Systematics, population genetic structure and phylogeography of Zacco (Cypriniformes: Cyprinidae) in Taiwan

初級淡水魚(primary freshwater fishes)為一群耐鹽度低、終生棲息在淡水水域中的純淡水魚類，其遷移、擴散及分佈均受到河川水系聯結的影響。因此，地區性歷史事件所引發的水文條件改變，常反映在當地初級淡水魚類族群的遺傳結構及種化事件上。族群遺傳結構的釐清及物種的界定同為演化生物學及保育生物學的關注重點。因此，初級淡水魚不僅為研究物種演化的極佳材料，其研究成果亦可豐富淡水魚保育相關資訊，作為制定保育政策之參考。本實驗即以台灣初級淡水魚—鱲屬魚類(Zacco)為主題，探討分類、演化及保育等相關問題。 鱲屬魚類，俗稱溪哥、苦槽仔、闊嘴郎或紅貓(雄性個體)，在分類上屬於鲤形目(Cypriniformes)，鲤科(Cyprinidae)，魚丹亞科 (Rasboninae)。本屬在此研究開始之初約有7-10種，主要分佈於東亞地區，包括日本、韓國、中國大陸及台灣，為各地淡水生態系中常見魚類。但其物種數目隨研究者所依據之分類標準而時有更動：溪哥在台灣過去咸認為有兩種，分別是東亞廣佈種—平頷鱲 (Z. platypus)和台灣特有種—粗首鱲(Z. pachycephalus)，原本分布於西部及蘭陽平原各河川。然而，近年來已成為假日垂釣者最喜好的魚種之一，因此，經常有放流人工養殖魚苗的事件，導致在花東各主要河川出現人為野放族群。 我在野地觀察研究初期，注意到台灣溪哥呈現複雜的形態多型性，似乎不只兩種。因此，我檢視計數(meristic) 的形質，並利用形質測量(morphometric)的方法去分析其形態多型性。接著再配合Amplified fragment length polymorphism (AFLP)分子遺傳標記，對台灣溪哥魚類進行系統分類研究(CHAPER II)。結果發現在過往認為的平頷鱲 (Z. platypus)和粗首鱲(Z. pachycephalus)中，不但各有兩種明顯的形態型(morphotypes)，而且4個形態型之間的AFLP亦呈現顯著差異，因此推測台灣溪哥魚類具有4個種(Z. pachycephalus、Z. sp、Z. platypus及Z. evolans)。 在CHAPER III中，我利用粒線體DNA D-loop序列，針對日本平頷鱲(Z. platypus)與台灣Z. platypus作親源關係分析，證實台灣Z. platypus為源自日本琵琶湖的平頷鱲族群，推測是經由多次人為放流所致。 在CHAPER IV中，我利用粒線體DNA D-loop序列及微隨體基因座(Microsatellite loci)，證實先前所發現的4種台灣溪哥，在此兩種分子遺傳標記上亦均有明顯分化，支持台灣有4種溪哥的推論。此外，本章節也針對此4種溪哥的族群遺傳結構、地理分布、及demographic history進行研究分析，結果顯示台灣溪哥的種間及種內之遺傳差異，可能同時源於「多次入侵」與「島內隔離種化」所致。此外亦證實東部人為放流的溪哥族群屬於來自北臺灣及中台灣的Z. pachycephalus。 在CHAPER V中，我簡單總結此論文的實驗結果：台灣有4種鱲屬魚類或是俗稱的溪哥，而非向來認為的2種。而且其中的一種屬於人為意外引進的種類。這4種的地理分佈與族群遺傳結構皆不相同，顯示台灣的溪哥族群受到歷史地質事件的影響。此外，依據目前染色體核型分析的初步結果與mtDNA所建構出來的演化樹顯示，在現行分類下的鱲屬(Zacco)魚類，並非屬於單系群(monophyletic group)，而是與其它近緣屬(i.e. Opsariichthys and Candidia)形成並系群(paraphyletic group)，因此，未來研究方向將著重在釐清鱲屬魚類與其他近緣屬間的親緣關係。The studies of population structures and speciation are major concerns of evolutionary and conservation biology. Primary freshwater fishes have little or no tolerance to saltwater. Their migration, dispersal and distribution depend upon direct connections between drainage basins. Thus, the population structure dynamics and species formation are associated with historical events which resulted from changes of hydrological conditions. The investigation of freshwater fishes can lead to important insights in evolutionary biology, and the results can provide useful information for conservation and management. Therefore, in this study, I use the primary freshwater, Zacco spp, in Taiwan as a model to elucidate several important issues, such as systematics, evolutionary history, and conservation in freshwater fishes. The Asian common minnow, Zacco, is a cyprinid genus of small and colorful fishes, which are dominant components in the freshwater ecosystems of East Asia, including Japan, Korea, China and Taiwan. There were 7-10 species recognized in this genus. Two species were formerly known in Taiwan; Z. pachycephalus (Günther) is endemic to Taiwan, and Z. platypus (Temmick &Schlegel) is wide-spread in East Asia. However, no Zacco were recorded in the eastern part of Taiwan until 17 years ago. The recent studies showed that the eastern populations of Z. pachycephalus were artificially introduced from unknown origins by anglers. In my initial field survey, I noticed apparent intraspecific morphological polymorphisms in currently recognized Zacco species, which prompted the thought that more than two species might occur in Taiwan. Consequently, I tackled the problems of Zacco species complex by two different approaches: morphologically and genetically. For morphological analyses, I used both meristic and morphometric characters (CHAPTER II). Furthermore, I integrated several genetic markers in identifying genetically distinct morphotypes: (1) AFLP (CHAPTER II) (2) mtDNA D-loop (CHAPTER III) (3) microsatellites (CHAPTER IV). Both morphological analyses and AFLP suggest two genetically distinct morphotypes in each of the two Taiwanese Zacco (Z. pachycephalus and Z. platypus). Therefore, I proposed that there are four Zacco species in Taiwan, which are referred herein as Z. pachycephalus,Z. sp, Z. platypus and Z. evolans. Furthermore, I analyzed the mtDNA D-loop sequences for Taiwanese and Japanese Z. platypus. Phylogenetic analyses confirmed that the Taiwanese Z. platypus originated from Lake Biwa of Japan through multiple introductions. Besides, I examined additional molecular markers, microsatellite loci, in Taiwanese Zacco. The results again supported the genetic divergence of the four newly identified Zacco species. I also examined the geographic distribution and demographic histories of these Zacco species. It is evident that the basis and origin of the intra- and interspecific divergences of Taiwanese Zacco resulted from “multiple invasions” and “insular vicariances”. It is also evidenced that the eastern artificially introduced populations originated from both northern and central populations of Z. pachycephalus. Finally, I summarized the results of my studies in CHAPTER V: (1) Four Zacco species are recognized in Taiwan and one of them was introduced by human activity. (2) Distinct geographical distribution and population genetic structure of these four species indicate the influences of historical geographic events on Zacco populations. In addition, I also showed preliminary results of karyoptypic studies and mtDNA phylogenetic analyses. I proposed that the genus Zacco is not a monophyletic group, but is paraphyletic with the genera Opsariichthys and Candidia. Therefore, my further study aims to clarify the taxonomic status of the genus Zacco.中文摘要……………………………………………………4 Abstract……………………………………………………6 CHAPTER I: General introduction 遺傳變異的來源……………………………………………8 生物親緣地理學……………………………………………9 初級淡水魚…………………………………………………11 鱲屬魚類Zacco……………………………………………12 論文架構…………………………………………………13 參考文獻…………………………………………………16 CHAPTER II: AFLPs congruent with morphological differentiation of Asian common minnow Zacco (Pisces: Cyprinidae) in Taiwan Abstract…………………………………………………20 Introduction……………………………………………21 Materials and methods………………………………24 Results…………………………………………………28 Discussion………………………………………………32 References………………………………………………39 Tables……………………………………………………44 Figures…………………………………………………48 CHAPTER III: Mitochondrial phylogeny reveals the artificial introduction of the pale chub Zacco platypus (Cyprinidae) in Taiwan Abstract………………………………………………53 Introduction…………………………………………54 Materials and methods……………………………56 Results………………………………………………58 Discussion……………………………………………60 References……………………………………………62 Tables…………………………………………………64 Figures…………………………………………………66 CHAPTER IV: Phylogeny, population structure and biogeography of the Asian freshwater minnow Zacco in Taiwan based on mitochondrial DNA and microsatellite variations Introduction……………………………………………69 Materials and methods………………………………72 Results……………………………………………………77 Discussion………………………………………………81 References………………………………………………85 Tables……………………………………………………89 Figures……………………………………………………93 CHAPTER V: Conclusion and future directions Conclusion………………………………………………97 Future directions………………………………………97 References………………………………………………99 Tables……………………………………………………100 Figures……………………………………………………101 Appendix 1: 台灣四種溪哥的雜交實驗………………103 Appendix 2: 主組織相容性複合體(Major Histocompatibility Complex; MHC)在四種台 灣溪哥的遺傳分析……………………………108 Appendix 3: 溪哥魚類染色重複性序列之選殖與分析………11

A Recurrent Itga9 Missense Mutation in Human Fetuses with Severe Chylothorax: Possible Correlation with Poor Response to Fetal Therapy

OBJECTIVES: To assess the possible correlations between the reported candidate genes (VEGFR3, FOXC2, ITGA9 and ITGB1) and the clinical response in fetuses with severe congenital chylothorax (CC) treated by prenatal OK -432 pleurodesis. METHODS: We studied 12 unrelated fetuses with severe CC, receiving fetal therapy by OK-432 pleurodesis. Genotyping of the candidate genes and the clinical parameters of these 12 fetuses were investigated. Additional 96 control individuals were enrolled to evaluate the possible polymorphisms at these candidate genes in population. RESULTS : A recurrent heterozygous missense mutation (c.1210G >A, p.G404S) was identified in the beta-propeller domain of integrin alpha(9) (ITGA9), a cell adhesion receptor, in four of the five fetuses who failed to respond to the OK-432 treatment. Computer modeling of the p.G404S substitution supported the deleterious nature of this mutation. Family analyses in three affected fetuses demonstrated that the heterozygous mutant allele is of parental origin, suggesting an autosomal recessive inheritance of this genetic defect. CONCLUSIONS: To the best of our knowledge, this is the first insight into the possible link between ITGA9 and CC in human fetuses . The identification of pathogenetic mutations and their possible link to the clinical responses of particular treatments may contribute to better pregnancy counseling and management

The Spectrum of the Factor 8 (F8) Defects in Taiwanese Patients with Haemophilia A

Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by various types of pathological defects in the factor VIII gene (F8), which encodes coagulation factor VIII (FVIII). To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we report the distribution of the mutations within the F8 gene in 31 Taiwanese unrelated HA patients (19 severe and 10 moderate/ mild males and two severe females ). Of these, 12 (38.7%) and one (3.2%) severe males were genotyped with the recurrent IVS22 and IVS1 inversion, respectively, similar to that in general populations (IVS22: 40-50%; IVS1: 2-5%). The F8 defects in the remaining 18 inversion-negative patients cover a wide spectrum, in which 17 different mutations were identified (10 missense and three nonsense mutations, and two small and two large deletions). Eleven of these mutations are novel: seven caused missense substitutions and four resulted in truncated proteins. To assess the putative pathogenetic impacts of the newly amino acid substitutions, computer analyses were performed based on molecular 3D modelling. The degree of conservation in cross-species FVIIIs and the position in known functional FVIII regions were studied. The novel missense mutations found in our series all occurred at evolutionary conserved residues that may carry a functional importance in our analyses. The results of this study add the short list of Taiwanese/ Chinese F8 mutations, and will enhance our understanding of the molecular basis of FVIII function and the mechanism underlying HA

Rapid Prenatal Confirmation of Lit1 Hypomethylation Using a Novel Quantitative Method (E-Q-Pcr) in Fetuses with Beckwith-Wiedemann Syndrome Impressed with Ultrasonography

We described a simplified and high-performance test (E-Q-PCR ) for rapid assessment of the DNA methylation status at LIT1 , a major genetic locus of Beckwith-Wiedemann syndrome (BWS) . The E-Q-PCR test can detect and quantify the methylation changes between BWS fetuses and unaffected individuals in aminocytes as well as in lymphocytes and can be completed in I working day, and thus is a useful method for prenatal molecular diagnosis of BWS