Gut content metabarcoding of specialized feeders is not a replacement for environmental DNA assays of seawater in reef environments

In tropical marine ecosystems, the coral-based diet of benthic-feeding reef fishes provides a window into the composition and health of coral reefs. In this study, for the first time, we compare multi-assay metabarcoding sequences of environmental DNA (eDNA) isolated from seawater and partially digested gut items from an obligate corallivore butterflyfish (Chaetodon lunulatus) resident to coral reef sites in the South China Sea. We specifically tested the proportional and statistical overlap of the different approaches (seawater vs gut content metabarcoding) in characterizing eukaryotic community composition on coral reefs. Based on 18S and ITS2 sequence data, which differed in their taxonomic sensitivity, we found that gut content detections were only partially representative of the eukaryotic communities detected in the seawater based on low levels of taxonomic overlap (3 to 21%) and significant differences between the sampling approaches. Overall, our results indicate that dietary metabarcoding of specialized feeders can be complimentary to, but is no replacement for, more comprehensive environmental DNA assays of reef environments that might include the processing of different substrates (seawater, sediment, plankton) or traditional observational surveys. These molecular assays, in tandem, might be best suited to highly productive but cryptic oceanic environments (kelp forests, seagrass meadows) that contain an abundance of organisms that are often small, epiphytic, symbiotic, or cryptic.</p

The Molecular Biogeography of the Indo-Pacific: Testing Hypotheses With Multispecies Genetic Patterns

Aim: To test hypothesized biogeographic partitions of the tropical Indo-Pacific Ocean with phylogeographic data from 56 taxa, and to evaluate the strength and nature of barriers emerging from this test. \u3eLocation: The Indo-Pacific Ocean. Time Period: Pliocene through the Holocene. Major Taxa Studied: Fifty-six marine species. Methods: We tested eight biogeographic hypotheses for partitioning of the Indo-Pacific using a novel modification to analysis of molecular variance. Putative barriers to gene flow emerging from this analysis were evaluated for pairwise ΦST, and these ΦST distributions were compared to distributions from randomized datasets and simple coalescent simulations of vicariance arising from the Last Glacial Maximum. We then weighed the relative contribution of distance versus environmental or geographic barriers to pairwise ΦST with a distance-based redundancy analysis (dbRDA). Results: We observed a diversity of outcomes, although the majority of species fit a few broad biogeographic regions. Repeated coalescent simulation of a simple vicariance model yielded a wide distribution of pairwise ΦST that was very similar to empirical distributions observed across five putative barriers to gene flow. Three of these barriers had median ΦST that were significantly larger than random expectation. Only 21 of 52 species analysed with dbRDA rejected the null model. Among these, 15 had overwater distance as a significant predictor of pairwise ΦST, while 11 were significant for geographic or environmental barriers other than distance. Main Conclusions: Although there is support for three previously described barriers, phylogeographic discordance in the Indo-Pacific Ocean indicates incongruity between processes shaping the distributions of diversity at the species and population levels. Among the many possible causes of this incongruity, genetic drift provides the most compelling explanation: given massive effective population sizes of Indo-Pacific species, even hard vicariance for tens of thousands of years can yield ΦST values that range from 0 to nearly 0.5

The molecular biogeography of the Indo‐Pacific: Testing hypotheses with multispecies genetic patterns

Aim: To test hypothesized biogeographic partitions of the tropical Indo‐Pacific Ocean with phylogeographic data from 56 taxa, and to evaluate the strength and nature of barriers emerging from this test. Location: The Indo‐Pacific Ocean. Time period: Pliocene through the Holocene. Major taxa studied: Fifty‐six marine species. Methods: We tested eight biogeographic hypotheses for partitioning of the Indo‐ Pacific using a novel modification to analysis of molecular variance. Putative barriers to gene flow emerging from this analysis were evaluated for pairwise ΦST, and these ΦST distributions were compared to distributions from randomized datasets and simple coalescent simulations of vicariance arising from the Last Glacial Maximum. We then weighed the relative contribution of distance versus environmental or geographic barriers to pairwise ΦST with a distance‐based redundancy analysis (dbRDA). Results: We observed a diversity of outcomes, although the majority of species fit a few broad biogeographic regions. Repeated coalescent simulation of a simple vicariance model yielded a wide distribution of pairwise ΦST that was very similar to empirical distributions observed across five putative barriers to gene flow. Three of these barriers had median ΦST that were significantly larger than random expectation. Only 21 of 52 species analysed with dbRDA rejected the null model. Among these, 15 had overwater distance as a significant predictor of pairwise ΦST, while 11 were significant for geographic or environmental barriers other than distance. Main conclusions: Although there is support for three previously described barriers, phylogeographic discordance in the Indo‐Pacific Ocean indicates incongruity between processes shaping the distributions of diversity at the species and population levels. Among the many possible causes of this incongruity, genetic drift provides the most compelling explanation: given massive effective population sizes of Indo‐Pacific species, even hard vicariance for tens of thousands of years can yield ΦST values that range from 0 to nearly 0.5

Population connectivity of neon damsel, Pomacentrus coelestis, inferred from otolith microchemistry and mtDNA

Understanding dispersal patterns and population connectivity is crucial to the conservation and management of fish assemblages in reef ecosystems. To reveal the population connectivity of reef fishes in the northern West Pacific, we examined the otolith chemistry and the mtDNA control region of Pomacentrus coelestis collected from six localities between Hainan Island (China) and Okinawa (Japan). The results of otolith chemistry analyses on pre-settlement signatures showed that fishes in north-west Taiwan may have a similar origin, whereas those in southern Taiwan might have a separate origin from the South China Sea. Furthermore, the elemental ratios of post-settlement signatures showed clear separation among localities, reflecting their sedentary behaviour after settlement and the influence of local environment. Population genetic analyses revealed that significant genetic differentiation occurred between populations in north-west Taiwan and populations in the South China Sea and the Kuroshio ecosystems. This study demonstrated that otolith chemistry and mtDNA analyses revealed consistent and complementary results of the dispersal and connectivity of P. coelestis populations, and provided evidence supporting that fish populations around Taiwan should be considered as two stocks. Thus, two marine protected area networks should be established for the conservation and management of reef fishes in this area

Population connectivity of Pomacentrus coelestis in Northern West-Pacific

了解海洋生物族群間的連通性是漁業管理與保護區設立必須具有的基礎資訊，藉由比較不同地理族群間的遺傳差異，可間接估算族群間過去或正在進行的基因交流，而耳石中的化學元素濃度變化也可用於研究魚類的遷徙與幼生散佈的途徑，因此藉由此兩種方法可以了解過去至現在族群之間的動態關係，本論文即利用上述兩種方法研究西太平洋變色雀鯛族群的連通性，並探討族群新添量的來源。去的研究發現，日本本島與琉球群島的變色雀鯛族群的生殖特徵如卵徑大小、卵塊大小與產卵頻率，會隨著不同緯度而呈現梯度變化，這些特徵被認為可能與遺傳變異有關，本研究以粒線體基因控制區的片段，分析台灣南部至日本本島共8族群的遺傳結構，結果顯示，日本本島與琉球群島族群間的基因交流很低，而族群間分化的型式與生殖特徵的變異型式相符，意味著生殖特徵的變異很可能具有遺傳基礎；另一方面經由族群變動係數的估算與族群擴張的檢測發現，各變色雀鯛族群可能在2萬七千到七萬年前遭受過嚴重的瓶頸效應與之後的族群快速擴張，因此推測更新世的冰河期海平面變遷可能對變色雀鯛歷史族群變動有很重大的影響。由估算台灣鄰近海域變色雀鯛族群間的Φst值，本研究發現台灣周圍海域的變色雀鯛族群可以分成兩大群，一群包括北台灣與澎湖族群，另一群則包括海南島、南台灣與琉球族群，此一分群的模式與過去研究台灣海域珊瑚礁生物族群結構的結果相似，本實驗進一步使用多基因座基因型和貝氏分配方法判斷新添入幼生的可能來源，結果顯示台灣南部族群新添入的幼生約有50%可能來自海南族群，且台灣南部與海南島變色雀鯛成體族群之間的基因交流非常強，此幼生散佈途徑與過去的推論並不符合，另一方面北部與澎湖的新添量組成則較為均質，對成體Φst 與幼生Fst分析中發現，台灣南部族群與澎湖—台灣北部族群間的基因交流皆很有限，因此推論在台灣周圍的變色雀鯛族群入添可能有兩個來源。了進一步驗證此幼生散佈路徑，分別自北部的卯澳及野柳、澎湖的青灣外灣與墾丁的眺石，每地點各採集十隻，共40隻變色雀鯛成體，分析不同地點魚體內扁平石(sagittae)的化學元素組成，結果顯示，Mg/Ca, Mn/Ca, Sr/Ca, Ba/Ca 與 δ18O等元素在不同採樣點之間皆有顯著差異(p &lt; 0.05)，主成分分析(PCA)結果顯示，眺石族群的化學元素組成明顯可與卯澳、野柳及青灣外灣區隔，而分成兩大群。此結果與先前的族群結構分析結果相符，且眺石族群的平均δ18O的數值為四個採樣點中最高(-2.239 ± 0.104)，表示在幼生漂浮期所接觸到的水團性質為高溫低鹽，過去研究顯示南中國海表層流水團的性質是台灣鄰近海域中溫度最高鹽度最低，因此符合先前以基因型分析法所得結果，也就是南部新添入的幼生可能源自南中國海。此外，在耳石微量元素分析過程中發現，Mn與Ba元素的累積可能受到體內生理調節的影響因此無法正確反映水體元素濃度的變化，因此Mn與Ba兩種元素必須謹慎使用。研究結果顯示，變色雀鯛的族群結構受到更新世冰河期海水面變遷的影響甚大，而現今族群間的連通性則主要受到當今洋流系統的影響。本研究藉由了解變色雀鯛不同地理族群間的連通性，可提供未來西太平洋珊瑚礁魚類資源管理與海洋保護區設立的參考資料。Analysis of genetic data can reveal past and ongoing connectivity among reef fish populations, and using geochemical signatures in calcified structures provide another indirect means of tracing movements and determining the population origin of reef fishes. These methods can be applied to determine which populations are connected by larval transport, and the results of these studies are crucial for effective management of fisheries and the design of marine reserves. In this dissertation, I used both methods to study the connectivity of Pomacentrus coelestis among populations in the West-Pacific. he results of genetic analysis indicated that a genetic break existed between mainland Japan and Okinawa populations, and this break was congruent with the pattern of phenotypic variations documented in previous studies. This evidence supports that the latitudinal variation of reproductive traits among P. coelestis populations is likely being genetically based. The historical demography of P. coelestis revealed by molecular data suggested that drastic changes in sea level and sea surface temperatures during the last glaciations might have resulted in dramatic reduction of their population size in the northwestern Pacific. Among populations around Taiwan, a genetic break was found between the Penghu-northern Taiwan group and the southern Taiwan group (Kenting and Hainan populations). However, the results of Bayesian assignments revealed a larval transportation pattern differed from that proposed by previous studies. The recruits in southern Taiwan were mainly from the South China Sea (approximately 50%), while the recruits in Penghu and northern Taiwan were from mixed sources. Moreover, both Φst of adult populations and Fst of recruit populations indicated limited gene flow between Penghu-northern Taiwan and southern Taiwan groups. It suggests that the recruits in southern Taiwan may have different origins comparing with those of northern Taiwan and Penghu populations.he geochemical signatures in sagittae showed that significant differences were found in Mg/Ca, Mn/Ca, Sr/Ca, Ba/Ca and δ18O values among sites in pre-settlement region of otoliths. By using the combined data of trace element ratios and stable isotopic compositions, the results of PCA (principal component analysis) showed that the otoliths collected from Tiaoshi, southern Taiwan and other three sites (Maoao, Yehliu and Chinwan Outer Bay) around Taiwan could be separated. This indicates that the population at Tiaoshi may have different source which is congruent with the results of genetic studies. Overall, it suggests P. coelestis larvae may be transported by the South China Sea Surface Water (SCSSW) from Hainan Island to southern Taiwan, and the population at Chinwan Outer Bay in Penghu and Yehliu in northern Taiwan may share the same gene pool (Fst = -0.0001, p &gt; 0.05). In addition, the results also indicated that the incorporation of Ba/Ca and Mn/Ca element ratios in P. coelestis otolith could control by physiological regulations. Thus, caution is warranted when using these two element ratios. hese results suggest that the last glaciations had profound effect on the historical demography of P. coelestis, and the recent demographic connectivity was mainly determined by local hydrodynamic regimes. Since self retention is unlikely the case in P. coelestis populations, the connectivity patterns of P. coelestis could be used as a reference for designing effective management strategies on marine organisms with low degree of self retention in the West-Pacific.Acknowledgements……………………………………………………………….. Ibstract in Chinese……………………………………………………………….. IIbstract in Englaish……………………………………………………………… IVable of contents….……………………………………………………………… Vhapter 1………………………………………………………………………….. 1.1 Reef fish connectivity and implications………………………………………… 1.2 The relationship between PLD and population connectivity…………………….…. 2.3 Larval fish sensory and behavior………………………………………………. 2.4 Biogeographic barriers and population connectivity…………………………..….. 3.5 The hydrodynamic regime in the West-Pacific and the population connectivity……… 4.6 Basic biology of P. coelestis……………...…………………………………… 4.7 Aims of this dissertation………………………………………………….…... 5hapter 2…………………………………………………………………….….… 8.1 Introduction…………………………………………………………...….… 8.2 Materials and Methods………………………………………………….….… 9.3 Results………………………………………………………………….…. 11 .4 Discussion…………………………………………………………………. 13.5 Tables………………………………………………………………….….. 20.6 Figures…………………………………………………………………….. 24hapter 3………………………………………………………………………….. 30.1 Introduction………………………………………………………………… 30.2 Materials and Methods…………………………………………………..…… 33.3 Results…………………………………………………………………..…. 36.4 Discussion……………………………………………………………….…. 39.5 Tables…………………………………………………………………..….. 44.6 Figures……………………………………………………………………... 49hapter 4……………………………………………………………………….….. 52.1 Introduction……………………………………………………………….… 52.2 Materials and Methods……………………………………………..……….… 55.3 Results……………………………………………….…………………..…. 58 .4 Discussion…………………………………………….………………….…. 59.5 Tables……………………………………………….…………………….... 64.6 Figures…………………………………………….………………………... 68hapter 5……………………………………………………………….……..…… 73.1 Historical demography……………………………………………………...… 73.2 Linkage between genetic differentiations and reproductive traits………………….… 73.3 Stock origin……………………………………………………………...…... 74 .4 The application on reef fish fisheries…………………………………………..... 74.5 Future studies………………………………………………………………... 75.6 Tables…………………………………………………………………......... 76.7 Figures…………………………………………………………………..….. 77eference…………………………………………………………………………... 78ppendix 1…………………………………………………………………………. 91 ppendix 2……………………………………………………………………....…. 92 ppendix 3…………………………………………………………………………. 9

Genetic structure and relatedness of juvenile sicklefin lemon shark (Negaprion acutidens) at Dongsha Island

Abstract Negaprion acutidens (sicklefin lemon shark) is distributed in the Indo-Pacific and in close association with coral reefs. Under the protection of the Dongsha Atoll National Park, a small but well-established juvenile population of N. acutidens inhabiting coastal areas of Dongsha Island was recently observed to display site fidelity by using acoustic telemetry. This study was designed to reveal the fine scale genetic structure and relatedness within and among 5 juvenile shark cohorts inhabiting 3 sampling sites at Dongsha Island. A total 188 juveniles were caught and sampled between 2016 and 2017, and genotyped with twelve loci. They were assigned to 5 year cohorts (2013–2017) based on the body length and date they were caught, also assigned to 3 sites based on where they were caught. Among five cohorts, the percentage of unrelated pairs within a cohort is more than 62% in average, suggesting a potential high mortality during their early life stage. The results of Fst and assignment testing showed that there was no significant genetic structure between sites and cohorts indicating that there was no fine scale genetic structure, even though the juveniles possessed strong site fidelity. A small effective population size (Ne) was detected (Ne = 86.7) which indicates the presence of a potentially isolated and vulnerable population at Dongsha. These results provide the genetic diversity as a baseline for future management and conservation of N. acutidens in the South China Sea

Genetic identification and hybridization in the seagrass genus Halophila (Hydrocharitaceae) in Sri Lankan waters

Seagrasses, as marine angiosperms, play important roles in coastal ecosystems. With increasing anthropogenic impacts, they are facing dramatic declines on a global scale. Halophila is well-known as a complex taxonomic challenge mainly due to high morphological plasticity. By using only a morphological approach, the genus could be over-split or similar species could be erroneously lumped, thus masking its true biodiversity. In the present study, we incorporated genetic identification with morphological examination to reveal the identity of Halophila plants in southern and northwestern Sri Lankan waters. The nuclear ribosomal internal transcribed spacer (ITS) region and chloroplast ribulose-bisphosphate carboxylase gene (rbcL) were used to identify plants collected from the Gulf of Mannar, Puttalam Lagoon, and Matara, Sri Lanka. Based on genetic identification, H. major (Zoll.) Miquel is reported for the first time from Sri Lanka, which might have been misidentified as H. ovalis in previous literature based on morphology alone. We also observed a first hybridization case of Halophila cross between H. ovalis and H. major. Two potential cryptic species were found, herein designated Halophila sp. 1 (allied to H. minor) and Halophila sp. 2 (closely related to H. decipiens). In order to clarify taxonomic ambiguity caused by morphological plasticity and the low resolution of genetic markers, further comparative phylogenomic approaches might be needed to solve species boundary issues in this genus