Genetic variability and phylogeography of the tropical brackish water species, Gracilaria tenuistipitata (Gracilariales, Rhodophyta)

Genetic, Linguistic and Archaeological Perspectives on Human Diversity in Southeast Asia

Polyploidization (whole genome duplication – WGD) is a recurrent process in plants and provides greater potential for diversification. Neopolyploids in natural populations should go under substantial structural changes in their genetics, reproductive mode (e.g. apomixis – asexual reproduction via seeds), and ecological preferences to ensure their successful establishment. Apomixis in plants provides reproductive assurance, and superior colonizing abilities respect to sexuals, but it also constrains genetic variation and clonal plants are expected to have restricted adaptive capabilities. These complex rearrangement processes and adaptations in polyploid complexes are well reflected by their genetic variation. However, there is a lack of non-model systems that exhibit successful changes with pronounced reflection for studies. Paspalum intermedium is a grass species with diverging genetic systems (diploidy vs. autopolyploidy, allogamy vs. autogamy and sexuality vs. apomixis) with substantial ecological differentiation between cytotypes occurring in allopatry, sympatry and parapatry, hence provides an ideal platform to study polyploidization, apomixis and their ecological and genetic importance in plant evolution. Therefore, in this thesis, I used P. intermedium as a model system to recognize the causality of biogeographic patterns, adaptation and ecological flexibility of cytotypes, to study variations in the expression of sexuality and apomixis, to analyze developmental competition between reproductive modes, and their effects on reproductive fitness, and to study genetic variation and its significance in polyploid complexes. I used chromosome counts, flow cytometry, and embryological analyses to characterize within-species genetic systems diversity. Environmental niche modelling was performed to evaluate intraspecific ecological attributes and to assess correlations among ploidy, and ecological conditions ruling species’ population dynamics, range expansion, adaptation and evolutionary history. Proportions of sexuality and apomixis in situ were analyzed against local climatic conditions to study the influence of environmental factors on reproductive modes. Total seed set and germinability analyses were used to estimate the reproductive fitness. Analysis of genetic markers AFLPs was used to assess the genetic variation between and within cytotypes and within and among populations. To get insights into the genetic structure variation depending on the reproductive mode and how it explains the niche variation between cytotypes, the results were compared with the geographical distribution patterns and different ecological preferences of the cytotypes. My results show that the two dominant cytotypes of P. intermedium are non-randomly distributed along local and regional geographical scales and displayed niche differentiation. Polyploidy and contrasting reproductive traits between cytotypes have promoted shifts in niche optima, and increased ecological tolerance and niche divergence. Ecologically specialized diploids maintain cytotype stability in core areas by displacing tetraploids, while broader ecological preferences and a shift from sexuality to apomixis favored polyploid colonization in peripheral areas promoting range expansion. The expression of sex and apomixis in tetraploid populations shows high variation both within and among populations. Even though ovule and seed analyses show apomictic development has higher competitive ability, fitness of apomictic individuals is depleted compared to sexual individuals and populations, indicating asexuality suffering higher seed abortion. Environmental modulation of reproduction was evident at population level where sex increased with higher mean diurnal range (MDR) while apomixis decreased. Thus, a Tug of War situation was identified between factors intrinsic to apomixis and environmental stressors promoting sex, suggesting a crucial role of local ecological conditions in sexual expression and adaptation of apomictic populations. Population structure analyses show that apomictic autotetraploids are of multiple independent origin. Although diploids show higher genetic variation, within and among population genetic variation equally make up the observed variation in all cytotypes. All individuals fall into three genetic clusters with substantial genetic admixture, and geographical distribution of genetic variation is in accordance with niche differentiation. The contact zone of the two cytotypes is primary in origin where tetraploids may frequently occur in mix ploidy populations. Polyploidization in P. interemedium is a recurring phenomenon and the newly arisen polyploids successfully establish themselves by acquiring enough genetic variation that allows them to adapt to new environments. Genetic variation analysis points to a slight deviation from the known General Purpose Ghenotype and the Frozen Niche Variation concepts as there is neither a common genotype nor are the diploids occupying a part of diploid sexuals’ niche.

We investigated genetic variation in mitochondrial cytochrome b within the long-tailed rice field mouse, Mus caroli Bonhote, 1902, across its entire geographic range in Southeast and East Asia with a view to: 1) assessing the pattern and causality of phylogeographic structure in a terrestrial small mammal from continental Southeast Asia; and 2) distinguishing genuine insular relics from cases of human-assisted translocation. We identified five main mtDNA lineages which show a similar level of differentiation as the subspecies of M. musculus and probably diverged during the Middle Pleistocene. Two of the lineages are restricted to large islands (Taiwan and Java) and their existence is explicable in terms of regional palaeogeographic factors including changes in sea level and climate. The remaining lineages are distributed in different regions on mainland Southeast Asia but vicariant explanations are inappropriate given the relatively short time frame. Dispersal across barriers followed by local differentiation probably explains the observed phylogeographic patterning on the mainland. A close genetic link between Okinawan M. caroli and populations in Laos confirms previous suggestions that people carried this species to the Ryukyu Archipelago. However, more intensive regional sampling is needed to identify a precise source area.

BackgroundBigfin squid is one of the economically important seafood resources in Vietnam’s fisheries and the waters around Con Dao and Phu Quoc islands are two major fishing grounds where this species has been actively exploited. The start codon targeted polymorphism (SCoT) and CAAT box–derived polymorphism (CBDP) techniques were used to generate DNA fingerprinting data to analyze the genetic diversity, variation, and structure of the two populations in the waters surrounding Phu Quoc and Con Dao islands together with mitochondrial cytochrome C oxidase subunit I (COI) gene sequence data. ResultsCon Dao population possessed a higher diversity [expected heterozygosity (He) = 0.2254, Shannon index (I) = 0.3459, percentage of polymorphic bands (PPB) = 80.14%, nucleotide diversity (π) = 0.0336, haplotype diversity (h) = 0.910 with 16 haplotypes] than Phu Quoc population (He = 0.1854, I = 0.2873, PPB = 70.38%, π = 0.0246, h = 0.838 with 14 haplotypes). The genetic diversity at species level in the investigated region was at level of He = 0.2169, I = 0.3399, PPB = 86.41, π = 0.0289, and h = 0.892 with 24 haplotypes. Based on DNA fingerprinting data, the pairwise genetic similarity coefficients among individuals of the Con Dao population were lower (average of 0.7977) than the Phu Quoc population (average of 0.8316). Based on mitochondrial COI data, the pairwise genetic distances among individuals of the Con Dao population were higher (average of 0.0361) than the Phu Quoc population (average of 0.0263). Gene differentiation (GST) between two investigated populations was 0.0316 and 0.0310 leading to the genetic distance was 0.0573 and 0.0213 and the gene flow between them was Nm = 8.2209 and 11.4700 migrants per generation among populations based on DNA fingerprinting and based on COI gene sequence data, respectively. Genetic variation within individuals of both populations (WP) played the key role in total genetic variation at species level in surveyed region. ConclusionsFor the bigfin reef squid species in the surveyed region, the Con Dao population had the higher genetic diversity than the Phu Quoc population, between them existed a low to moderate genetic differentiation and a genetic exchange via gene flow. The DNA fingerprinting data better revealed the genetic differentiation between the two surveyed populations while the mitochondrial COI gene sequence data could show the phylogenetic relationship among the surveyed individuals and the other from the sea regions in Southeast Asia. Based on the results obtained, fisheries management strategies are suggested toward the conservation and sustainable exploitation of this species.

SUMMARYThe effects of salinity, light intensity and sediment on Gracilaria tenuistipitata C.F. Chang & B.M. Xia on growth, pigments, agar production, and net photosynthesis rate were examined in the laboratory under varying conditions of salinity (0, 25 and 33 psu), light intensity (150, 400, 700 and 1000 µmol photons m−2 s−1) and sediment (0, 0.67 and 2.28 mg L−1). These conditions simulated field conditions, to gain some understanding of the best conditions for cultivation of G. tenuistipitata. The highest growth rate was at 25 psu, 700 µmol photons m−2 s−1 with no sediments, that provided a 6.7% increase in weight gain. The highest agar production (24.8 ± 3.0 %DW) was at 25 psu, 150–400 µmol photons m−2 s−1 and no sediment. The highest pigment contents were phycoerythrin (0.8 ± 0.5 mg g−1FW) and phycocyanin (0.34 ± 0.05 mg g−1 FW) produced in low light conditions, at 150 µmol photons m−2 s−1. The highest photosynthesis rate was 161.3 ± 32.7 mg O2 g−1 DW h−1 in 25 psu, 400 µmol photons m−2 s−1 without sediment in the short period of cultivation, (3 days) and 60.3 ± 6.7 mg O2 g−1 DW h−1 in 25 psu, 700 µmol photons m−2 s−1 without sediment in the long period of cultivation (20 days). The results indicated that salinity was the most crucial factor affecting G. tenuistipitata growth and production. This would help to promote the cultivation of Gracilaria cultivation back into the lagoon using these now determined baseline conditions. Extrapolation of the results from the laboratory study to field conditions indicated that it was possible to obtain two crops of Gracilaria a year in the lagoon, with good yields of agar, from mid‐January to the end of April (dry season), and from mid‐July to the end of September (first rainy season) when provided sediment was restricted.

Extinction in the Anthropocene.

The mandarinfish Synchiropus splendidus is extensively collected in Southeast Asia (mainly in the Philippines) and highly favoured for the marine aquarium trade. Males are more popular than females for their large first dorsal fins and the fishery is not managed. To examine possible population replenishment dynamics arising as a result of selective fishing, the effects of sex-selective fishing on sex ratios and population connectivity were considered. This study determined the sex ratios and analyzed the population genetic structure from mandarinfish collected at six locations: one from Palau, where the species is not exploited, and five from Bohol in the Philippines, where the species has long been heavily fished. The findings reported very low male to female ratios (0.12 to 0.30) from four of the five locations in Bohol, with relatively more males to females in the specimens collected from Palau (2.3). The analyses from allozymes (43 alleles from 10 loci) and microsatellites (118 alleles from 5 loci) revealed that genetic connectivity was high among the five locations in the Philippines as well as with the specimens collected from the more-distant Palau. The genetic homogeneity observed across the geographical range considered is inconsistent with the hypothesized limited dispersal ability of the species and could be explained by recent species range expansion associated with sea level rise in the region. The results suggest that the present genetic structure, at least in the geographic region considered, may not be determined by current patterns of gene flow, but may, instead, be driven by recent sea-level changes associated with periods of glaciation. Caution is suggested to ensure that heavily localized fishing does not produce excessively biased adult sex ratios.

The effects of Pleistocene environmental fluctuations on the distribution and diversity of organisms in Southeast Asia are much less well known than in Europe and North America. In these regions, the combination of palaeoenvironmental reconstruction and inferences about population history from genetic data has been very powerful. In Southeast Asia, mosquitoes are good candidates for the genetic approach, with the added benefit that understanding the relative contributions of historical and current processes to population structure can inform management of vector species. Genetic variation among populations of Anopheles minimus was examined using 144 mtDNA COII sequences from 23 sites in China, Thailand and Vietnam. Haplotype diversity was high, with two distinct lineages that have a sequence divergence of over 2% and exhibit different geographical distributions. We compare alternative hypotheses concerning the origin of this pattern. The observed data deviate from the expectations based on a single-panmictic population with or without growth, or a stable but spatially structured population. However, they can be readily accommodated by a model of past fragmentation into eastern and western refugia, followed by growth and range expansion. This is consistent with the palaeoenvironmental reconstructions currently available for the region.

Uncovering the hidden diversity and evolutionary history of arthropods of medico-veterinary importance could have significant implications for vector-borne disease control and epidemiological intervention. The buffalo fly Haematobia exigua is an obligate bloodsucking ectoparasite of livestock. As an initial step towards understanding its population structures and biogeographic patterns, we characterized partial cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) sequences of H. exigua from three distinct geographic regions in Southeast Asia. We detected two distinct mitochondrial haplogroups of H. exigua in our surveyed geographic regions. Haplogroup I is widespread in the Southeast Asian mainland whereas haplogroup II is generally restricted to the type population Java Island. Both haplogroups were detected co-occurring on Borneo Island. Additionally, both haplogroups have undergone contrasting evolutionary histories, with haplogroup I exhibited a high level of mitochondrial diversity indicating a population expansion during the Pleistocene era dating back to 98,000 years ago. However, haplogroup II presented a low level of mitochondrial diversity which argues against the hypothesis of recent demographic expansion.

Fossil evidence indicates that the globally distributed brown rat (Rattus norvegicus) originated in northern China and Mongolia. Historical records report the human-mediated invasion of rats into Europe in the 1500s, followed by global spread because of European imperialist activity during the 1600s–1800s. We analyzed 14 genomes representing seven previously identified evolutionary clusters, and tested alternative demographic models to infer patterns of range expansion, divergence times, and changes in effective population (Ne) size for this globally important pest species. We observed three range expansions from the ancestral population that produced the Pacific (diverged ∼16.1 kya), eastern China (∼17.5 kya), and Southeast (SE) Asia (∼0.86 kya) lineages. Our model shows a rapid range expansion from SE Asia into the Middle East and then continued expansion into central Europe 788 yr ago (1227 AD). We observed declining Ne within all brown rat lineages from 150–1 kya, reflecting population contractions during glacial cycles. Ne increased since 1 kya in Asian and European, but not in Pacific, evolutionary clusters. Our results support the hypothesis that northern Asia was the ancestral range for brown rats. We suggest that southward human migration across China between the 800s–1550s AD resulted in the introduction of rats to SE Asia, from which they rapidly expanded via existing maritime trade routes. Finally, we discovered that North America was colonized separately on both the Atlantic and Pacific seaboards, by evolutionary clusters of vastly different ages and genomic diversity levels. Our results should stimulate discussions among historians and zooarcheologists regarding the relationship between humans and rats.

Anopheles baimaii (Diptera: Culicidae) significantly contributes to the transmission of parasites causing malaria in Southeast Asia and South Asia. This study examined the morphological (wing shape) and molecular (mitochondrial gene) variations of An. baimaii in four of Thailand's border islands, and also investigated the presence of Plasmodium parasites in these mosquitoes. No Plasmodium infections were detected in the samples. Significant differences in wing shape were observed in most island populations (p < 0.05). A single-linkage tree, constructed using Mahalanobis distances, clustered the populations into two groups based on geographical locations. Genetic variation in An. baimaii was also analyzed through cytochrome c oxidase subunit I (COI) gene sequences. This analysis identified 22 segregating sites and a low nucleotide diversity of 0.004. Furthermore, 18 distinct haplotypes were identified, indicating a high haplotype diversity of 0.825. Neutrality tests for the overall population revealed a significantly negative Fu's Fs value (-5.029), indicating a population expansion. In contrast, Tajima's D yielded a negative value (-1.028) that did not reach statistical significance. The mismatch distribution analysis exhibited a bimodal pattern, and the raggedness index was 0.068, showing no significant discrepancy (p = 0.485) between observed and expected distributions. Pairwise genetic differentiation assessments demonstrated significant differences between all populations (p < 0.05). These findings provide valuable insights into the COI gene and wing morphometric variations in An. baimaii across Thailand's islands, offering critical information for understanding the adaptations of this malaria vector and guiding future comprehensive research.

Development of landscape connectivity and spatial population models is challenging, given the uncertainty of parameters and the sensitivity of models to factors and their interactions over time. Using spatially and temporally explicit simulations, we evaluate the sensitivity of population distribution, abundance and connectivity of tigers in Southeast Asia to variations of resistance surface, dispersal ability, population density and mortality. Utilizing a temporally dynamic cumulative resistant kernel approach, we tested (1) effects and interactions of parameters on predicted population size, distribution and connectivity, and (2) displacement and divergence in scenarios across timesteps. We evaluated the effect of varying levels of factors on simulated population, cumulative resistance kernel extent, and kernel sum across nine timesteps, producing 24,300 simulations. We demonstrate that predicted population, range shifts, and landscape connectivity are highly sensitive to parameter values with significant interactions and relative strength of effects varying by timestep. Dispersal ability, mortality risk and their interaction dominated predictions. Further, population density had intermediate effects, landscape resistance had relatively low impacts, and mitigation of linear barriers (highways) via lowered resistance had little relative effect. Results are relevant to regional, long-term tiger population management, providing insight into potential population growth and range expansion across a landscape of global conservation priority.

Mitochondrial DNA (mtDNA) can be a powerful genetic marker for tracing origins and history of invasive populations. Here, we use mtDNA to address questions relevant to the understanding of invasion pathways of common starlings (Sturnus vulgaris) into Western Australia (WA) and discuss the utility of this marker to provide information useful to invasive species management. Mitochondrial sequence data indicate two geographically restricted genetic groups within Australia. Evidence of dispersal from genetically distinct sources outside the sampled range of starlings in Australia suggests increased vigilance by management agencies may be required to prevent further incursions from widely separated localities. Overall, genetic diversity in Australia was lower than in samples from the native range. Within Australia, genetic diversity was lowest in the most recently colonized area in the west, indicating that demographic bottlenecks have occurred in this area. Evidence of restricted dispersal between localities on the edge of the range expansion (ERE) in WA and other Australian sampling localities suggests that localized control within the ERE may be effective in preventing further range expansion. Signatures of spatial and demographic expansion are present in mismatch analyses from sampling localities located at the ERE, but neutrality indices did not support this finding, suggesting that the former may be more sensitive to recent expansion. Additionally, mismatch analyses support the presence of admixture, which is likely to have occurred pre-introduction. We compare our findings with those from a microsatellite study of the same samples and discuss how the mtDNA analyses used here offer valuable and unique insights into the invasion history of introduced species.

Simple SummaryIslands provide unique opportunities to study how geographic isolation influences genetic diversity and evolutionary processes. The Oriental Garden lizard (Calotes versicolor) is a widespread reptile found across mainland and island habitats in Thailand. By analyzing mitochondrial CO1 sequences from populations in the Andaman Sea and Gulf of Thailand regions, we assessed their genetic diversity and population structure. The results revealed a mixture of shared and unique haplotypes, with some island and mainland populations being genetically similar, while others showed clear divergence. These patterns suggest that both historical connections and current geographic barriers have shaped the genetic landscape of C. versicolor. Our findings contribute to the understanding of reptile biogeography in Southeast Asia and provide valuable information for biodiversity conservation.Geographic isolation can shape genetic variation and structure, leading to divergence between island and mainland populations. The Oriental Garden lizard (Calotes versicolor Daudin, 1802) is a widespread agamid reptile in Asia, occurring across diverse habitats from continental Southeast Asia to offshore islands. We examined mitochondrial cytochrome c oxidase subunit I (CO1) sequence variation in 143 individuals from 23 localities across the Andaman Sea and Gulf of Thailand to assess genetic diversity and structure between insular and mainland populations. Forty-six haplotypes (Cve1–Cve46) were identified, with haplotype diversity (Hd) ranging from 0.500 to 1.000 and nucleotide diversity (π) from 0.0057 to 0.0265. AMOVA revealed low to moderate differentiation between island and mainland groups in the Andaman Sea (FCT = 0.075, p > 0.05) and negligible differentiation in the Gulf of Thailand (FCT = 0.009, p > 0.05). Haplotype networks and PCoA showed clustering of most island and mainland populations within regions, with some localized divergence. Divergence-time analysis indicated that lineages split within the last 0.5 million years ago (Ma), coinciding with late Pleistocene climatic oscillations and sea-level changes. Species delimitation analyses supported three major lineages, including a geographically restricted clade confined to Trat Province and Phuket Island. These results suggest that C. versicolor populations are structured more by regional geography than strict island–mainland separation, reflecting historical connectivity and contemporary gene flow. The findings contribute to understanding reptile biogeography in Southeast Asia and highlight populations of conservation value.

Genetic variation and relationships of four species of medically important echinostomes (Trematoda: Echinostomatidae) in South-East Asia