary of the MCMC chains was evaluated
by inspecting whether the standard deviation of split frequencies approached zero
and the potential scale reduction factor (PSRF) reached 1.0 for all parameters. We
also investigated the convergence using Tracer 1.5 (Rambaut and Drummond 2009]), and the 25% initial phase of the Markov chain was discarded as a burn-in. A congeneric
R. pearsoni (GenBank accession number JN106201) was used as an out group in the phylogenetic
analysis of D-loop in order to examine the monophyletic lineage of R. affinis.

To estimate the time to the most recent common ancestor (TMRCA) among the observed
clades, D-loop was analyzed in BEAST version 1.8 (Rambaut and Drummond 2007]). The gene was selected for the analysis as its divergence rate is known and has
been used in previous publications (Mao et al. 2010]; Chen et al. 2006]; Salgueiro et al. 2004]). Based on jModel test, HKY+I was selected as the best substitution model and relaxed-clock
model with an uncorrelated lognormal distribution was used to estimate the substitution
rate. We performed two independent runs of MCMC chains with 60 million generation
each with parameters logged every 1,000 generation. Tracer version 1.5 (Rambaut and
Drummond 2009]) was used to combine the two runs as well as to examine the effective sample size
(ESS) for the parameters. Trees were collated using Tree Annotator version 1.8 where
the maximum clade credibility tree and Median heights were selected; and 10% (6,000
trees) of the sample trees were selected as burn-in. To convert the estimates scaled
by mutation rate to calendar years, we used the divergence rate of 20%/Myr for control
region which was previously calibrated in the noctule bat (Petit et al. 1999]) and used in R. affinis (Mao et al. 2010]) and other bats (Chen et al. 2006]; Salgueiro et al. 2004]).