Detection and Characterisation of Endosymbiont Wolbachia (Rickettsiales: Anaplasmataceae) in Elaeidobius kamerunicus (Coleoptera: Curculionoidea), Pollinating Agent of Oil Palm, and Its Relationships between Populations.

Elaeidobius kamerunicus is the most efficient pollinator of oil palm. Wolbachia is an endosymbiotic bacteria associated with E. kamerunicus that has a potential to affect the fecundity and fitness of the E. kamerunicus. Despite their importance, no studies have been conducted to investigate its prevalence in E. kamerunicus. The objectives of this study were to detect and characterise Wolbachia in E. kamerunicus and determine the phylogenetic relationship of Wolbachia strains that infect E. kamerunicus by using three genetic markers namely Filamenting temperature-sensitive mutant Z (ftsZ), Chaperonin folding protein (groEL), and Citrate Synthase Coding Gene (gltA). DNA was extracted from 210 individuals of E. kamerunicus and the Wolbachia infections were detected using the wsp marker. The infected samples (n = 25, 11.9%) were then sequenced using ftsZ, gltA and groEL markers for strain characterization. In this study, a combination of four markers was used to construct the phylogeny of Wolbachia. Similar topologies were shown in all trees; Neighbour-Joining (NJ), Maximum Parsimony (MP), and Bayesian Inference (BI), which showed the mixing of individuals that harbor Wolbachia between populations. Interestingly, Wolbachia on E. kamerunicus was claded together with the species Drosophila simulans under supergroup B. This is the first report of Wolbachia infecting E. kamerunicus which is very valuable and significant as one of the parameters to evaluate the quality of the E. kamerunicus population for sustaining its function as a great pollinator for oil palm.

Metagenomic profile of the bacterial communities associated with Ixodes granulatus (Acari: Ixodidae): a potential vector of tick-borne diseases.

Ixodes granulatus Supino, 1897 (Acari: Ixodida) is one of Malaysia's most common hard ticks and is a potential vector for tick-borne diseases (TBDs). Despite its great public health importance, research on I. granulatus microbial communities remains largely unexplored. Therefore, this study aimed to investigate the bacterial communities of on-host I. granulatus collected from three different recreational areas on the East Coast of Peninsular Malaysia using high throughput Next Generation Sequencing (NGS). A total of 9 females on-host I. granulatus were subjected to metabarcoding analysis targeting V3-V4 regions of 16S ribosomal RNA (rRNA) using the Illumina MiSeq platform. This study identified 15 bacterial phyla corresponding to 19 classes, 54 orders, and 90 families from 435 amplicon sequence variants (ASVs), revealing a diverse bacterial community profile. Together with 130 genera assigned, local I. granulatus harbored 4 genera of pathogens, i.e., Rickettsia da Rocha Lima, 1916 (Rickettsiales: Rickettsiaceae) (58.6%), Borrelia Swellengrebel 1907 (Spirochaetales: Borreliaceae) (31.6%), Borreliella Adeolu and Gupta 2015 (Spirochaetales: Borreliaceae) (0.6%), and Ehrlichia Cowdria Moshkovski 1947 (Rickettsiales: Ehrlichiaceae) (39.9%). Some endosymbiont bacteria, such as Coxiella (Philip, 1943) (Legionellales: Coxiellaceae), Wolbachia Hertig 1936 (Rickettsiales: Ehrlichiaceae), and Rickettsiella Philip, 1956 (Legionellales: Coxiellaceae), were also detected at very low abundance. Interestingly, this study reported the co-infection of Borrelia and Ehrlichia for the first time, instilling potential health concerns in the context of co-transmission to humans, especially in areas with a high population of I. granulatus. This study successfully characterized the tick microbiome and provided the first baseline data of I. granulatus bacterial communities in Malaysia. These results support the need for way-forward research on tick-associated bacteria using NGS, focusing on medically important species toward TBD prevention.

Development of Nuclear DNA Markers for Applications in Genetic Diversity Study of Oil Palm-Pollinating Weevil Populations.

The oil palm-pollinating weevil (Elaeidobius kamerunicus Faust) was introduced from Cameroon, West Africa, to Malaysia in 1981, and subsequently, to other oil palm-growing countries as well. This study aims to develop a set of robust E. kamerunicus-specific nuclear DNA markers to directly assess the genetic diversity of the weevil populations. A total of 19,148 SNP and 223,200 SSR were discovered from 48 weevils representing three origins (Peninsular Malaysia, Sabah, and Riau) using RAD tag sequencing. Subsequent filtering steps further reduced these to 1000 SNP and 120 SSR. The selected 220 SNP exhibited a polymorphism information content (PIC) of 0.2387 (±0.1280), and 8 SSR had the PIC of 0.5084 (±0.1928). These markers were found to show sufficient polymorphism, making it possible to assign 180 weevils into three major clusters from Ghana, Cameroon, and Southeast Asia (mainly in Malaysia and Indonesia). These DNA markers successfully confirmed the Cameroon origin of the Southeast Asian cluster. However, the presence of null alleles in the SSR markers, due to limited flexibility of the probe design on the short RAD tags, led to an underestimation of heterozygosity within the populations. Hence, the developed SNP markers turned out to be more efficient than the SSR markers in the genetic diversity assessment of the E. kamerunicus populations. The genetic information provides useful insight into developing guidelines for the genetic monitoring and conservation planning of E. kamerunicus.

Metabarcoding of Parasitic Wasp, Dolichogenidea metesae (Nixon) (Hymenoptera: Braconidae) That Parasitizing Bagworm, Metisa plana Walker (Lepidoptera: Psychidae).

Microbiome studies of the parasitoid wasp, Dolichogenidea metesae (Nixon) (Hymenoptera, Braconidae) are important because D. metesae has potential as a biological control agent to suppress the pest, Metisa plana Walker (Lepidoptera, Psychidae). Three field populations of parasitic wasps with different Integrated Pest Management (IPM) practices to control M. plana collected from Perak state (Tapah) and Johor state (Yong Peng and Batu Pahat districts) in Peninsular Malaysia were studied. Bacterial community composition and structure were analysed using α and ß diversity metrics. Proteobacteria (83.31%) and Bacteroidetes (6.80%) were the most dominant phyla, whereas unknown family from order Rhizobiales was the most abundant family found in all populations followed by Pseudomonadaceae. Family Micrococcaceae was absent in Tapah. Rhizobiales gen. sp. and Pseudomonas sp. were abundant in all populations. Pearson's correlation analysis showed the strongest correlation between individuals of Batu Pahat and Yong Peng (r = 0.89827, p < 0.05), followed by Tapah and Yong Peng with r = 0.75358, p < 0.05 and Batu Pahat and Tapah (r = 0.69552, p < 0.05). We hypothesise that low diversity and richness in Tapah might be due to direct and indirect effect of insecticides application. This preliminary data was the first study to do inventory of the microbiomes in the gut of the D. metesae.

Kajian mikrobiom ke atas spesies penyengat parasitoid, Dolichogenidea metesae (Nixon) (Hymenoptera, Braconidae) adalah penting kerana spesies ini berpotensi sebagai agen kawalan biologi untuk mengawal spesies perosak, Metisa plana Walker (Lepidoptera, Psychidae). Tiga populasi penyengat parasitoid yang mengaplikasikan Pengurusan Perosak Bersepadu (IPM) yang berbeza untuk mengawal M. plana iaitu daripada negeri Perak (Tapah) dan negeri Johor (Yong Peng dan Batu Pahat) di Semenanjung Malaysia telah dikaji. Komposisi dan struktur komuniti bakteria telah dianalisis menggunakan metrik kepelbagaian α dan ß. Proteobacteria (83.31%) dan Bacteroidetes (6.80%) merupakan filum yang paling dominan, manakala famili yang tidak diketahui daripada order Rhizobiales ialah famili yang paling melimpah ditemui dalam kesemua populasi diikuti oleh Pseudomonadaceae. Famili Micrococcaceae didapati tidak hadir di populasi Tapah. Rhizobiales gen. sp. dan Pesudomonas sp. adalah paling melimpah di dalam semua populasi. Analisis korelasi Pearson menunjukkan korelasi yang paling tinggi di antara individu daripada Batu Pahat dan Yong Peng (r = 0.89827, p < 0.05) diikuti oleh Tapah dan Yong Peng dengan nilai r = 0.75358, p < 0.05 dan Batu Pahat dan Tapah (r = 0.69552, p < 0.05). Kami menghipotesis bahawa kepelbagaian dan kelimpahan yang rendah di Tapah mungkin disebabkan oleh kesan langsung dan tidak langsung penggunaan racun serangga. Data awalan ini merupakan kajian pertama yang melakukan inventori mikrobiom ke atas usus D. metesae.

First Record of the Tortoise Tick, Amblyomma geoemydae (Cantor, 1847) (Acari: Ixodidae) Parasitizing a Tree Shrew, Tupaia glis (Scandentia: Tupaiidae) in West Malaysia.

The tick genus Amblyomma Koch, 1844 (Acari: Ixodidae) has received little attention in Malaysia; therefore, its associated hosts and distribution records are poorly known. In this study, we collected six Amblyomma sp. individuals (two larvae and four adults) that infested a common treeshrew, Tupaia glis (Diard, 1820) (Scandentia: Tupaiidae) caught in a recreational area in Sungai Lembing, Pahang (West Malaysia). The adult female ticks were morphologically identified according to taxonomic keys prior to molecular identification using cytochrome oxidase subunit I (COI) and 16S rDNA genes. The ticks were genetically verified as Amblyomma geoemydae (Cantor, 1847) with 98%-99% similarity to the available GenBank sequences. Neighbor-joining (NJ) trees indicated that A. geoemydae was clearly distinguished from other Amblyomma ticks and this was supported with high bootstrap values. This paper is the first to report A. geoemydae ticks infesting T. glis and provides a new tick-host record from West Malaysia. This information is significant for further investigation, specifically on this tick species as potential vector of tick-borne disease (TBD) agents.

A new genus Anamalysia van Achterberg (Hymenoptera, Braconidae, Alysiinae), six new species, and two new combinations from India, Indonesia, Malaysia, Singapore, Thailand, and Vietnam.

A new genus of the tribe Alysiini (Hymenoptera, Braconidae, Alysiinae) is described with specimens from India, Indonesia, Malaysia, Singapore, Thailand, and Vietnam, and six new species are described: Anamalysiaidiastimorpha sp. nov. (type species), A.knekosoma sp. nov., A.mellipes sp. nov., A.transversator sp. nov., A.vandervechti sp. nov., and A.vanhengstumi sp. nov.. We transfer one species from Coelalysia to Anamalysia: A.urbana (Papp, 1967) comb. nov. from Singapore and one species from Alysiasta to Anamalysia: A.triangulum (Fischer, 2006) comb. nov. from Malaysia, Laos, Indonesia and Vietnam. A key to the genus of Anamalysia is included.

First report on metabarcoding analysis of gut microbiome in Island Flying Fox (Pteropushypomelanus) in island populations of Malaysia.

Flying fox (Pteropushypomelanus) belongs to the frugivorous bats, which play a crucial role in maintaining proper functioning of an ecosystem and conservation of the environment. Bats are well-known carriers of pathogenic viruses, such as BatCov RaTG13 from the coronavirus family that share 90.55% with SARS-CoV-2, the pathogen causing recent global pandemic coronavirus disease 19 (COVID-19). However, bats' possible role as a carrier of pathogenic bacteria is less explored. Here, using metabarcoding analysis through high-throughput sequencing, we explored the gut microbiome composition of different island populations on the east and west coasts of Peninsula Malaysia. The 16S rRNA gene in samples from Redang Island, Langkawi Island, Pangkor Island and Tinggi Island was amplified. Bacterial community composition and structure were analysed with α and ß diversity metrics. A total of 25,658 operational taxonomic units at 97% similarity were assigned to eight phyla, 44 families, 61 genera and 94 species of microbes. The Proteobacteria was the dominant phylum in all populations. Meanwhile, the genera Enterobacter, Pseudomonas and Klebsiella, isolated in this study, were previously found in the rectum of other fruit bats. Our analyses suggest that Redang Island and Langkawi Island have high bacteria diversity. Thus, we found geographic locality is a strong predictor of microbial community composition and observed a positive correlation between ecological features and bacterial richness.

Metabarcoding data analysis revealed the plant dietary variation of long-tailed macaque Macacafascicularis (Cercopithecidae, Cercopithecinae) living in disturbed habitats in Peninsular Malaysia.

The long-tailed macaque (Macacafascicularis) has a wide range in both Peninsular Malaysia and Borneo. Although the primates are especially vulnerable to habitat alterations, this primate lives in disturbed habitats due to human-induced land-use. Thus, this study presents a faecal metabarcoding approach to clarify the plant diet of long-tailed macaques from five locations in Peninsular Malaysia to represent fragmented forest, forest edge, island and recreational park habitats. We extracted genomic DNA from 53 long-tailed macaque faecal samples. We found 47 orders, 126 families, 609 genera and 818 species across these five localities. A total of 113 plant families were consumed by long-tailed macaques in Universiti Kebangsaan Malaysia, 61 in the Malaysia Genome and Vaccine Institute, 33 in Langkawi Island, 53 in Redang Island and 44 in the Cenderawasih Cave. Moraceae (33.24%) and Fabaceae (13.63%) were the most common families consumed by long-tailed macaques from the study localities. We found that habitat type impacted diet composition, indicating the flexibility of foraging activities. This research findings provide an understanding of plant dietary diversity and the adaptability of this macaque with the current alteration level that applies to long-tailed macaque conservation management interest in the future.

Morphological and molecular identification of medically important questing Dermacentor species collected from some recreational areas of Peninsular Malaysia.

Questing is a situation when a tick is seeking to get closer or ambush its potential host. However, information on questing tick species in Malaysia is still lacking, thus the association with tick-borne diseases (TBD) is not completely understood. The aim of this study was to investigate the tick species from five most frequently visited recreational areas in Pahang and Terengganu states, which were recorded to have high potential of TBD cases. By implementing handpick method, a total of 18 males and 15 females belonging to five Dermacentor Koch, 1844 species, were collected, namely D. compactus Neumann 1901, D. tricuspis (Schulze, 1933), D. auratus Supino 1897, D. steini (Schulze, 1933), and D. falsosteini Apanaskevich, Apanaskevich & Nooma respectively. The specimens were collected and identified based on morphological characters prior to obtaining the molecular data of COI and 16S rDNA. The D. compactus was the most abundant species collected in this study, while D. falsosteini was the least. All species were distinctly separated on the Neighbor Joining and Maximum Parsimony tree topologies and supported with high bootstrap values. Furthermore, a low intraspecific variation (0.00 - 0.01) was observed amongst the individuals of the same species in both genes. Meanwhile, each Dermacentor species was genetically different, with interspecific values ranging from 0.13-0.19 and 0.11-0.20 for COI and 16S rDNA. These findings had successfully recorded the tick species that were potentially associated with TBD, and which might be circulated among humans and animals. This study also has some implications on the diversity and geographical extension of Dermacentor ticks, thus should warrant further investigation as a potential vector of tick-borne diseases and public health importance.

Metabarcoding in Diet Assessment of Heterotrigona itama Based on trnL Marker towards Domestication Program.

Honey quality is the main criterion used for evaluating honey production in the stingless bee Heterotrigona itama, and it is correlated with the plant species consumed as its main diet. The objective of this study was to obtain the metabarcode data from 12 populations of H. itama species throughout Malaysia (Borneo and Peninsular Malaysia) using the trnL marker. A total of 262 species under 70 families and five phyla of plants were foraged by H. itama in the studied populations. Spermatophyta and Magnoliophyta were recorded as the two most abundant phyla foraged, at 55.95% and 32.39%, respectively. Four species, Garcinia oblongifolia, Muntingia calabura, Mallotus pellatus, and Pinus squamata, occurred abundantly and were consumed by H. itama in all the populations. These data are considered as a fundamental finding that is specific to the diet of H. itama for strategizing the management of the domestication process specifically in a mono-cropping system and in a netted structure. Thus, based on these findings, we recommend Momordica charantia, Melastoma sp., and Cucumis sativa as the best choices of food plant species to be planted and utilized by H. itama in meliponiculture.

Diet Composition of the Wild Stump-Tailed Macaque (Macaca arctoides) in Perlis State Park, Peninsular Malaysia, Using a Chloroplast tRNL DNA Metabarcoding Approach: A Preliminary Study.

Understanding dietary diversity is a fundamental task in the study of stump-tailed macaque, Macaca arctoides in its natural habitat. However, direct feeding observation and morphological identification using fecal samples are not effective and nearly impossible to obtain in natural habitats because this species is sensitive to human presence. As ecological methods are challenging and time-consuming, DNA metabarcoding offers a more powerful assessment of the diet. We used a chloroplast tRNL DNA metabarcoding approach to identify the diversity of plants consumed by free-ranging M. arctoides in the Malaysia-Thailand border region located in Perlis State Park, Peninsular Malaysia. DNA was extracted from three fecal samples, and chloroplast tRNL DNA was amplified and sequenced using the Illumina MiniSeq platform. Sequences were analyzed using the CLC Genomic Workbench software. A total of 145 plant species from 46 families were successfully identified as being consumed by M. arctoides. The most abundant species were yellow saraca, Saraca thaipingensis (11.70%), common fig, Ficus carica (9.33%), aramata, Clathrotropis brachypetala (5.90%), sea fig, Ficus superba (5.44%), and envireira, Malmea dielsiana (1.70%). However, Clathrotropis and Malmea are not considered Malaysian trees because of limited data available from Malaysian plant DNA. Our study is the first to identify plant taxa up to the species level consumed by stump-tailed macaques based on a DNA metabarcoding approach. This result provides an important understanding on diet of wild M. arctoides that only reside in Perlis State Park, Malaysia.

Effect of Irradiating Puparia of Oriental Fruit Fly (Diptera: Tephritidae) on Adult Survival and Fecundity for Sterile Insect Technique and Quarantine Purposes.

The aim of this study was to investigate the effect of gamma irradiation on survivability (adult emergence, sex ratio, adult longevity), fecundity (eggs hatchability, number of eggs produced), and morphological differences in the size of the ovary and testes of unirradiated and irradiated adults of Oriental fruit fly [Bactrocera dorsalis (Hendel)]. A dose of 100 Gy was determined as the minimum needed for inhibitory effects against B. dorsalis that would not deter adult emergence, sex ratio (male:female), and adult longevity, with 82.6 ±7.02, 1:1.09, and 107 ± 24.5, respectively. Doses from 50 to 400 Gy range did not completely prevent the adult emergence; however, the emergence decreased by increasing the radiation dose. Adult survivability significantly decreased among all the treatment groups of B. dorsalis, except for the 50 Gy and unirradiated flies. At a dose of 50 Gy, fertile females showed a significant reduction in fecundity by not producing eggs after mating with the sterile males. Meanwhile, the number of eggs laid decreased with increasing dose and no fertile egg was hatched starting at 100 Gy of irradiation. Testes and ovaries of 20-d old flies irradiated as pupae were smaller than those of control flies. In this study, 100 Gy was concluded as the minimum effective dose for the disinfestation and sterilization of B. dorsalis puparia. Results represent new findings used as a basis for sterile insect technique and quarantine programs for managing B. dorsalis, particularly in Malaysia.

Sequence variation data of the mitochondrial DNA D-loop region of the captive Malayan Gaur (Bos gaurus hubbacki).

This article contains data of the sequence variation in the mitochondrial DNA D-loop region of the Malayan gaur (Bos gaurus hubbacki), locally known as the seladang, from two captive centers. Thirty fecal samples of Malayan gaur were collected from Jenderak Selatan Wildlife Conservation Center (Pahang) and the Sungkai Wildlife Reserve (Perak) for DNA extraction and amplification with polymerase chain reactions. DNA sequences were then analyzed using neighbor joining (NJ) and maximum parsimony (MP) methods. Based on the 652 base pairs obtained, we found seven variable characters with a value of 1%. The genetic distance between the two captive centers was 0.001. Haplotype analyses detected only four haplotypes between these two captive centers. Both NJ and MP trees demonstrate that all individuals in the Jenderak and Sungkai captive centers are in the same clade. Genetic variation of the Malayan gaur in these centers is considered low, possibly because individuals share the same common parent. This sequence variation data are of paramount importance for designing a proper breeding and management program of the Malayan gaur in the future.

Phylogenetic inferences of Nepenthes species in Peninsular Malaysia revealed by chloroplast (trnL intron) and nuclear (ITS) DNA sequences.

BACKGROUND: The chloroplastic trnL intron and the nuclear internal transcribed spacer (ITS) region were sequenced for 11 Nepenthes species recorded in Peninsular Malaysia to examine their phylogenetic relationship and to evaluate the usage of trnL intron and ITS sequences for phylogenetic reconstruction of this genus. RESULTS: Phylogeny reconstruction was carried out using neighbor-joining, maximum parsimony and Bayesian analyses. All the trees revealed two major clusters, a lowland group consisting of N. ampullaria, N. mirabilis, N. gracilis and N. rafflesiana, and another containing both intermediately distributed species (N. albomarginata and N. benstonei) and four highland species (N. sanguinea, N. macfarlanei, N. ramispina and N. alba). CONCLUSIONS: The trnL intron and ITS sequences proved to provide phylogenetic informative characters for deriving a phylogeny of Nepenthes species in Peninsular Malaysia. To our knowledge, this is the first molecular phylogenetic study of Nepenthes species occurring along an altitudinal gradient in Peninsular Malaysia.

Precise identification of different stages of a tick, Ixodes granulatus Supino, 1897 (Acari:Ixodidae)

Objective: To identify different stages of Ixodes granulatus (I. granulatus) based on morphological characters prior to molecular identification which is significant for con-firming and identifying the nymphal stages of I. granulatus. Methods: A total of 14 individuals of adult, engorged and nymphal ticks collected from three different localities were examined morphologically using taxonomic keys, followed by PCR using cytochrome oxidase subunit I (COI). Clustering analysis based on COI sequences was carried out by constructing neighbor-joining and maximum parsimony tree to clarify the genetic variation and diversity of local I. granulatus. Results: Based on external morphological characterizations, nine individuals (64.3%) were successfully identified as I. granulatus, while five individuals were recognized only as Ixodes sp. due to lack of morphological characters visible and development during that stage. Molecular analysis of local I. granulatus using COI gene revealed 93%–94%sequence homology from available sequence in GenBank and was in concordance with the morphological identification. Furthermore, a low intraspecific variation was observed among the species of I. granulatus collected from different localities (0%–3.7%). Conclusions: These findings demonstrated for the first time the establishment of COI gene for identifying I. granulatus nymphal tick which is of paramount importance to the control of potential tick-borne infections in Malaysia. Moreover, this study provides evidence that a combination of morphology and molecular data was corroborated as an accurate tool for tick identification.

Molecular identification of blood meal sources of ticks (Acari, Ixodidae) using cytochrome b gene as a genetic marker.

Blood meal analysis (BMA) from ticks allows for the identification of natural hosts of ticks (Acari: Ixodidae). The aim of this study is to identify the blood meal sources of field collected on-host ticks using PCR analysis. DNA of four genera of ticks was isolated and their cytochrome b (Cyt b) gene was amplified to identify host blood meals. A phylogenetic tree was constructed based on data of Cyt b sequences using Neighbor Joining (NJ) and Maximum Parsimony (MP) analysis using MEGA 5.05 for the clustering of hosts of tick species. Twenty out of 27 samples showed maximum similarity (99%) with GenBank sequences through a Basic Local Alignment Search Tool (BLAST) while 7 samples only showed a similarity range of between 91-98%. The phylogenetic trees showed that the blood meal samples were derived from small rodents (Leopoldamyssabanus, Rattustiomanicus and Sundamysmuelleri), shrews (Tupaiaglis) and mammals (Tapirusindicus and Prionailurusbengalensis), supported by 82-88% bootstrap values. In this study, Cyt b gene as a molecular target produced reliable results and was very significant for the effective identification of ticks' blood meal. The assay can be used as a tool for identifying unknown blood meals of field collected on-host ticks.

Continental monophyly and molecular divergence of Peninsular Malaysia's Macaca fascicularis fascicularis.

The phylogenetic relationships of long-tailed macaque (Macaca fascicularis fascicularis) populations distributed in Peninsular Malaysia in relation to other regions remain unknown. The aim of this study was to reveal the phylogeography and population genetics of Peninsular Malaysia's M. f. fascicularis based on the D-loop region of mitochondrial DNA. Sixty-five haplotypes were detected in all populations, with only Vietnam and Cambodia sharing four haplotypes. The minimum-spanning network projected a distant relationship between Peninsular Malaysian and insular populations. Genetic differentiation (F(ST), Nst) results suggested that the gene flow among Peninsular Malaysian and the other populations is very low. Phylogenetic tree reconstructions indicated a monophyletic clade of Malaysia's population with continental populations (NJ = 97%, MP = 76%, and Bayesian = 1.00 posterior probabilities). The results demonstrate that Peninsular Malaysia's M. f. fascicularis belonged to Indochinese populations as opposed to the previously claimed Sundaic populations. M. f. fascicularis groups are estimated to have colonized Peninsular Malaysia ~0.47 million years ago (MYA) directly from Indochina through seaways, by means of natural sea rafting, or through terrestrial radiation during continental shelf emersion. Here, the Isthmus of Kra played a central part as biogeographical barriers that then separated it from the remaining continental populations.

Phylogenetic relationships of Malaysia's long-tailed macaques, Macaca fascicularis, based on cytochrome b sequences.

Phylogenetic relationships among Malaysia's long-tailed macaques have yet to be established, despite abundant genetic studies of the species worldwide. The aims of this study are to examine the phylogenetic relationships of Macaca fascicularis in Malaysia and to test its classification as a morphological subspecies. A total of 25 genetic samples of M. fascicularis yielding 383 bp of Cytochrome b (Cyt b) sequences were used in phylogenetic analysis along with one sample each of M. nemestrina and M. arctoides used as outgroups. Sequence character analysis reveals that Cyt b locus is a highly conserved region with only 23% parsimony informative character detected among ingroups. Further analysis indicates a clear separation between populations originating from different regions; the Malay Peninsula versus Borneo Insular, the East Coast versus West Coast of the Malay Peninsula, and the island versus mainland Malay Peninsula populations. Phylogenetic trees (NJ, MP and Bayesian) portray a consistent clustering paradigm as Borneo's population was distinguished from Peninsula's population (99% and 100% bootstrap value in NJ and MP respectively and 1.00 posterior probability in Bayesian trees). The East coast population was separated from other Peninsula populations (64% in NJ, 66% in MP and 0.53 posterior probability in Bayesian). West coast populations were divided into 2 clades: the North-South (47%/54% in NJ, 26/26% in MP and 1.00/0.80 posterior probability in Bayesian) and Island-Mainland (93% in NJ, 90% in MP and 1.00 posterior probability in Bayesian). The results confirm the previous morphological assignment of 2 subspecies, M. f. fascicularis and M. f. argentimembris, in the Malay Peninsula. These populations should be treated as separate genetic entities in order to conserve the genetic diversity of Malaysia's M. fascicularis. These findings are crucial in aiding the conservation management and translocation process of M. fascicularis populations in Malaysia.

Chremistica ribhoi sp. n. (Hemiptera: Cicadidae) from North-East India and its mass emergence.

The genus Chremistica Stål, 1870 is reported from the northeastern region of India for the first time. A new species, Chremistica ribhoi, is described from this region and male genitalia illustrations and scanning electron microscope photographs of the pygofer for C. ribhoi are presented. Observations on mass emergence of C. ribhoi are described.