Identification and characterization of microsatellite DNA markers developed in an endangered cyprinid of the Mekong River,the seven‐line barb (Probarbus jullieni Sauvage, 1880)

Microsatellite markers for an endangered cyprinid species of the Mekong River, the seven‐line barb (Probarbus jullieni Sauvage, 1880) were developed from the wild caught samples using (GT)₁₅ probe. The number of alleles per locus ranged from seven to 16. The expected heterozygosities ranged from 0.47 to 0.91. Also, these primers were successfully amplified in the two closely related species, P. labeamajor and P. labeaminor. These markers have proven to be very useful for the population genetic structure study in this species and other related cyprinids.     

Genetic characterization of <Emphasis Type="Italic">Perna viridis</Emphasis> L. in peninsular Malaysia using microsatellite markers

A total of 19 polymorphic microsatellite loci were used to analyse levels of genetic variation for 10 populations of Perna viridis L. collected from all over peninsular Malaysia. The populations involved in this study included Pulau Aman in Penang, Tanjung Rhu in Kedah, Bagan Tiang in Perak, Pulau Ketam in Selangor, Muar, Parit Jawa, Pantai Lido and Kampung Pasir Puteh in Johore, and Kuala Pontian and Nenasi in Pahang state. The number of alleles per locus ranged from two to seven, with an average of 3.1. Heterozygote deficiencies were observed across all the 10 populations. Characterization of the populations revealed that local populations of P. viridis in peninsular Malaysia were genetically similar enough to be used as a biomonitoring agent for heavy metal contamination in the Straits of Malacca. Cluster analysis grouped the P. viridis populations according to their geographical distributions with the exception of Parit Jawa. The analysis also revealed that P. viridis from the northern parts of peninsular Malaysia were found to be the most distant populations among the populations of mussels investigated and P. viridis from the eastern part of peninsular Malaysia were closer to the central and southern populations than to the northern populations.     

Isolation and characterization of microsatellite DNA loci in sea bass,Lates calcarifer Bloch

Polymerase chain reaction (PCR)‐based isolation of microsatellite arrays (PIMA) technique was used to isolate seven polymorphic microsatellite loci in sea bass, Lates calcarifer Bloch. A total of 62 samples of wild and cultivated sea bass collected from a few populations within Peninsular Malaysia were used in the study. For seven polymorphic loci, the number of alleles ranged from four to nine and locus heterozygosities ranged from 0.710 to 1.000. The loci will be useful for studying population structure, genetic variability of wild and hatchery stocks of L. calcarifer and broodstock management purposes.     

Anadromy and heterogenous population of a tropical shad Tenualosa ilisha in Malaysia,as revealed by otolith microchemistry and molecular evidence

Tenualosa ilisha was found recently in the Perak River in western Peninsular Malaysia. Molecular phylogenetic and haplotype network analyses suggest that T. ilisha has two genetically distinct populations/groups: (i) Peninsular Malaysia (Malaysia population), and (ii) Peninsular Malaysia, Thailand, India and Bangladesh (Indian Ocean population). The results also suggest that the T ilisha population in Peninsular Malaysia is genetically heterogeneous with a typical anadromous migration pattern.     

Morphological and allozyme studies of small terrestrial snails (Opeas sp., Subulina sp., and Huttonella bicolor) collected from peninsular malaysia

Shell morphological characters and allozyme electrophoresis were used to study the relationships among six geographical populations of land snails collected from Peninsular Malaysia. Allozyme electrophoresis was used to study the genetic variations to complement the morphological features studied that included shell lengths, numbers of whorls and shell colour. Ten loci coding for six enzymes (MDH, LAP, ALP, PGM, G6P DH, and EST) could be reliably scored in samples from the six populations studied. The dendrogram showed two major clusters with one cluster comprising Subulinidae populations from Perak, Selangor, Johor, Terengganu, and Pahang while the other cluster included only the Streptaxidae Huttonella bicolor (red) population. The Subulinidae populations were grouped into two subclusters: one subcluster included the Subulina sp. populations from Perak, Selangor, and Johor, while the other subcluster included the Opeas sp. populations from Terengganu and Pahang. Morphological features can identify the different families ant therefore they can complement the allozyme genetic studies on the land snail populations. Like other reports in the literature, our results also underline the importance of a genetic approach in conjunction with a morphological approach, for discriminating land snail species. The present results suggest that small land snails, which were similar in colour but different in sizes, were not of the same family/genus. The text was submitted by the authors in English.     

Detection of morphometric differentiation between isolated up- and downstream populations of Siah Mahi (<Emphasis Type="Italic">Capoeta capoeta gracilis</Emphasis>) (Pisces: Cyprinidae) in the Tajan River (Iran)

It has been postulated that the building of the Shahid-Rajaei dam on the Tajan River around 1995 has lead to the morphological divergence of Siah Mahi Capotes capoeta gracilis (Pisces) of up- and downstream populations due to the isolation. A 13-landmark morphometric truss network system was used to investigate the hypothesis. Univariate analysis of variance showed significant differences between the means of the two groups for 45 standardized morphometric measurements out of 78 characters studied. In linear discriminant function analysis (DFA), the overall assignment of individuals into their original groups was 87.6%. The proportion of individuals correctly classified into their original groups was 90.3% for upstream and 83.7% for downstream population. The principal component analysis (PCA) scatter plot of individual component scores between PC1 and PC2 showed 121 fish specimens grouped into two areas but with a relativity high degree of overlap between two populations. Clustering analysis based on Euclidean square distances among groups of centroids using an UPGMA resulted into two main clusters indicating two populations of C. c. gracilis. The present study indicated the presence of two morphologically different populations of C. c. gracilis in the Tajan River across the Shahid-Rajaei dam, probably due to their limited downstream dispersal and the elimination of upstream migration altogether, due the construction of the dam.     

Genetic structure of the common terrestrial pulmonate snail,Cryptozona siamensis (Pfeiffer, 1856), in Thailand

Allozyme variation was examined in 432 specimens of the pulmonate snail, Cryptozona siamensis, from 24 localities across three regions of Thailand plus an additional population from Malaysia. Although this snail is a cosmopolitan species it was predicted that C. siamensis should exhibit a large degree of genetic subdivision across these regions due to a low level of inter-population gene flow. To test this hypothesis, 10 enzyme systems were screened using horizontal starch gel electrophoresis, from which 14 presumed allozyme loci were scored and 13 found to be polymorphic. For these 13 polymorphic loci the expected heterozygosity levels were moderate (H_exp = 0.036–0.183, mean = 0.101). Genetic differentiation among the samples was low, with a low F_ST value (0.254) and genetic distance (D = 0.000–0.124, mean = 0.034) and no significant isolation by distance (IBD) across the whole sampled range of Thailand or Thailand plus Malaysia. However, an IBD pattern in the population substructure in the central Thailand region was detected. The low F_ST and moderate H_exp values in various populations of C. siamensis suggests a high degree of gene flow/migration between populations across the three geographic regions of Thailand (northern, central and southern) as well as the sampled population in Malaysia, perhaps associated with the anthropochoric effect of snail dispersal. The apparent IBD in the central Thailand region could be the consequence of local dynamics though this region.     

Systematics,genetics, and biogeography of intertidal mites (Acari,Oribatida) from the Andaman Sea and Strait of Malacca

This study demonstrates for the first time the presence of marine‐associated mites in the Andaman Sea and Strait of Malacca and reveals a relatively high diversity of these taxa with six species from two different families: Selenoribatidae and Fortuyniidae. Indopacifica, a new genus of Selenoribatidae, is described from Thailand and Malaysia, with two new species, Indopacifica pantai n. sp. and Indopacifica parva n. sp. The genus is characterized by the unique combination of following characters: lacking lamellar ridges, incomplete dorsosejugal suture, fourteen pairs of notogastral setae, and presence of epimeral foveae. A phylogenetic reconstruction based on 18S ribosomal RNA sequences clearly confirms the distinctness of the new genus Indopacifica and places it close to the genus Rhizophobates. The lack of molecular genetic data of possible relatives impedes a clear assessment, and hence, we emphasize the need for further combined approaches using morphological and molecular genetic sequence data. All species show wide distribution areas within this geographic region suggesting that these taxa are good dispersers despite their minute size and wingless body. Molecular genetic data demonstrate recent gene flow between far distant populations of I. pantai n. sp. from the coasts of Thailand and two islands of Malaysia and hence confirm this assumption. The seasonally changing surface currents within this geographic area may favor hydrochorous dispersal and hence genetic exchange. Nevertheless, morphometric data show a slight trend to morphological divergence among the studied populations, whereas this variation is suggested to be a result of genetic drift but also of habitat differences in one population of Alismobates pseudoreticulatus.     

Mitochondrial DNA Diversity of the Asian Moon Scallop, <Emphasis Type="Italic">Amusium pleuronectes</Emphasis> (Pectinidae), in Thailand

Sequence variation of the mitochondrial DNA 16S rRNA region of the Asian moon scallop, Amusium pleuronectes, was surveyed in seven populations along the coast of Thailand. A total of 16 unique haplotypes were detected among 174 individuals with a total 27 variable sites out of 534 bp sequenced. The mitochondrial haplotypes grouped into two distinct arrays (estimated to differ by about 2.62% to 2.99% nucleotide divergence) that characterized samples collected from the Gulf of Thailand versus the Andaman Sea. Low levels of intrapopulation variation were observed, while in contrast, significant divergence was observed between populations from the Gulf of Thailand and Andaman Sea. Results of AMOVA reveal a high F _ST value (0.765) and showed that the majority of the total genetic variance (76.03%) occurred among groups (i.e., Andaman Sea and the Gulf of Thailand) and little among populations within the group (0.52%) and within populations (23.45%). The genetic differentiation between the populations recorded in the present study is similar to that observed in a variety of marine species in the Indo-Pacific. The implications of the findings for management of A. pleuronectes genetic resources in Thailand are discussed.     

First report of paralytic shellfish poisoning (PSP) caused by Alexandrium tamiyavanichii in Kuantan Port,Pahang, East Coast of Malaysia

Harmful algal bloom (HAB) is a proliferation of algae, which naturally produce biotoxins and cause harmful effects to humans, the environment and organisms associated with it. Paralytic shellfish poisoning (PSP) was reported for the first time in Kuantan Port, Pahang, Malaysia, in November 2013, followed by a second episode in August 2014. The toxicity level reported during the second event was as high as 3500 μg of STX equiv./100 g shellfish. Ten people were hospitalized with PSP symptoms after consuming contaminated shellfish. This study was conducted at Kuantan Port to identify the organisms responsible for these events. Water samples were collected monthly for a period of 12 months beginning in September 2014. HAB species were identified based on their morphology using light and fluorescence microscopes, and their classification was supported by molecular evidence based on internal transcribed spacer (ITS) sequences. Monthly cell abundance of Alexandrium tamiyavanichii was measured at four sampling stations. Toxin production by three strains isolated from the area was determined using HPLC. Our results revealed the presence of several HAB species, including the PSP‐producing species A . tamiyavanichii . The highest cell density of A . tamiyavanichii was 840 cells L^?1. The presence of GTX components was detected in these strains. However, other toxin components could not be determined. This study reported, for the first time, the presence of PSP‐producing A . tamiyavanichii on the Pahang coast of east Peninsular Malaysia and confirmed that the PSP events in Kuantan Port were attributable to this species. The presence of this species further indicates that several safety measures need to be considered to safeguard public health, particularly in Pahang coastal waters.     

Morphological and molecular characterization of the Landes honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.) ecotype for genetic conservation

A population of honey bees (Apis mellifera mellifera L.) with an annual colony brood cycle adapted to a locally abundant floral source in the Landes region of Southwest France is the subject of genetic conservation efforts. This population is maintained by local beekeepers in an area that experiences both an annual seasonal influx of non-local colonies and the permanent culture of imported stock. However, some colonies native to the Landes do not have the adapted brood cycle and their status as ecotypic are in question. The present study used morphology, mitochondrial DNA and microsatellites to characterize the endemic population and suggests further genetic conservation strategies. These methods yielded different degrees of discrimination of native and imported colonies and provided a powerful suite of tools for local resource managers. Colonies from the Landes could be differentiated from non-local French A. m. mellifera populations using morphometric analysis, and from non-native and reference populations using mtDNA and microsatellites. Seven morphological characters were identified by discriminant analysis as informative for delineating the Landes ecotype from other A. m. mellifera populations. Mitochondrial haplotypes for the population were characterized and five microsatellite loci were found to be informative in characterizing the Landes population. Asymmetric gene flow detected with microsatellite alleles was observed to be 5.5–5.9% from imported to native stocks of honey bees while introgression of native microsatellite alleles into imported colonies was 21.6%.     

Differential abundance and core members of the bacterial community associated with wild male Zeugodacus cucurbitae fruit flies (Insecta: Tephritidae) from three geographical regions of Southeast Asia

Zeugodacus cucurbitae (Coquillet) is one of the most significant and widespread tephritid pest species of agricultural crops. This study reports the bacterial communities associated with Z. cucurbitae from three geographical regions in Southeast Asia (Thailand, Peninsular Malaysia, and Sarawak). The bacterial microbiota were investigated by targeted 16S rRNA gene (V3–V4 region) sequencing using the Illumina Mi-Seq platform. At 97% similarity and filtering at 0.001%, there were seven bacterial phyla and unassigned bacteria, comprising 11 classes, 23 orders, 39 families and 67 genera. The bacterial diversity and richness varied within and among the samples from the three geographical regions. Five phyla were detected for the Sarawak sample, and six each for the Thailand and Peninsular Malaysia samples. Four phyla—Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria—were represented in all the fruit fly specimens, forming the core members of the bacterial community. Proteobacteria was the predominant phylum, followed by Bacteroidetes, Firmicutes, and Actinobacteria. Fifty-three genera were represented in the Thailand sample, 56 in the Peninsular Malaysia sample, and 55 in the Sarawak sample. Forty-two genera were present in all the three geographical regions. The predominant core members were order Enterobacteriales (Proeteobacteria), and family Enterobacteriaceae (Enterobacteriales). Klebsiella (Enterobacteriaceae) was the predominant genus and K. oxytoca the predominant species with all specimens having?>?10% relative abundance. The results indicate the presence of a great diversity as well as core members of the bacterial community associated with different populations of Z. cucurbitae.

     

Genetic variation within and between populations of hermaphroditic <Emphasis Type="Italic">Bulinus truncatus</Emphasis> tetraploid freshwater snails of the Albertine Rift,East Africa

Genetic variability within and among Bulinus truncatus of the Albertine Rift freshwater bodies were assessed to investigate the degree of inbreeding and gene flow in the snail populations. The effect of ploidy on the genetic structuring of B. truncatus is also described. We characterized the genetic structure of seven B. truncatus populations from Lake Albert, Lake Kivu, and Katosho swamp in Tanzania using five polymorphic microsatellite loci. Genetic differentiation was quantified using pairwise FST values and Nei’s standard genetic distances. Different alleles were observed across all loci and genetic diversity was low although it varied greatly across populations; observed heterozygosity was, however, higher than the expected heterozygosity in three of the populations studied. Significant heterozygote deficiencies were observed coupled with significant linkage disequilibria in five populations for all the five loci examined in this study. We found significant genetic differentiation among the seven freshwater bodies; private alleles were observed across all loci indicating restricted or absence of gene flow between populations. Limited snail dispersal and the reproductive biology of B. truncatus are the major forces shaping the genetic variation observed. Low genetic variation within B. truncatus populations exposes them to a high parasite infection risk as predicted in the Red Queen hypothesis.     

Comparative analysis of morphometric characters of juvenile sterlet Acipenser ruthenus L. from natural population and aquaculture

A comparative analysis was performed on sixteen morphometric characters, in three different juvenile sterlet Acipenser ruthenus L. populations. Specimens were collected from a wild population in the Serbian part of the Danube River (n = 46), from aquaculture stocks in the Czech Republic originating from Russia (n = 40), and aquaculture stocks in the Slovak Republic originating from Slovakian part of the Danube River (n = 28). Average values for total length were 29·9 ± 3·9 cm, 29·1 ± 3·7 cm and 27·3 ± 7·7 cm for Serbia, Czech Republic and Slovak Republic, respectively. Populations were compared using t‐test and sequential Bonferroni correction for multiple comparisons was applied in order to determine significant differences between them. Results of analysis showed that all three populations differed in prebarbel length, interocular distance and maximum head width. Although all these characters are head‐related, head length itself was very uniform among all populations. The natural population from the Serbian part of the Danube River differed from the populations reared in aquaculture in seven morphometric characters. The two populations reared in aquaculture consistently showed lower morphological variability than the wild population, even though they had different genetic backgrounds (Russia and Slovakia). Future genetic studies will show if this tendency is caused by a reduction in genetic variability.     

Inter- and Intraspecific Variation in Wild-type and Single Female-derived Populations of Xiphinema americanum-group Nematodes

Ten populations of Xiphinema americanum-group nematodes were reared from individual females to evaluate inter- and intraspecific variation under identical host and environmental conditions. Data indicated that morphometric variability of X. americanum was the result of genetic variation rather than phenotypic plasticity and that genetic heterogeneity was greater than previously thought. Morphometrics of single female derived (SFD) populations identified different genotypes present in the field populations. Stylet length was the least variable morphometric character of SFD populations, but collectively stylet measurements of all individuals formed an uninterrupted continuum ranging from 107-148 μm. Range and frequency of stylet measurements of field populations could be accounted for by the relative proportion of different genotypes in the population. Nine SFD populations were identified as X. americanum sensu stricto, and one SFD population was similar to X. californicum.     

Genetic Differences Between Hatchery Stocks and Natural Populations in Pacific Abalone (<Emphasis Type="Italic">Haliotis discus</Emphasis>) Estimated Using Microsatellite DNA Markers

Genetic variations within and between nine hatchery stocks and seven natural populations of abalone including Ezo-abalone (Haliotis discus hannai) and Kuro-abalone (H. d. discus) were assayed with nine microsatellite markers. Marked reductions of genetic variability in the hatchery stocks were recognized in the allelic diversity and mean heterozygosity compared with the natural populations. Thirteen of 16 significant HWE deviations in hatchery stocks revealed heterozygotes excess, while all natural populations did not show such a tendency. Highly significant F _ST values were observed for all cases between the hatchery stocks, and between the hatchery stocks and natural populations. Genetic distance (D _A) between each hatchery stock and the geographically proximal population (mean ± SD, 0.108 ± 0.035) were similar to those estimated for between the natural Ezo-abalone and Kuro-abalone (0.101 ± 0.021). The self-assignment test, which allocated individuals to their own stock with a high success rate, provided evidence of solid genetic differences among the nine hatchery stocks. These results suggests that the allelic composition and diversity in the natural populations was not necessarily reflected in the hatchery stocks owing to population bottleneck and genetic drift through seedling process, and thus the seedling and stocking practice of these hatchery stocks should take much notice of the results to conserve the genetic diversity of natural populations.     

Isolation and characterization of the first microsatellite loci from the order Psocoptera in the economically important pest insect Liposcelis decolor (Pearman) and cross‐species amplification

A total of 11 microsatellite loci from the invasive insect pest Liposcelis decolor were isolated and characterized of which six loci were polymorphic. A population survey involving a total of 30–192 individuals per locus from five populations revealed a range of four to seven alleles per locus and moderate observed heterozygosities (0.183–0.565), highlighting the utility of these loci in further population genetic studies. Cross‐species amplifications were successful for two to 11 loci in five other Liposcelis species also of international economic importance.     

Phenotypic and genetic differentiation between diadromous and landlocked puyen Galaxias maculatus

This study reports the phenotypic and genetic differences between individuals of puyen Galaxias maculatus from two sites in the same river basin in Tierra del Fuego National Park, southern South America. Individuals from the two sampling sites presented morphometric and genetic differences. The morphometric differences indicated that individuals from Laguna Negra (LN) were short and more robust and had large eyes, whereas those from Arroyo Negro (AN) were thin and elongated and had small eyes. Genetic differences showed that AN individuals had a greater genetic structuration and an older demographic history than LN individuals. The results of this study affirmed that the individuals from the two sampling sites belong to different populations with a high degree of isolation. The demographic history could indicate that the individuals of G. maculatus which migrated to northern areas during the last glaciation settled in the Beagle Channel after its formation. The LN population could have originated after the retreat of the glaciers, migrating from AN.     

POPULATION DIFFERENTIATION AND RACIAL ADMIXTURE IN THE AFRICANIZED HONEYBEE (APIS MELLIFERA L.)

To study the degree of interpopulational differentiation and racial admixture in Africanized honeybees, we collected worker bees from three regions of Brazil (the northeast, the state of Sao Paulo, and Porto Alegre) and from Uruguay and determined their genotypes for 10 enzyme loci. We also performed a morphometric analysis on forewing measurements of worker bees from the northeast and Porto Alegre regions of Brazil and from Paysandu, Uruguay. Comparative analysis of interpopulational heterogeneity snowed that there are significant differences, especially at the Mdh locus, among the populations from different regions. An increase in the frequency of the Mdh^B allele was observed from north to south, with predominance in the Uruguayan populations. A small component of interpopulational variability was detected in the populations studied. Racial admixture was calculated from information obtained for Mdh in Africa and Europe. The percentages of racial admixture differed slightly but significantly among Brazilian regions. The morphometric study based on canonical variables exhibited a similar pattern. The greater proportions of Apis mellifera adansonii alleles in the admixture may be explained by selection during the initial stage of migration of Africanized bees and by preferential mating between individuals of the same race. Differences in the proportions of A. m. adansonii alleles between regions indicate incipient populational differentiation of Africanized bees. We suggest that greater gene flow from the European races in the south of Brazil could be one of the causes of this phenomenon.     

Reduced MHC class II diversity in island compared to mainland populations of the black-footed rock-wallaby (<Emphasis Type="Italic">Petrogale lateralis lateralis</Emphasis>)

Many animal populations that are endangered in mainland areas exist in stable island populations, which have the potential to act as an “ark” in case of mainland population declines. Previous studies have found neutral genetic variation in such species to be up to an order of magnitude lower in island compared to mainland populations. If low genetic variation is prevalent across fitness-related loci, this would reduce the effectiveness of island populations as a source of individuals to supplement declining mainland populations or re-establish extinct mainland populations. One such species, the black-footed rock-wallaby (Petrogale lateralis lateralis), exists within fragmented mainland populations and small island populations off Western Australia. We examined sequence variation in this species within a fitness-related locus under positive selection, the MHC class II DAB β1 locus. The mainland populations displayed greater levels of allelic diversity (4–7 alleles) than the island population, despite being small and isolated, and contained at least two DAB gene copies. The island population displayed low allelic diversity (2 alleles) and fewer alleles per individual in comparison to mainland populations, and probably possesses only one DAB gene copy. The patterns of DAB diversity suggested that the island population has a markedly lower level of genetic variation than the mainland populations, in concordance with results from microsatellites (genotyped in a previous study), but preserved unique alleles which were not found in mainland populations. Where possible, conservation actions should pool individuals from multiple populations, not only island populations, for translocation programs, and focus on preventing further declines in mainland populations.