# Phylogeographic evidence for 2 genetically distinct zoonotic *Plasmodium knowlesi* parasites, Malaysia

Ruhani Yusof, Md Atique Ahmed, Jenarun Jelip, Hie Ung Ngian, Sahlawati Mustakim, Hani Mat Hussin, Mun Yik Fong, Rohela Mahmud, Frankie Anak Thomas Sitam, J. Rovie-Ryan Japning, et al.

<figure><img src="image_1.png" /></figure>

► To cite this version:

Ruhani Yusof, Md Atique Ahmed, Jenarun Jelip, Hie Ung Ngian, Sahlawati Mustakim, et al. Phylogeographic evidence for 2 genetically distinct zoonotic *Plasmodium knowlesi* parasites, Malaysia. Emerging Infectious Diseases, 2016, 22 (8), pp.1371-1380. 10.3201/eid2208.151885. hal-04254936

HAL Id: hal-04254936

https://hal.science/hal-04254936v1

Submitted on 23 Oct 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# Phylogeographic Evidence for 2 Genetically Distinct Zoonotic *Plasmodium knowlesi* Parasites, Malaysia

Ruhani Yusof,<fnref n="1" /> Md Atique Ahmed,<fnref n="1" /> Jenarun Jelip, Hie Ung Ngian, Sahlawati Mustakim, Hani Mat Hussin, Mun Yik Fong, Rohela Mahmud, Frankie Anak Thomas Sitam, J. Rovie-Ryan Japning, Georges Snounou, Ananias A. Escalante, Yee Ling Lau

Infections of humans with the zoonotic simian malaria parasite *Plasmodium knowlesi* occur throughout Southeast Asia, although most cases have occurred in Malaysia, where *P. knowlesi* is now the dominant malaria species. This apparently skewed distribution prompted an investigation of the phylogeography of this parasite in 2 geographically separated regions of Malaysia, Peninsular Malaysia and Malaysian Borneo. We investigated samples collected from humans and macaques in these regions. Haplotype network analyses of sequences from 2 *P. knowlesi* genes, type A small subunit ribosomal 18S RNA and cytochrome c oxidase subunit I, showed 2 genetically distinct divergent clusters, 1 from each of the 2 regions of Malaysia. We propose that these parasites represent 2 distinct *P. knowlesi* types that independently became zoonotic. These types would have evolved after the sea-level rise at the end of the last ice age, which separated Malaysian Borneo from Peninsular Malaysia.

The number of malaria cases in Malaysia steadily decreased from a peak of 59,208 in 1995 to 3,850 confirmed cases in 2013; of these, 80% were reported in the 2 states of Malaysian Borneo and the remainder in 6 of the 11 states of Peninsular Malaysia (Figure 1) (1). In Malaysia, the simian malarial parasite species *Plasmodium knowlesi* is now the dominant species infecting humans and is >2 times more prevalent than *P. falciparum* or *P. vivax*.

Author affiliations: University of Malaya Faculty of Medicine, Kuala Lumpur, Malaysia (R. Yusof, M.A. Ahmed, M.Y. Fong, R. Mahmud, Y.L. Lau); Sabah State Health Department, Sabah, Malaysia (J. Jelip); Hospital Kapit, Sarawak, Malaysia (H.U. Ngian); Hospital Tengku Microbiology Laboratory, Ampuan Rahimah, Selangor, Malaysia (S. Mustakim); Kelantan State Health Department, Kelantan, Malaysia (H.M. Hussin); Department of Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur, Malaysia (F.A.T. Sitam, J. Rovie-Ryan); Sorbonne Universités, INSERM, Paris, France (G. Snounou); Temple University, Philadelphia, PA, USA (G. Snounou, A.A. Escalante)

DOI: http://dx.doi.org/10.3201/eid2208.151885

Humans were found to be susceptible to *P. knowlesi* when this species was experimentally transmitted to man in 1932, the year in which it was first described (2,3). In 1965, the first confirmed case of a naturally acquired infection in humans was recorded (4). The next naturally acquired confirmed cases were reported in 2004, when a stable focus of *P. knowlesi* was discovered in Sarawak, 1 of 2 states that make up Malaysian Borneo (5). Thereafter, transmission of *P. knowlesi* to humans occurred in the second state, Sabah (6,7), and in neighboring countries (8,9).

The natural hosts of *P. knowlesi* are principally the long-tailed (*Macaca fascicularis*) and pig-tailed (*M. nemestrina*) macaques (10), 2 species that are widely distributed in the Southeast Asia countries in which cases of *P. knowlesi* have been recorded. To date, human-to-human transmission has not been observed. Infections in humans can cause severe disease that can be fatal (9,11), underscoring the public health concern raised by this zoonotic simian parasite.

The 2 states of Malaysian Borneo appear to be the epicenter of zoonotic *P. knowlesi* infections: 1,391 cases in Malaysian Borneo and 423 cases in Peninsular Malaysia were recorded in 2012. A total of 1,407 PCR-confirmed cases were reported during 2004–2013 in Malaysia, which contrasts with the low number of cases (n = 136) reported from neighboring countries (9): Cambodia (n = 1), China (n = 36), Indonesia (n = 1), Myanmar (n = 14), the Philippines (n = 5), Singapore (n = 2), Thailand (n = 36), and Vietnam (n = 32). The reasons for this uneven distribution remain unclear. Geographic variation in mosquito species and human social factors could be an explanation; it is also possible that the parasite populations circulating on the island of Borneo are distinct from those found in continental Malaysia. The *P. knowlesi* strains that had been studied in earlier years displayed distinct biologic characteristics, which in some cases led malariologists to propose distinct *P. knowlesi* subspecies (12). Such differences

<footnote n="1">¹These authors contributed equally to this article.</footnote>
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Geographic origin of the genetic sequences generated during study of Plasmodium knowlesi parasite populations, Malaysia. The numbers in each circle refer to the number of sequences (macaque or human) obtained for the genes P. knowlesi type A small subunit ribosomal 18S RNA (numbers in white) and P. knowlesi cytochrome oxidase subunit I (numbers in yellow).</figcaption></figure>

could indicate that local ecologic factors are influential
and that each *P. knowlesi* subspecies became a zoonosis
independently in each geographic area.

To explore whether the *P. knowlesi* populations in
Malaysia differed and independently became zoonoses,
we focused on 2 genes that have been extensively used for
phylogenetic studies (13–16): 1 nuclear, encoding the type
A small subunit ribosomal 18S RNA (*PkA*-type 18S rRNA),
and 1 mitochondrial, encoding the cytochrome oxidase
subunit I protein (*PkCOX1*). Using samples collected from
humans and macaques in both regions of the country, we
generated the relevant sequences, compared them to those
published previously (17), and conducted phylogenetic and
population genetic analyses.

Materials and Methods

Sample Collection

The Medical Research Ethic Committee of the Ministry
of Health Malaysia, Sabah State Director of Health, Kel-
antan State Director of Health, and directors of state and

district hospitals approved this study. Ethical approval was
granted by the Medical Research and Ethics Committee
of the Malaysian Ministry of Health (Reference Number:
KKM/NIHSEC/800/-2/2/2/P13-316), the Medical Ethics
Committee of University Malaya Medical Centre, and the
Department of Wildlife and National Parks.

We used previously collected human blood samples for
this study (7): 78 microscopically confirmed *P. knowlesi*-
positive blood samples from patients in 8 states in Malaysia,
including Sabah and Sarawak in Malaysian Borneo (Figure
1). We also examined blood samples from 8 long-tailed ma-
caques collected during routine surveys by the Department
of Wildlife and National Parks in the Peninsular Malaysia
states of Pahang, Selangor, and Negeri Sembilan. All sam-
ples were collected during September 2012–December 2013
(Tables 1, 2). In addition to these samples, we included pre-
viously published sequences deposited into GenBank during
2003–2015 in the analyses; these sequences were derived
from samples collected from humans and macaques (on-
line Technical Appendix Table, http://wwwnc.cdc.gov/EID/
article/22/8/15-1885-Techapp1.pdf).
<table><caption>**Table 1.** Samples and sequences obtained for the *PkA-type 18S rRNA* gene used to distinguish 2 distinct *Plasmodium knowlesi* parasite populations in Malaysia*</caption><thead><tr><th rowspan="3">Population location</th><th colspan="2">No. samples†</th><th colspan="4">No. sequences</th><th rowspan="3">Total sequences obtained</th></tr><tr><th colspan="2">Human</th><th colspan="2">Human</th><th colspan="2">Macaque</th></tr><tr><th>Human</th><th>Macaque</th><th>This study‡</th><th>GenBank§</th><th>This study‡</th><th>GenBank§</th></tr></thead><tbody><tr><td colspan="8"><strong>Peninsular Malaysia</strong></td></tr><tr><td>Kelantan</td><td>13</td><td>ND</td><td>27</td><td>ND</td><td>ND</td><td>ND</td><td>27</td></tr><tr><td>Johor</td><td>3</td><td>ND</td><td>7</td><td>ND</td><td>ND</td><td>ND</td><td>7</td></tr><tr><td>Selangor</td><td>7</td><td>5</td><td>11</td><td>ND</td><td>15</td><td>ND</td><td>26</td></tr><tr><td>Terengganu</td><td>2</td><td>ND</td><td>3</td><td>ND</td><td>ND</td><td>ND</td><td>3</td></tr><tr><td>Pahang</td><td>14</td><td>1</td><td>21</td><td>1</td><td>3</td><td>ND</td><td>25</td></tr><tr><td>Negeri Sembilan</td><td>4</td><td>2</td><td>10</td><td></td><td>6</td><td>1</td><td>17</td></tr><tr><td colspan="8"><strong>Malaysian Borneo</strong></td></tr><tr><td>Sarawak</td><td>22</td><td>ND</td><td>48</td><td>8</td><td>ND</td><td>18</td><td>74</td></tr><tr><td>Sabah</td><td>13</td><td>ND</td><td>30</td><td>ND</td><td>ND</td><td></td><td>30</td></tr><tr><td>Total samples</td><td colspan="2">86</td><td>78</td><td>8</td><td>8</td><td>6</td><td>86 human + 14 macaque</td></tr><tr><td>Total sequences</td><td colspan="2">NA</td><td>157</td><td>9</td><td>24</td><td>19</td><td>166 human + 43 macaque</td></tr></tbody></table>

## Amplification and Sequencing of Gene Fragments

Genomic DNA was extracted from the human and macaque blood samples by using the DNeasy Blood Tissue Kit (QIA-GEN, Hilden, Germany), according to the manufacturers' protocol. An established nested PCR protocol was used to test the samples; all tested positive for *P. knowlesi* only (5).

The *PkA-type 18S rRNA* and *PkCOX1* genes were then amplified (MyCycler, Bio-Rad, Hercules, CA). In a primary amplification reaction, a *PkA-type 18S rRNA* fragment of 1.1. kb was obtained by using the oligonucleotide primer pair rPLU5+rPLU6 (18). The amplification reaction was completed in a mixture containing 1X Green Go Taq Flexi Buffer (Promega, Madison, WI, USA); 4.0 mol/L magnesium chloride solution; 0.2 mol/L dNTP Mix (Promega), 0.2 mM of each primer; 1 U GoTaq Flexi DNA Polymerase (Promega); and 4 mL of DNA template combined with nuclease-free water to obtain a final volume of 25 mL. The PCR amplification was initiated at 95°C for

10 min, then by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. For the secondary amplification, 4 mL of the primary amplification product were used as a template by using forward primer Pkl1 (5'-ACATAACTGATGCCTCCGCGTA) and reverse primer Pkl2 (5'-CACACATCGTTCCTCTAAGAAGC) to obtain a 986 990-bp fragment. The reaction mixture and the cycling conditions were as above with minor modification for the 35 cycles: denaturation at 94°C for 1 min, annealing at 53°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 10 min. The amplified PCR fragments were cloned into the pGEM-T Vector (Invitrogen, Carlsbad, CA, USA). Plasmids purified from ≥2 positive clones from each ligation mixture were selected for sequencing (First Base Laboratories Sdn Bhd, Malaysia). Any polymorphism that was not observed in ≥2 samples was only included in the analysis if its validity

<table><caption>**Table 2.** Samples and sequences obtained for the *PkCOX1* gene used to distinguish the 2 distinct *Plasmodium knowlesi* parasite populations in Malaysia*</caption><thead><tr><th rowspan="3">Location</th><th colspan="2">No. samples†</th><th colspan="4">No. sequences</th><th rowspan="3">Total sequences obtained</th></tr><tr><th colspan="2">Human</th><th colspan="2">Human</th><th colspan="2">Macaque</th></tr><tr><th>Human</th><th>Macaque</th><th>This study‡</th><th>GenBank§</th><th>This study‡</th><th>GenBank§</th></tr></thead><tbody><tr><td colspan="8"><strong>Peninsular Malaysia</strong></td></tr><tr><td>Kelantan</td><td>13</td><td>ND</td><td>15</td><td>ND</td><td>ND</td><td>ND</td><td>15</td></tr><tr><td>Johor</td><td>3</td><td>ND</td><td>4</td><td>ND</td><td>ND</td><td>ND</td><td>4</td></tr><tr><td>Selangor</td><td>7</td><td>5</td><td>5</td><td>ND</td><td>5</td><td>1</td><td>11</td></tr><tr><td>Terengganu</td><td>2</td><td>ND</td><td>1</td><td>ND</td><td>0</td><td>ND</td><td>1</td></tr><tr><td>Pahang</td><td>14</td><td>1</td><td>18</td><td>ND</td><td>2</td><td>1</td><td>21</td></tr><tr><td>Negeri Sembilan</td><td>4</td><td>2</td><td>ND</td><td>1</td><td>1</td><td>ND</td><td>2</td></tr><tr><td colspan="8"><strong>Malaysian Borneo</strong></td></tr><tr><td>Sarawak</td><td>22</td><td>ND</td><td>23</td><td>21</td><td>ND</td><td>22</td><td>66</td></tr><tr><td>Sabah</td><td>13</td><td>ND</td><td>18</td><td>ND</td><td>ND</td><td>ND</td><td>18</td></tr><tr><td>Total samples</td><td colspan="2">NA</td><td>78</td><td>22</td><td>8</td><td>12</td><td>100 human + 20 macaque</td></tr><tr><td>Total sequences</td><td colspan="2">NA</td><td>84</td><td>22</td><td>8</td><td>24</td><td>106 human + 32 macaque</td></tr></tbody></table>

<footnote>*NA, not applicable; ND, no data; PkA-type 18S rRNA, P. knowlesi type A small subunit ribosomal 18S RNA.</footnote>

<footnote>†Previously collected samples used for this study.</footnote>

<footnote>‡GenBank accession nos. KT852845–KT852938 and KJ917815–KJ917903.</footnote>

<footnote>§GenBank accession numbers listed in online Technical Appendix Table (http://wwwnc.cdc.gov/EID/article/22/8/15-1885-Techapp1.pdf).</footnote>

<footnote>*NA, not applicable; ND, no data; PkCOX1 *P. knowlesi* cytochrome oxidase subunit I.</footnote>

<footnote>†Previously collected samples used for this study.</footnote>

<footnote>‡GenBank accession nos. KT900705–KT900797).</footnote>

<footnote>§GenBank accession numbers listed in online Technical Appendix Table (http://wwwnc.cdc.gov/EID/article/22/8/15-1885-Techapp1.pdf).</footnote>
was confirmed by a repeated cycle of amplification, clon-
ing, and sequencing.

For the *PkCOX1* gene, amplification was achieved by
using forward (5'-GCCAGGATTATTTGGAGG) and re-
verse (5'-CAGGAATACGTCTAGGCA) primers to obtain
a 1,116-bp fragment. These primers were designed based
on a published gene sequence (GenBank accession no.
AY598141). The amplification reaction was achieved as
above. The PCR amplification was initiated at 95°C for 3
min, then denatured for 35 cycles at 94°C for 1 min, an-
nealed at 52°C for 1 min, extended at 72°C for 1 minute,
and put through final extension at 72°C for 10 min. The
purified amplified fragments were then sent for sequencing.

Sequence Editing and Alignment

We analyzed the DNA sequences using BioEdit Sequence
Alignment Editor Software (http://www.mbio.ncsu.edu/
BioEdit/bioedit.html) on the reference *P. knowlesi* H-strain
(GenBank accession no. AM910985) for the *PkA-type 18S*
*rRNA* and the *P. knowlesi* mitochondrial sequence (Gen-
Bank accession no. NC 00723244) for the *PkCOX1* gene.
Results were exported to MEGA 5.6 software (http://www.
megasoftware.net) for further alignment and analysis. We
performed similarity searches using BLAST (http://blast.
ncbi.nlm.nih.gov/Blast.cgi). We obtained 28 additional
*PkA-type 18S RNA* sequences derived from *P. knowlesi*-
infected samples (9 from humans and 19 from macaques)
and 46 additional *PkCOX1* sequences derived from *P.
knowlesi*-infected samples (22 from humans and 24 from
macaques) from GenBank and included these sequences in
the analysis (online Technical Appendix Table).

Haplotype Network Analysis

We estimated polymorphism of the *PkA-type 18S rRNA* and *PkCOX1* genes by computing haplotype diversity (Hd), number of haplotypes (h), nucleotide diversity (p), number of polymorphic sites, and the average number of pairwise nucleotide differences using DnaSP version 5.10.01 software (BioSoft http://en.bio-soft.net/). We constructed haplotype networks for *PkA-type 18S rRNA* and *PkCOX1* genes based on their polymorphic sites by using the median-joining method in NETWORK version 4.6.1.2 software (Fluxus Technology Ltd, Suffolk, UK). We inferred the genealogical haplotype network using the sequences of *P. knowlesi* human and macaque isolates from Peninsular Malaysia and Malaysian Borneo. Where available, we included sequences from the *P. knowlesi* H and Nuri strains as references.

Population Genetic Structure Analysis

To define genetic structure of the *P. knowlesi* parasite
population in Malaysia, we used STRUCTURE version
2.3.4 software (The Pritchard Lab, Stanford University,

Stanford, CA, USA) that deploys the Bayesian model-
based clustering approach. We estimated the most proba-
ble number of populations (K) using an admixture model.
All sample data (for both genes) were run for values K =
1–8, each with a total of 15 iterations. We used 500,000
Markov Chain Monte Carlo generations for each run af-
ter a burn-in of 50,000 steps. The most likely number K
in the data was estimated by calculating ΔK values and
identifying the K value that maximizes the log probability
of data, lnP(D) (19). The most probable K value was then
calculated according to Evanno's method (20) by using
the webpage interface STRUCTURE Harvester (21). We
also used ARLEQUIN version 3.5.1.3 software (Univer-
sity of Berne, Berne, Switzerland) to compute pairwise
differences (*F*<sub>ST</sub>) between populations (i.e., humans and
macaques from Peninsular Malaysia and Malaysian Bor-
neo) (22) from haplotypes that showed 10,100 permuta-
tions. *F*<sub>ST</sub> is a comparison of the sum of genetic variability
within and between populations on the basis of the dif-
ferences in allelic frequencies. We interpreted *F*<sub>ST</sub> values
as no (0), low (>0–0.05), moderate (0.05–0.15), and high
(0.15–0.25) genetic differentiation.

Neutrality and Demographic Analysis

We examined departure from a strict neutral model, includ-
ing demographic expansions, on the basis of pairwise mis-
match distribution, the Tajima D test (23), Fu and Li D (24),
Fu and Li F, and Fu Fs statistics (25) using DnaSP version.
5.10.01 software (26). Significant negative values for these
tests indicate either a purifying selection or population ex-
pansion, and positive values indicate balancing selection.

Results

The sequences analyzed in this study were derived from
130 *P. knowlesi*-infected blood samples obtained from 23
macaques and 107 humans. We analyzed a total of 209
*PkA-type 18S RNA* sequences (105 from Peninsular Ma-
laysia and 104 from Malaysian Borneo) and 138 *PkCOX1*
sequences (54 from Peninsular Malaysia and 84 from Ma-
laysian Borneo).

Gene Diversity Indices

Analysis of the molecular polymorphism within the
209 partial *PkA-type 18S rRNA* sequences (945 bp) re-
vealed moderately polymorphic sequences (p = 0.00324 ±
0.00019). Overall, 137 polymorphic sites yielded 93 hap-
lotypes. Nucleotide and haplotype diversities were broadly
similar for both Peninsular Malaysia and Malaysian Bor-
neo samples (Table 3). Single-nucleotide polymorphisms
were scattered throughout the gene; most the Peninsular
Malaysia sequences displayed a distinct single nucleotide
polymorphism (G→A at position 830) (online Technical
Appendix Table).
<table><caption>**Table 3.** Genetic characteristics of the 2 genetically distinct zoonotic *Plasmodium knowlesi* parasite populations in Malaysia*</caption><thead><tr><th>Gene and location</th><th>No. samples</th><th>No. haplotypes</th><th>No. polymorphic sites</th><th>Haplotype diversity ±SD</th><th>Nucleotide diversity ±SD</th><th>Average. no. nucleotide differences</th></tr></thead><tbody><tr><td><strong>PkA-type 18S rRNA</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Peninsular Malaysia</td><td>105</td><td>47</td><td>62</td><td>0.906 ±0.0.025</td><td>0. 0.00284 ±0.00024</td><td>2.66667</td></tr><tr><td>Malaysian Borneo</td><td>105</td><td>48</td><td>85</td><td>0.871 ±0.031</td><td>0.00276 ±0.0.00029</td><td>2.59341</td></tr><tr><td>Overall</td><td>210</td><td>93</td><td>137</td><td>0.941 ±0.0.011</td><td>0.00324 ±0.00019</td><td>3.03550</td></tr><tr><td><strong>PkCOX1</strong></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Peninsular Malaysia</td><td>53</td><td>20</td><td>20</td><td>0.827 ±0.039</td><td>0.00141 ±0.00018</td><td>1.52685</td></tr><tr><td>Malaysian</td><td>84</td><td>25</td><td>25</td><td>0.676 ±0.057</td><td>0.00115 ±0.00014</td><td>1.24498</td></tr><tr><td>Borneo</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Overall</td><td>137</td><td>44</td><td>39</td><td>0.848 ±0.025</td><td>0.00215 ±0.00013</td><td>2.33104</td></tr></tbody><tfoot><tr><td colspan="7"><em>*PkA-type 18S rRNA, P. knowlesi type A small subunit ribosomal 18S RNA; PkCOX1 P. knowlesi cytochrome oxidase subunit I.</em></td></tr></tfoot></table>

Analysis of the molecular polymorphism within the 138 partial *P. knowlesi* mitochondrial *COX1* sequences (1,082 bp) revealed low instances of polymorphism (p = 0.00215 ± 0.00013). Overall, 61 polymorphic sites yielded 44 haplotypes. Although nucleotide diversities were similar for sequences from both regions, haplotype diversity was higher for the sequences from Peninsular Malaysia (h = 19, Hd = 0.827 ± 0.039) than for those from Malaysian Borneo (h = 25, Hd = 0.676 ± 0.057) (Table 3). Sequences from Malaysian Borneo were distinguished from those from Peninsular Malaysia by 2 distinct single nucleotide polymorphisms (G→A at position 166 and T→C at position 659) (online Technical Appendix figure).

## Haplotype network

DNA sequence variation in our phylogeographic study is more clearly observed in a haplotype network. The network tree for the *PkA-type 18S RNA* haplotypes (Figure 2) showed 2 distinct *P. knowlesi* populations that, with 1 exception, clustered exclusively to 1 of the 2 regions of Malaysia: Peninsular Malaysia (n = 47) and Malaysian Borneo (n = 48). The exception was of 2 haplotypes derived from 1 macaque sample (haplotypes 88 and 89) from Peninsular Malaysia that clustered with the Malaysian Borneo haplotypes. In each cluster, only 2 haplotypes were shared by humans and macaques, but in each case 1 was dominant (in Malaysian Borneo, haplotype 1: n = 38, human = 27,

<figure><img src="image_4.png" /></figure>

**Figure 2.** Median-joining networks of *Plasmodium knowlesi* type A small subunit ribosomal 18S RNA haplotypes from Malaysia. The genealogical haplotype network shows the relationships among the 93 haplotypes present in the 209 sequences obtained from human and macaque samples from Peninsular Malaysia and Malaysian Borneo. Each distinct haplotype has been designated a number (H_n). Circle sizes represent the frequencies of the corresponding haplotype (the number is indicated for those that were observed >1×). Small red nodes are hypothetical median vectors created by the program to connect sampled haplotypes into a parsimonious network. Distances between nodes are arbitrary.
macaque = 11; and in Peninsular Malaysia, haplotype 9: n = 31, human = 27, macaque = 4). The network tree for the *PkCOX1* genes showed a similar pattern to that of *PkA-type 18S rRNA*: it had 2 geographically distinct *P. knowlesi* populations (n = 19 for Peninsular Malaysia and n = 25 for Malaysian Borneo) (Figure 3), and dominant haplotypes in each cluster were shared between humans and macaques (Figure 3).

The excess of unique *PkA-type 18S rRNA* and *PkCOX1* haplotypes observed for the *P. knowlesi* populations in humans (Figures 2, 3) is indicative of an evolutionarily recent population expansion. A signature of population expansion was also evident from the unimodal shape of the pairwise mismatch distribution of the *PkA-type 18S rRNA* and *PkCOX1* genes (Figure 4). Calculations by using Tajima D, Fu and Li D and F, and Fu Fs statistics also showed significant negative values (p = 0.05–0.001; Table 4). However, the low number of samples and consequent sequences from macaques precludes any meaningful comparison.

## Population Structure

We used a Bayesian admixture model implemented in STRUCTURE to calculate the potential number of *P. knowlesi* parasite populations within Malaysia. Because the study samples were collected from 8 different states of Malaysia (Figure 1), we used K values from 1 to 8 for the analysis. For both genes, significant genetic structure was found between the parasite populations when K = 2 (Figure

5), indicating 2 distinct populations clustered to 1 of the 2 main regions of Malaysia (*PkA-type 18S rRNA*, K = 2, ΔK = 121.79; *PkCOX1*, K = 2, ΔK = 481.27). In *PkA-type 18S rRNA* and *PkCOX1* sequences, we also estimated pairwise *F*<sub>ST</sub> values using ARLEQUIN software to determine to what extent population differentiation exists within *P. knowlesi* in Malaysia on the basis of host and geographic origin, i.e., between humans and macaques and between Peninsular Malaysia and Malaysian Borneo. This analysis revealed particularly high population differentiation *F*<sub>ST</sub> values (>0.21 for *PkA-type 18S rRNA* and >0.60 for *PkCOX1*) for samples originating from Peninsular Malaysia and Malaysian Borneo irrespective of the host (human or macaque) from which they were collected (Table 5). For the macaque and human population within the same geographic region, the *F*<sub>ST</sub> values were low (<0.05) (Table 5), suggesting that parasitic transmission was confined to each of the regions. These results are concordant with the haplotype network analysis.

## Discussion

The results of the various analyses conducted on the *P. knowlesi* parasites collected from Peninsular Malaysia and Malaysian Borneo strongly support the conclusion that the 2 geographically separated regions of this country harbor genetically distinct *P. knowlesi* populations. Haplotype diversity, a measure of species evenness (low values indicate skewing toward a few predominant haplotypes), was high

<figure><img src="image_5.png" /></figure>

**Figure 3.** Median-joining networks of *Plasmodium knowlesi* cytochrome oxidase subunit I haplotypes from Malaysia. The genealogical haplotype network shows the relationships among the 44 haplotypes present in the 138 sequences obtained from human and macaque samples from Peninsular Malaysia and Malaysian Borneo. Each distinct haplotype has been designated a number (H_n). Circle sizes represents the frequencies of the corresponding haplotype (the number is indicated for those that were observed more than once). Small red nodes are hypothetical median vectors created by the program to connect sampled haplotypes into a parsimonious network. Distances between nodes are arbitrary.
<figure><img src="image_6.png" /><figcaption>**Figure 4.** Pairwise mismatch distribution of *Plasmodium knowlesi* parasite populations, Malaysia. A) Type A small subunit ribosomal 18S RNA; B) cytochrome oxidase subunit I. Red dotted lines represent the observed frequencies of the pairwise differences among mitochondrial DNA sequences; blue lines represent the expected curve for a population that has undergone a demographic expansion.</figcaption></figure>

for both the Peninsular Malaysia (0.906 ± 0.0.025) and the Malaysian Borneo (0.871 ± 0.031) isolates, which may indicate a sustained transmission of *P. knowlesi* in both regions of Malaysia over long periods. Similar high haplotype diversity values have been reported for the *P. knowlesi* *csp* gene in isolates from Sarawak (17). Nucleotide diversity, however, was low for the genes analyzed in this study, irrespective of the samples' geographic or host origins, indicating that only minor differences occurred between the haplotypes observed (online Technical Appendix Table, Figure). A similar pattern has been observed for geographically separated *P. vivax* populations between which gene flow is limited (14).

The relatively large sample group size and the short genetic distances between the intraspecific sequences in this study are not suited for phylogenetic studies that aim to reconstruct genealogies. Therefore, we subjected the sequences to a median-joining haplotype network analysis, which showed that the network consisted mostly of unique haplotypes that clearly form 2 clusters: 1 comprised the samples obtained from Peninsular Malaysia, and the other the samples from Malaysian Borneo (Figures 2, 3). Within each cluster, the dominant haplotypes were shared

between humans and macaques; a similar observation was previously reported for a sample set collected in Sarawak (17). Additional population structure analyses showed very high genetic differentiation between 2 distinct *P. knowlesi* populations from the 2 geographic regions and very low genetic differentiation between the human and macaque parasites within each of these regions (Figure 5). These observations strongly support the conclusion that humans are susceptible to infection by any of the *P. knowlesi* types circulating in macaques.

The question arises as to how these 2 distinct populations arose. Analyses of the complete mitochondrial DNA revealed that *P. knowlesi* parasites were present in macaques around 65,000 years ago, before human settlement in Southeast Asia (17). Although DNA sequences isolated in our study are too short to perform a comparable analysis, it has been proposed that the macaque populations of Borneo became isolated from those of Peninsular Malaysia, Java, and Sumatra around 15,000 years ago, when the rise in the level of the South China Sea at the end of the last ice age submerged parts of Sundaland (27). Thus, *P. knowlesi* populations likely became isolated, along with their natural vertebrate and insect hosts, and consequently

<table><caption>**Table 4.** Results of statistical testing for neutrality of *Plasmodium knowlesi* parasite populations in Malaysia</caption><thead><tr><th>Gene and location</th><th>Tajima D (23)</th><th>Fu and Li D (24)</th><th>Fu and Li F (25)</th><th>Fu Fs (26)</th></tr></thead><tbody><tr><td><strong>PkA-type 18S rRNA</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Peninsular Malaysia</td><td>-2.49764*</td><td>-3.71579†</td><td>-3.85230†</td><td>-56.133</td></tr><tr><td>Malaysian Borneo</td><td>-2.73586*</td><td>-6.47796†</td><td>-5.87592†</td><td>-59.698</td></tr><tr><td>Overall</td><td>-2.71801*</td><td>-7.08484†</td><td>-5.97285†</td><td>-151.882</td></tr><tr><td><strong>PkCOX1</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Peninsular Malaysia</td><td>-2.06554‡</td><td>-3.20519‡</td><td>-3.33283†</td><td>-18.092</td></tr><tr><td>Malaysian Borneo</td><td>-2.28662†</td><td>-3.60846†</td><td>-3.71725†</td><td>-27.990</td></tr><tr><td>Overall</td><td>-2.02607‡</td><td>-4.40748†</td><td>-4.11218†</td><td>-48.131</td></tr></tbody></table>

<footnote>*PkA-type 18S rRNA, *P. knowlesi* type A small subunit ribosomal 18S RNA; *PkCOX1* *P. knowlesi* cytochrome oxidase subunit I.</footnote>

<footnote>†p<0.001.</footnote>

<footnote>‡p<0.02.</footnote>

<footnote>‡p<0.05.</footnote>
<figure><img src="image_7.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 5.** Most likely number of *Plasmodium knowlesi* parasite subpopulation haplotype clusters (K) in Malaysian Borneo (gray) and Peninsular Malaysia (white). A) Type A small subunit ribosomal 18S RNA (K = 2, ΔK = 121.79), including comparison of K and ΔK; B) cytochrome oxidase subunit I (K = 2, ΔK = 481.27), including comparison between K and DK. Relationships were determined by using Bayesian model-based STRUCTURE version 2.3.4 software (The Pritchard Laboratory, Stanford University, Stanford, CA, SUA). ΔK = mean (|L"(K)|)/sd(L(K)). Vertical axes represent membership coefficient values.</figcaption></figure>

evolved separately. This mechanism of geographic isolation and host demography is considered important in the differentiation and origin of *P. knowlesi* parasites and other closely related *Plasmodium* species, for which diversity is probably underestimated (16,28). We propose that the 2 distinct *P. knowlesi* populations currently circulating in Peninsular Malaysia and Malaysian of Borneo

correspond to 2 independently evolving populations. The *PkCOX1* haplotypes available for the *P. knowlesi* isolates from Peninsular Malaysia (Malayan, GenBank accession no. AB444106.1; Hackeri, accession no. AB444107.1; H, accession no. AB444108) fall within the Peninsular Malaysia cluster; corresponding *PkA-type 18S rRNA* sequences are not available.

<table><caption>**Table 5.** *F*<sub>ST</sub> results for pairwise population comparisons of 2 genetically distinct zoonotic *Plasmodium knowlesi* parasite populations and associated significance, Malaysia*†</caption><thead><tr><th rowspan="2">Gene and location</th><th rowspan="2">Haplotype</th><th colspan="4">F<sub>ST</sub> values</th></tr><tr><th>HuPen</th><th>MaPen</th><th>HuBor</th><th>MaBor</th></tr></thead><tbody><tr><td colspan="6"><strong>PkA-type 18S rRNA</strong></td></tr><tr><td rowspan="2">Peninsular Malaysia</td><td>HuPen</td><td>NA</td><td>‡</td><td>‡</td><td>‡</td></tr><tr><td>MaPen</td><td>0.0483</td><td>NA</td><td>‡</td><td>‡</td></tr><tr><td rowspan="2">Malaysian Borneo</td><td>HuBor</td><td>0.2710</td><td>0.2167</td><td>NA</td><td>NS</td></tr><tr><td>MaBor</td><td>0.3134</td><td>0.3090</td><td>0.0008</td><td>NA</td></tr><tr><td colspan="6"><strong>PkCOX1</strong></td></tr><tr><td rowspan="2">Peninsular Malaysia</td><td>HuPen</td><td>NA</td><td>§</td><td>‡</td><td>‡</td></tr><tr><td>MaPen</td><td>0.0461</td><td>NA</td><td>‡</td><td>‡</td></tr><tr><td rowspan="2">Malaysian Borneo</td><td>HuBor</td><td>0.6008</td><td>0.6303</td><td>NA</td><td>NS</td></tr><tr><td>MaBor</td><td>0.6219</td><td>0.6925</td><td>0.0007</td><td>NA</td></tr></tbody></table>

<footnote>*HuBor, human haplotypes from Malaysian Borneo; HuPen, human haplotypes from Peninsular Malaysia; MaBor, Macaque haplotypes from Malaysian Borneo; MaPen, macaque haplotype from Peninsular Malaysia; NA, not applicable; NS, not significant; *PkA-type 18S rRNA*, *P. knowlesi* type A small subunit ribosomal 18S RNA; *PkCOX1 P. knowlesi* cytochrome oxidase subunit I.</footnote>

<footnote>†ARLEQUIN (University of Berne, Berne, Switzerland) software package version 3.5.1.3 was used to compute pairwise differences between populations (i.e., humans and macaques from Peninsular Malaysia and Malaysian Borneo).</footnote>

<footnote>‡p<0.001; p values computed with 10,100 permutations.</footnote>

<footnote>§p<0.05.</footnote>
The 3 *P. knowlesi* subspecies proposed to date (12) were found in distinct locations (Taiwan, Java, and Peninsular Malaysia). These parasites were not studied further and confirmation of their subspecific status remains to be confirmed, but no material suitable for genetic analysis is available. We note that evidence of potential hybrid forms was obtained from a single sample from a macaque from Sabah and for the H strain. The low frequency of such forms suggests a recent admixture through increased human movement between the 2 geographic regions of Malaysia, biologic factors that limit their fitness, or both. The clear geographic genetic differentiation and the indiscriminate distribution between human and macaque hosts for the parasites within each cluster strongly indicate that *P. knowlesi* became zoonotic independently in the 2 regions. Given that *P. knowlesi* has long been zoonotic, the data also indicate that human-to-human transmission has not been established over time.

While our study was underway, *P. knowlesi* populations from Sarawak were shown to be dimorphic across their genome (29,30). Furthermore, analyses based on microsatellite diversity identified 2 distinct parasite groups across the whole of Malaysia that strongly cluster with the macaque host species but not in the human samples: 1 was dominant in *M. fascicularis* macaques and the other in *M. nemestrina* macaques (31). These distinct groups do not correspond to the 2 distinct clusters we propose because their pattern of distribution is incongruent with the marked geographic distribution we report. It is likely that they represent a more recent population structure driven by adaptation to the macaque as vector and host. Considering the differences in the mutation rates between mitochondrial and nuclear genes and microsatellites (32,33), the data from the microsatellite analyses would be expected to detect recent population structuring, and our data from the nuclear and mitochondrial genes would reveal a more ancient geographic isolation.

In conclusion, it is clear that the *P. knowlesi* malaria parasites are not a homogeneous group but form a complex of related types, possibly including emerging subspecies, of which evolution and distribution has been shaped by past and recent events. It is equally clear that further analyses encompassing a larger number of genetic loci or whole genomes from sample sets collected in Malaysia, as well as in neighboring countries, will be needed to obtain a more comprehensive picture of the phylogeographic distribution and population structure of *P. knowlesi*. The zoonotic potential of these parasites constitutes a threat to the efforts to eliminate malaria in Southeast Asia. Thus, more epidemiologic and biologic investigations are necessary to help devise strategies that will minimize if not eliminate this threat.

## Acknowledgments

The authors thank Noradilah Marzuki, Marlindawati Mohd Ali, Hanapi Seli, Isma Lyna Ismail, Lin Bee Bee, Fatimah Haslina Abdullah, Azizah Mustafa, Kartina Md. Noor, Azizon Othman, Masturah Che Abdullah Hairolafizah Sebeki, and Sarimah Hashim for their assistance in collecting samples and in microscopic observation.

This study was funded by University of Malaya High Impact Research Fund (UM-MOHE UM.C/625/1/HIR/MOHE/MED/09) and Post-graduate Research Grant (PV044/2012A).

Ms. Yusof is enrolled in the Master of Medical of Science program at Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. Her research interests include evolutionary genetics of malaria parasites.

## References

1. World Health Organization. World malaria report 2014. Geneva, Switzerland: World Health Organization; 2014 [cited 2/19/2015 Feb 19]. http://www.who.int/malaria/publications/world_malaria_report_2014/en

2. Knowles R, Das Gupta BM. A study of monkey-malaria, and its experimental transmission to man. (A preliminary report). Ind Med Gaz. 1932;67:301-20 [cited 2/19/2015 Feb 19]. http://www.cabdirect.org/abstracts/19322901628.html

3. Sinton JA, Mulligan HW. A critical review of the literature relating to the identification of the malarial parasites recorded from monkeys of the families *Cercopithecidae* and *Colobidae*. Rec Mal Surv Ind. 1932;3:357-80 [cited 2/19/2015 Feb 19]. http://www.cabdirect.org/abstracts/19342900941.html

4. Chin W, Contacos PG, Coatney GR, Kimball HR. A naturally acquired quotidian-type malaria in man transferable to monkeys. Science. 1965;149:865. http://dx.doi.org/10.1126/science.149.3686.865

5. Singh B, Sung LK, Matusop A, Radhakrishnan A, Shamsul SSG, Cox-Singh J, et al. A large focus of naturally acquired *Plasmodium knowlesi* infections in human beings. Lancet. 2004;363:1017-24. PubMed http://dx.doi.org/10.1016/S0140-6736(04)15836-4

6. Barber BE, William T, Dhararaj P, Anderios F, Grigg MJ, Yeo TW, et al. Epidemiology of Plasmodium knowlesi malaria in north-east Sabah, Malaysia: family clusters and wide age distribution. Malar J. 2012;11:401. http://dx.doi.org/10.1186/1475-2875-11-401

7. Yusof R, Lau YL, Mahmud R, Fong MY, Jelip J, Ngian HU, et al. High proportion of Knowlesi malaria in recent malaria cases in Malaysia. Malar J. 2014;13:168. http://dx.doi.org/10.1186/1475-2875-13-168

8. Cox-Singh J. Zoonotic malaria: *Plasmodium knowlesi*, an emerging pathogen. Curr Opin Infect Dis. 2012;25:530-6. http://dx.doi.org/10.1097/QCO.0b013e3283558780

9. Singh B, Daneshvar C. Human infections and detection of *Plasmodium knowlesi*. Clin Microbiol Rev. 2013;26:165-84. http://dx.doi.org/10.1128/CMR.00079-12

10. Coatney GR, Collins WE, Warren M, Contacos PG. The primate malarias. Washington (DC): US Department of Health, Education and Welfare; 1971.

11. Ahmed MA, Cox-Singh J. Plasmodium knowlesi—an emerging pathogen. ISBT Sci Ser. 2015;10(Suppl 1):134-40. http://dx.doi.org/10.1111/voxs.12115

12. Garnham PCC. Malaria parasites and other haemosporidia. Oxford: Blackwell Scientific Publications; 1966.

13. Pacheco MA, Battistuzzi FU, Junge RE, Cornejo OE, Williams CV, Landau I, et al. Timing the origin of human
malarias: the lemur puzzle. BMC Evol Biol. 2011;11:299.
http://dx.doi.org/10.1186/1471-2148-11-299

14. Taylor JE, Pacheco MA, Bacon DJ, Beg MA, Machado RL, Fairhurst RM, et al. The evolutionary history of *Plasmodium vivax* as inferred from mitochondrial genomes: parasite genetic diversity in the Americas. Mol Biol Evol. 2013;30:2050–64.
http://dx.doi.org/10.1093/molbev/mst104

15. Joy DA, Feng X, Mu J, Furuya T, Chotivanich K, Krettli AU, et al. Early origin and recent expansion of *Plasmodium falciparum*. Science. 2003;300:318–2. http://dx.doi.org/10.1126/science.1081449

16. Muehlenbein MP, Pacheco MA, Taylor JE, Prall SP, Ambu L, Nathan S, et al. Accelerated diversification of nonhuman primate malarias in Southeast Asia: adaptive radiation or geographic speciation? Mol Biol Evol. 2015;32:422–39.
http://dx.doi.org/10.1093/molbev/msu310

17. Lee K-S, Divis PCS, Zakaria SK, Matusop A, Julin RA, Conway DJ, et al. *Plasmodium knowlesi*: reservoir hosts and tracking the emergence in humans and macaques. PLoS Pathog. 2011;7:e1002015. http://dx.doi.org/10.1371/journal.ppat.1002015

18. Singh B, Bobogare A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg. 1999;60:687–92.

19. Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155:945–59.

20. Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol. 2005;14:2611–20. http://dx.doi.org/10.1111/j.1365-294X.2005.02553.x

21. Earl DA, von Holdt BM. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour. 2012;4:359–61 http://dx.doi.org/10.1007/s12686-011-9548-7.

22. Excoffier L, Lischer HE. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour. 2010;10:564–7.
http://dx.doi.org/10.1111/j.1755-0998.2010.02847.x

23. Tajima F. The effect of change in population size on DNA polymorphism. Genetics. 1989;123:597–601.

24. Fu YX. Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics. 1997;147:915–25.

25. Fu YX, Li WH. Statistical tests of neutrality of mutations. Genetics. 1993;133:693–709.

26. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25:1451–2. http://dx.doi.org/10.1093/bioinformatics/btp187

27. Hanebuth T, Stattegger K, Grootes PM. Rapid flooding of the sunda shelf: A late-glacial sea-level record. Science. 2000;288:1033–5.
http://dx.doi.org/10.1126/science.288.5468.1033

28. Pacheco MA, Reid MJC, Schillaci MA, Lowenberger CA, Galdikas BMF, Jones-Engel L, et al. The origin of malarial parasites in orangutans. PLoS One. 2012;7:e34990.
http://dx.doi.org/10.1371/journal.pone.0034990

29. Ahmed AM, Pinheiro MM, Divis PCS, Siner A, Zainudin R, Wong IT, et al. Disease progression in *Plasmodium knowlesi* malaria is linked to variation in invasion gene family members. PLoS Negl Trop Dis. 2014;8:e3086.
http://dx.doi.org/10.1371/journal.pntd.0003086

30. Pinheiro MM, Ahmed MA, Millar SB, Sanderson T, Otto TD, Lu WC, et al. *Plasmodium knowlesi* genome sequences from clinical isolates reveal extensive genomic dimorphism. PLoS One. 2015;10:e0121303. http://dx.doi.org/10.1371/journal.pone.0121303

31. Divis PC, Singh B, Anderios F, Hisam S, Matusop A, Kocken CH, et al. Admixture in humans of two divergent *Plasmodium knowlesi* populations associated with different macaque host species. PLoS Pathog. 2015;11:e1004888. http://dx.doi.org/10.1371/journal.ppat.1004888

32. Haas RJ, Payseur BA. Multi-locus inference of population structure: a comparison between single nucleotide polymorphisms and microsatellites. Heredity. 2011;106:158–71.
http://dx.doi.org/10.1038/hdy.2010.21

33. Lehmann T, Blackston CR, Besansky NJ, Escalante AA, Collins FH, Hawley WA. The Rift Valley complex as a barrier to gene flow for *Anopheles gambiae* in Kenya: the mtDNA perspective. J Hered. 2000;91:165–8. http://dx.doi.org/10.1093/jhered/91.2.165

Address for correspondence: Yee Ling Lau, Tropical Infectious Disease Research & Education Centre (TIDREC), Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia; email: lauyeeling@um.edu.my

<figure><img src="image_11.png" /></figure>

Thank You EID Reviewers

We couldn't do it without your support.

All articles published in the Emerging Infectious Diseases journal are peer-reviewed by volunteers from around the globe, enabling us to bring you high-quality content about new and emerging infectious diseases and trends world-wide. To see a list of reviewers who contributed to articles published in 2015, visit: http://wwwnc.cdc.gov/eid/pages/reviewers.htm
# Phylogeographic Evidence for 2 Genetically Distinct Zoonotic *Plasmodium knowlesi* Parasites, Malaysia

## Technical Appendix

**Technical Appendix Table.** Accession numbers of sequences retrieved from GenBank database included in analyses of phylogeographic evidence for two genetically distinct zoonotic *Plasmodium knowlesi* parasites in Malaysia

<table><thead><tr><th rowspan="3">Location</th><th colspan="2">PkA-type 18S rRNA</th><th colspan="2">PkCOX1</th></tr><tr><th colspan="2">Source</th><th colspan="2">Source</th></tr><tr><th>Humans</th><th>Macaques</th><th>Humans</th><th>Macaques</th></tr></thead><tbody><tr><td>Malaysian Borneo</td><td>7 Samples<br>KH33_(AY327549)<br>KH35_(AY327550)<br>KH35_(AY327552)<br>KH43_(AY327551)<br>KH96_(AY327553)<br>KH107_(AY327554)<br>KH115_(AY327555)<br>KH131_(AY327556)</td><td>5 Samples<br>A2_(DQ350264)<br>LT3-B6_(DQ641524)<br>LT3-C7_(DQ350266)<br>LT4-A1_(DQ350267)<br>LT4-A7_(DQ350268)<br>LT4-B1_(DQ350269)<br>LT4-C18_(FJ619069.1)<br>LT4-C20_(DQ641525)</td><td>21 Samples<br>KH45H1_(EU880446.1)<br>KH54H4_(EU880447.1)<br>KH58H2_(EU880448.1)<br>KH100H1_(EU880449.1)<br>KH108H12_(EU880451.1)<br>KH137H15_(EU880452.1)<br>KH176H2_(EU880454.1)<br>KH185H3_(EU880455.1)</td><td>10 Samples<br>LT3H20_(EU880472.1)<br>LT4H5_(EU880474.1)<br>LT15H21_(EU880475.1)<br>LT15H22_(EU880476.1)<br>LT20H5_(EU880479.1)<br>LT20H23_(EU880480.1)<br>LT20H24_(EU880481.1)<br>LT20H25_(EU880482.1)</td></tr><tr><td></td><td>NA</td><td>LT20-C1_(FJ619088)<br>LT20-C5_(FJ619090)</td><td>KH225H11_(EU880457.1)<br>KH229H4_(EU880458.1)</td><td>LT26H26_(EU880483.1)<br>LT26H27_(EU880484.1)</td></tr><tr><td></td><td>NA</td><td>LT22-A1_(DQ641518)<br>LT22-A17_(DQ641519)<br>LT22-B2_(DQ641520)</td><td>KH273H19_(EU880459.1)<br>KH275H13_(EU880460.1)<br>KH294H10_(EU880461.1)</td><td>LT48H6_(EU880485.1)<br>LT48H28_(EU880486.1)<br>LT51H6_(EU880487.1)</td></tr><tr><td></td><td>NA</td><td>LT22-B18_(DQ641521)<br>LT22-C4_(DQ641522)</td><td>KH343H18_(EU880462.1)<br>KH369H14_(EU880463.1)<br>KH381H3_(EU880465.1)</td><td>LT51H29_(EU880488.1)<br>LT53H30_(EU880489.1)<br>LT53H31_(EU880490.1)</td></tr><tr><td></td><td>NA</td><td>LT22-C7_(DQ641523)<br>LT33-D1_(FJ619097)</td><td>KH396H16_(EU880466.1)<br>KH397H8_(EU880464.1)</td><td>LT53H32_(EU880491.1)<br>LT54H33_(EU880493.1)</td></tr><tr><td></td><td>NA</td><td>LT33-D12_(FJ619098)</td><td>KH431H9_(EU880467.1)<br>KH433H7_(EU880468.1)</td><td>LT57H34_(EU880495.1)<br>LT57H35_(EU880496.1)</td></tr><tr><td></td><td>NA</td><td>NA</td><td>KH468H17_(EU880470.1)</td><td>LT57H36_(EU880497.1)</td></tr><tr><td></td><td>NA</td><td>NA</td><td>NA</td><td>LT57H37_(EU880498.1)</td></tr><tr><td>Peninsular Malaysia</td><td>AM910985 (Pk H strain)<br>NA</td><td>AY327557 (Pk Nuri)<br>NA</td><td>AB444108 (Pk H strain)<br>NA</td><td>AB444107.1 (Pk Hackeri)<br>AB444106.1 (Pk Malayan)</td></tr></tbody></table>
<figure><img src="image_12.png" /><figcaption>**Technical Appendix Figure.** Nucleotide polymorphism in the cytochrome oxidase 1 (cox1) gene of *Plasmodium knowlesi* isolates from humans and macaques, Peninsular Malaysia and Malaysian Borneo.</figcaption></figure>