Kusza, Szilvia

Mar, Khyne U.

Pucher, Herwig

Susekov, Andrey

Zachos, Frank E.

University of Debrecen Electronic Archive

Published by Associazione Teriologica Italiana Volume 29 (1): 152–154, 2018
Hystrix, the Italian Journal of Mammalogy
Available online at:
http://www.italian-journal-of-mammalogy.it doi:10.4404/hystrix–00049-2018
Short Note
High levels of mitochondrial genetic diversity in Asian elephants (Elephas maximus) from Myanmar
Szilvia Kusza1,∗, Franz Suchentrunk2, Herwig Pucher3, Khyne U Mar4, Frank E. Zachos5
1Institute of Animal Science, Biotechnology and Nature Conservation, University of Debrecen, Debrecen, Hungary
2Research Institute of Wildlife Ecology, University of Veterinary Medicine, Vienna, Austria
3TIERplus, Linz, Austria
4Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield, United Kingdom
5Natural History Museum Vienna, Vienna, Austria
Keywords:
mtDNA
microsatellites
Myanmar
Asian elephant
Elephas maximus
Article history:
Received: 1 July 2016
Accepted: 7 August 2017
Acknowledgements
We would like to thank Anita Haiden (Vienna), Ann-Christin Honnen
and Britt Petersen (Basel, Switzerland and Kiel, Germany) for assistance
with the labwork. Szilvia Kusza was supported by a Campus Hungary
fellowship. The authors are grateful for the insightful comments made
by the referees and the associate editor on an earlier version of this
paper.
Abstract
We analysedmtDNA control region sequences and 11microsatellites in 78Asian working elephants
(Elephas maximus) from two camps in Myanmar (ca. 60 km apart), which holds the second largest
elephant population in Asia. We found limited heterozygosity (overall Ho and He of 0.55 and 0.59)
but high mtDNA diversity (overall haplotype and nucleotide diversities of 0.89 and 0.011, respect-
ively) due to the presence of both mitochondrial lineages (α and β ) known for Asian elephants.
The fact that 13 of the 23 haplotypes found in this study were novel emphasises the importance of
Myanmar for the conservation of this endangered species. Both markers support the occurence of a
single panmictic population in the region. Demographic tests produced some indication of a recent
bottleneck in the microsatellite dataset, but the mtDNA sequences did not show either a signature
of past expansion or bottlenecks.
TheAsian elephant (Elephas maximus) is the largest terrestrial mam-
mal of the Asian continent and of high ecological importance. Due
to anthropogenic impacts, however, it has vanished from approx.95%
of its former distribution range, with present total population size es-
timates of ca. 40000–50000 (http://www.iucnredlist.org/details/7140/0).
Population numbers are declining, and the species is listed as En-
dangered by the IUCN (http://www.iucnredlist.org/details/7140/0). Al-
though Myanmar is a stronghold of the species (4000–5000 animals),
very little is known about the genetic diversity of its elephants. To our
knowledge, there are only very few studies that included Myanma ele-
phants (Hartl et al. 1996; Vidya et al. 2009). None of these studies
analysed nuclear DNA (only mitochondrial loci), and none of them had
a sample size larger than 24.
Two distinct mtDNA haplogroups are found in Asian elephants, α
and β (or B and A). Unfortunately, the group called α by some authors
is called B by others, and accordingly β is the equivalent of A (Hartl
et al., 1996; Fernando et al., 2000, 2003; Fleischer et al., 2001). In the
present study, we follow the α and β terminology. The two lineages
are believed to have diverged between 1.6 and 2.1 Mya (Vidya et al.,
2009). They co-occur in large parts of the range, and Myanmar is be-
lieved to be an ancestral area of the α-clade (Vidya et al., 2009). More
detailed knowledge on the genetic make-up of the Myanma elephants
is important and timely. Our study aims at filling this gap by analys-
ing elephants from two camps in Myanmar with respect to sequences
of the mtDNA and for the first time, nuclear markers.
A total of 78 Asian elephants (38 males and 40 females, from the
age of several months to 57 years) were sampled in two working camps
∗Corresponding author
Email address: kusza@agr.unideb.hu (Szilvia Kusza)
in Myanmar: Myaing Hay Wun (MHW) (n=40) and Taikkyi (n=38).
Camp elephants are kept in semi-captivity; they are taken to work on
logging sites 4 to 6 hrs per day 5 days per week. At night they are
released into the forest for unsupervised foraging where also wild ele-
phants occur. It is supposed that most of the calves born in captivity
are sired by wild bulls. Both camps are located in the southeast of the
country, to the northwest of Yangon and geographically close to each
other (≈60 km).Additional information on Myanma elephants can be
found on Appendix A (Supplemental S1).
Blood samples were taken in March and April 1996 and stored in
DMSO buffer. Specific ethical requirements beyond those pertain-
ing to veterinary examinations did not exist. DNA was extracted us-
ing the GenElute™ Mammalian Genomic DNA Miniprep kit (Sigma-
Aldrich). A portion of the mitochondrial control region was amplified
using the primers LoxProL andHiCtrIIH (following Fickel et al., 2007),
sequencing both strands on an automated sequencer (3730xl DNAAna-
lyzer, Applied Biosystems). Individual genotypes were established
through amplification of 11 nuclear microsatellite loci (Nyakaana and
Arctander, 1988; Comstock et al., 2000; Eggert et al., 2000; Fernando
et al., 2001; Appendix B, Supplemental S2). PCR runs consisted of 5
min at 95 ◦C followed by 35 amplification cycles (30 s at 95 ◦C, 30 s at
the locus-specific annealing temperature and 30 s at 72 ◦C) and a final
extension step of 10 min at 72 ◦C. Annealing temperature was 60 ◦C
except for the loci LafMSO2 and LafMSO3 (55 ◦C) and FH102 (52 ◦C).
Sequences were aligned using BioEdit 7.2.3 (Hall, 1999). Number of
haplotypes, number of polymorphic sites (S), haplotype (Hd) and nuc-
leotide diversities (pi) were calculated with DnaSP 5.10.1 (Librado and
Rozas, 2009). Pairwise ΦST , Chakraborty’s amalgamation test, neut-
rality/demographic tests (Tajima’s D and Fu’s FS, based on 10000 per-
mutations) and mismatch distribution analyses were calculated using
Hystrix, the Italian Journal of Mammalogy ISSN 1825-5272 7th March 2018
©cbe2018 Associazione Teriologica Italiana
doi:10.4404/hystrix–00049-2018
Genetic status of Myanmar Elephas maximus
Figure 1 – Median-joining networks of the 23 haplotypes found in Myanmar in the present study (right, based on 563 bp) and of an enlarged dataset including 309 Asian elephant
sequences from GenBank (left, based on 336 bp). Circle sizes are proportional to haplotype frequencies. Numbers denote mutational steps, colours refer to our sampling sites — grey:
MHW, black: Taikkyi; GenBank sequences are white. The dashed line separates the two known mtDNA clades α and β . Red dots represent median vectors.
ARLEQUIN 3.5.2.(Excoffier and Lischer, 2010). NETWORK 5.0.0.1
(Bandelt et al., 1999) was used to construct two median-joining net-
works using default parameters: (1) based on the haplotypes of the
present study (563 bp); (2) including 309 previously published (Ap-
pendix C, Supplemental S3) Asian elephant sequences from GenBank
(336 bp).
Occurrence of microsatellite null alleles was tested for withMICRO-
CHECKER 2.2.3 (Van Oosterhout et al., 2004). Number of alleles
per locus, observed (Ho) and expected (He) heterozygosities and devi-
ations from Hardy-Weinberg equilibrium (HWE) were calculated with
ARLEQUIN 3.5.2 which was also used to calculate the pairwise Fst
value between the two camps. GENEPOP 3.4 (Raymond and Rousset,
1995) was used to test for pairwise linkage between loci. A factorial
correspondence analysis (FCA) and a Structure cluster analysis (Ap-
pendix D) were carried out for the total dataset. Effective population
size (Ne) was estimated using the bias-corrected linkage disequilibirum
approach (LDNe) as implemented in NeESTIMATOR 2.01 (Do et al.,
2014) with three different frequency cut-off levels (0.05, 0.02, 0.01)
below which alleles were not considered. Recent reduction in effective
population size was tested for with BOTTLENECK 1.2.0.2 (Cornuet
and Luikart, 1996), using a Wilcoxon test under a two-phase mutation
model of microsatellite evolution with 1000 iterations and 70% step-
wise mutations.
Our 78 control region sequences (563 bp) yielded 23 different hap-
lotypes (Em HT1-HT23; GenBank accession numbers: KX882107–
KX882129) based on 19 variable sites. In accordance with Vidya et
al. (2009), we found both α (6) and β (17) haplotypes in Myanmar
(Fig. 1), and the respective frequencies were also similar: the 24 My-
anmar elephants in Vidya et al. (2009) yielded 25% α and 75% β ,
while our study yielded 15 α (19.2%) and 63 β (80.8%) haplotypes.
This is in clear contrast to the results from the neighbouring popula-
tion in Thailand, where Fickel et al. (2007) found 50% of each clade in
their sample of 78 elephants from throughout the country based on 411
bp. Thirteen of our 23 haplotypes were novel. Only five haplotypes
were shared by both populations, while 15 haplotypes were private to
one of them, but differentiation between the camps was non-significant
(ΦST=0.23, p=0.127, Chakraborty’s amalgamation test: p=0.09). Hap-
lotype (Hd) and nucleotide diversities (pi) were high in both camps
and overall (Tab. 1). One-hundred-and-two Lao elephants (Ahlering
et al., 2011) only yielded six different control region haplotypes result-
ing in a lower haplotype diversity of 0.74, while nucleotide diversity
was identical to ours (0.011). In Thai elephants haplotype and nucle-
otide diversities were very similar (Hd=0.90 vs. our 0.89) or slightly
higher (pi of 0.016 vs. 0.011) than in our Myanma elephants. The com-
paratively high nucleotide diversity in Thailand (Fickel et al., 2007),
Sri Lanka and Myanmar (Vidya et al., 2009) is due to the presence
of substantial numbers of animals from both clades. The mismatch
distribution for our total population was bimodal, indicating a popula-
tion without past population expansion or bottleneck. This was sup-
ported by statistically insignificant values of Tajima’s D and Fu’s FS
(D=1.617, p=0.96; FS=-3.531, p=0.15). The median-joining network
confirmed the lack of conspicuous geographic structuring in that both
camps harboured haplotypes from both previously identified mtDNA
haplogroups. All major haplotypes in the global network were present
in Myanmar, which, together with the private haplotypes found in this
study, means that Myanma elephants harbour a large portion of the spe-
cies’ overall mtDNA diversity. Our network also showed that there are
elephants from two β -subclades inMyanmar, which Vidya et al. (2009)
interpreted as evidence of independent colonisation of elephants from
the southwest (India/Sri Lanka) and the southeast (Sunda region). My-
anmar seems to be a phylogeographic crossroads for both major clades
(α and β ) and two β -subclades that originated in allopatry.
Microsatellite genotyping was successful at six or more loci in 61
elephants (percentage of missing data: 11.47%), 35 from MHW and
26 from Taikkyi. All loci were in HWE in both camps and in the total
population, and no linkage disequilibrium between any two loci or null
153
Hystrix, It. J. Mamm. (2018) 29(1): 152–154
Table 1 – Sample size and genetic diversity indices for the mtDNA sequences and nuclear genotypes found in Myanmar elephants. Standard deviations are in parentheses.
Camp n
Number of
haplotypes
Number of
polymorphic sites
Singleton
haplotypes
Haplotype
diversity
Nucleotide
diversity
Mean observed
heterozygosity
Mean expected
heterozygosity
MHW 40 10 17 5
0.810
(0.035)
0.009
(0.002)
0.550
(0.169)
0.580
(0.198)
Taikkyi 38 18 19 10
0.929
(0.022)
0.012
(0.001)
0.541
(0.224)
0.591
(0.201)
Total 78 23 19 15
0.893
(0.019)
0.011
(0.001)
0.547
(0.186)
0.586
(0.191)
alleles were detected. No differentiation between the two camps was
found as revealed by pairwise Fst (0.001, p=0.41), FCA and Structure
analysis (Appendix D, Supplemental S4), suggesting a single genetic-
ally homogeneous population. Overall mean allelic diversity was 4.55,
and observed and expected heterozygosities averaged over all loci for
the total populationwere 0.55 and 0.59, respectively (Appendix B, Sup-
plemental S2). These values are within the range of previously reported
genetic variability for Asian elephants in Borneo, Sri Lanka, Vietnam,
Myanmar, Malaysia, Cambodia, Lao PDR, Thailand, Bhutan and India
(Fernando et al., 2000, 2001, 2003; Vidya et al., 2005, 2009; Fickel et
al., 2007; Thitaram et al., 2010), although expected heterozygosity was
found to be considerably higher in elephants from the Lao PDR (0.75 vs
our 0.59). For the total Myanma sample of our study Ne was estimated
at between 40.1 (CI 22.7–91.0) and 53.6 (31.8–117.0), depending on
the cut-off allele frequency. Thitaram et al. (2010) analysed diversity
and effective population sizes of 156 working elephants from Thailand
based on 14microsatellite loci (eight of whichwere also included in our
study). Heterozygosity values were similar to ours (between 0.58 and
0.60). Their estimate of overall effective population size based on the
LDNe approach was about 200 and thus four to five times higher than
ours, but the geographic origin of their elephants spanned the whole
of Thailand, which precludes a direct comparison. The bottleneck test
yielded significant p values for our total dataset (<0.001), suggesting a
relative excess of heterozygosity and a recent bottleneck.
Taken together, rather low levels of heterozygosity, but high levels of
mtDNA diversity and private haplotypes were found in Myanma ele-
phants. No evidence for deviation from panmixia was found in either
marker set for our total sample. The semicaptive status of our camp
elephants allows for sufficient gene flow in the region through mating
with wild bulls to consider the two camps being part of a single genetic
population. In contrast to Thai elephants, where nuclear differentiation
between animals carrying α and β haplotypes was found (Fickel et al.,
2007), no such separation exists in our Myanma elephants (Appendix
D, Supplemental S4). Although our study is the most comprehensive
to date on Myanmar elephants, we only covered a small part of their
range in the country, and generalisations of our demographic results
should not be made until other regions have been studied. What even
our limited dataset shows, however, is that Myanmar not only harbours
a significant number of the remaining Asian elephants but also a large
part of the species’ genetic diversity.
References
Ahlering M.A., Hedges S., Johnson A., Tyson M., Schuttler S.G., Eggert L.S., 2011. Ge-
netic diversity, social structure, and conservation value of the elephants of the Nakai
Plateau, Lao PDR, based on non-invasive sampling. Conserv. Genet. 12: 413–422.
Bandelt H.J., Forster P., Rohl A., 1999. Median-joining networks for inferring intraspecific
phylogenies. Mol. Biol. Evol. 16: 37–48.
Comstock K.E., Wasser S.K., Ostrander E.A., 2000. Polymorphic microsatellite DNA loci
identified in the African elephant (Loxodonta africana). Mol. Ecol. 9: 1004–1006.
Cornuet J.M., Luikart G., 1996. Description and power analysis of two tests for detecting
recent population bottlenecks from allele frequency data. Genetics 144: 2001–2014.
Do C., Waples R.S., Peel D., Macbeth G.M., Tillet B.J., Ovenden J.R., 2014. NeEstimator
v2: re-implementation of software for the estimation of contemporary effective popula-
tion size (Ne) from genetic data. Mol. Ecol. Resour. 14: 209–214.
Eggert L.S., Ramakrishnan U., Mundy N.I., Woodruff D.S., 2000. Polymorphic microsatel-
lite DNAmarkers in theAfrican elephant (Loxodonta africana) and their use in theAsian
elephant (Elephas maximus). Mol. Ecol. 9: 2223–2224.
Excoffier L., Lischer H.E.L., 2010. Arlequin suite ver. 3.5: A new series of programs to
perform population genetics analyses under Linux and Windows. Mol. Ecol. Res. 10:
564–567.
Fickel J., Lieckfeldt D., Ratanakorn P., Pitra C., 2007. Distribution of haplotypes and
microsatellite alleles among Asian elephants (Elephas maximus) in Thailand. Eur.
J.Wildlife Res. 53: 298–303.
Fernando P., Pfrender M., Encalada S., Lande R., 2000. Mitochondrial DNA variation,
phylogeography and population structure of the Asian elephant. Heredity 84: 362–372.
Fernando P., Vidya T.N.C., Melnick D.J., 2001. Isolation and characterization of tri- and
tetranucleotide microsatellite loci in the Asian elephant, Elephas maximus. Mol. Ecol.
Notes 1: 232–233.
Fernando P., Vidya T.N.C., Payne J., StueweM., Davison G., Alfred R.J., Andau P., Bosi E.,
Kilbourn A., Melnick A.J., 2003. DNA analysis indicates that Asian elephants are native
to Borneo and are therefore a high priority for conservation. PLoS Biol. 1: 110–115.
Fleischer R.C., Perry E.A., Muralidharan K., Stevens E.E., Wemmer C.M., 2001. Phylogeo-
graphy of the Asian elephant (Elephas maximus) based on mitochondrial DNA. Evolu-
tion 55: 1882–1892.
Hall T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis
program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95–98.
Hartl G.B., Kurt F., Tiedemann R., Gmeiner C., Nadlinger K., U Mar K., Rübel A., 1996.
Population genetics and systematics of Asian elephant (Elephasmaximus): a study based
on sequence variation at the Cyt b gene of PCR-amplified mitochondrial DNA from hair
bulbs. Z. Säugetierkd. 61: 285–294.
Librado P., Rozas J., 2009. DnaSP v. 5.: A software for comprehensive analysis of DNA
polymorphism data. Bioinformatics 25: 1451–1452.
Nyakaana S., Arctander P., 1998. Isolation and characterisation of microsatellite loci in the
African elephant (Loxodonta africana, Blumenbach 1797). Mol. Ecol. 7: 1436–1437.
Raymond M., Rousset F., 1995. Genepop (ver. 3.1): population genetics software for exact
tests and ecumenicism. J. Hered. 86: 248–249.
Thitaram C., Somgird C., Mahasawangkul S., Angkavanich T., Roongsri R., Thongtip N.,
Colenbrander B., van Steenbeek F.G., Lenstra J.A., 2010. Genetic assessment of captive
elephant (Elephas maximus) populations in Thailand. Conserv. Genet. 11: 325–330.
Van Oosterhout C., HutchinsonW.F., Wills D.P.M., Shipley P., 2004.Micro-checker: a soft-
ware for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol.
Notes 4: 535–538.
Vidya T.N.C., Fernando P., Melnick D.J., Sukumar R., 2005. Population genetic structure
and conservation of Asian elephants (Elephas maximus) across India. Anim. Conserv.
8: 377–388.
Vidya T.N.C., Sukumar R., Melnick D.J., 2009. Range-wide mtDNA phylogeography
yields insights into the origins of Asian elephants. Proc. R. Soc. 276: 893–902.
Associate Editor: M. Scandura
Supplemental information
Additional Supplemental Information may be found in the online version of this arti-
cle:
Supplemental S1 Appendix A. Additional information on Myanma elephants.
Supplemental S2 Appendix B. Observed number of alleles (Na), observed (Ho) and
expected (He) heterozygosities and p values for deviation from HWE in the two
camps and the total population.
Supplemental S3 Appendix C. Accession numbers and information of the Asian
elephant sequences downloaded from GenBank.
Supplemental S4 Appendix D. Test of genetic differentiation at 11 autosomal mi-
crosatellites.
154


High levels of mitochondrial genetic diversity in Asian elephants (Elephas maximus) from Myanmar

https://dea.lib.unideb.hu/dea/bitstream/handle/2437/269812/FILE_UP_2_High%20levels%20of.pdf?sequence=1&isAllowed=y

High levels of mitochondrial genetic diversity in Asian elephants (Elephas maximus) from Myanmar

Abstract

Similar works

Full text

Available Versions

University of Debrecen Electronic Archive