中文摘要
本研究以基因標誌(genetic markers)分析塘鱧以及蝦虎魚類之親緣關係，主要之成果可以分成五個部分。玆分述如後：
(1) 分析脊椎動物已發表的粒線體去氧核醣核酸序列，利用其中位於tRNAPHE 以及16S rRNA中保守的序列設計二個萬用引子。本研究利用這二個萬用引子以聚合連鎖反應(polymerase chain reaction; PCR)增幅放大脊椎動物的DNA。研究樣本包含脊椎動物的五個綱共14個物種，所有的PCR都成功的放大出約1.2kb的DNA片段。其中PCR產物長度的變化極微。顯示此一片段在脊椎動物中尚稱保守。進一步定序反應證明此一片段包含粒線體DNA中完整的12S rRNA，tRNAVAL 以及大約200bp的16S rRNA序列。由於12S rRNA基因廣泛的被運用在動物的親緣分析。大至哺乳類各目之間，小及不同屬之間的演化關係，均曾利用此基因加以探討。因此，這組萬用引子對於研究脊椎動物不同階層之親緣關係將非常具有價值。
(2) 利用上述萬用引子，增幅放大蝦虎亞目魚類的粒線體DNA。在本研究中一共採得43種蝦虎魚類，他們分別屬於33個不同的屬。這些魚類的粒線體12S rRNA以及tRNAVAL均被增幅並且定序。在親緣分析方面，利用最大儉約法(Maximum Parsimony)以及Neighbor-joining法所得到的結果非常相似，並大致上與過去型態分析所得的結論一致。其中沙塘鱧(Odontobutis)在兩種分析方法中都位於親緣樹根部(root)的位置，可以視為除溪鱧之外所有蝦虎魚類的外群。在傳統的塘鱧魚類之中，DNA分子證據強烈支持塘鱧亞科(Eleotrinae)的單一起源。而尖塘鱧(Butinae)應該是比較接近五個鰓被架(branchiostegal ray)的一群。在其餘的蝦虎魚類中，利用尾上骨(epural)的數目大致上可以將其區分為兩群。擁有一個尾上骨的魚類包括，Pezold (1993)的蝦虎亞科(Gobinae)與蚓蝦虎科(Microdesmidae)等是為一群。而彈塗魚亞科(Oxudercinae)，擬蝦虎亞科(Gobionellinae)等有二個尾上骨的魚類是為第二群。這兩群則互為姊妹群。這其中禿頭鯊亞科(Sicydiinae)是為例外。雖然它有一個尾上骨但是卻被歸為第二群。由於在第二群中同一種類不同個體之間常常有一個以及二個尾上骨的多型性現象。所以禿頭鯊亞科應可視為在第二類群中較衍生的種類。
(3) 進一步利用這些蝦虎魚類的12S rRNA序列來分析此一基因的二級結構。在此一分析中還另外加入其它九種魚類的12S rRNA序列，這九種魚類分屬八個不同的目(Order)。在本研究所提出的43個stem之中，至少有一個以上互補突變(compensatory mutation)出現在脊椎動物的共有41個。分析蝦虎魚類互補突變的比例為百分之68。依據此一數據根據Hillis and Dixon (1993)的方式計算可以得到stem的序列需給予的權數(weighting)為0.66。然而不同的loop與stem權數(分別為1比1、1比0.8、1比0.6、1比0.5等)去分析蝦虎魚類的親緣關係時卻得到相同的結果。顯示減輕stem的權數與親緣關係的分析沒有關聯。另外在loop之中，很明顯的可以發現核酸組成的不對稱性即：A% > T%以及C% > T%。相同的情況也在哺乳類的12S rRNA觀察到。這種不對稱核酸組成可以用粒線體DNA複製時在C與A鹼基上所產生自發性的去硝基化(deamination)的假說解釋。此一假說也同時可以解釋在stem以及loop上transition比例大過於transversion的現象。
(4) 為了解塘鱧魚類的分布與洋流的關係，本研究分析採自高屏溪口的塘鱧。以確定其生殖週期，以及漂浮性幼體(pelagic larvae)出現的時間。在雄性個體中，其雄性素(testosterone)在一年之中以六至八月為最高，與其GSI的變動相符。雌性個體的性激素(estradiol)，以及GSI與雄性個體的變化相同。進一步分析發現生殖週期的變化與當地光週期以及水溫的變動有很強的相關性。這兩項因子可能是影響塘鱧生殖週期的重要因素。
(5) 在塘鱧屬魚類的系統分類上，分別以同功電泳以及DNA序列分析台灣以及周圍離島採得的塘鱧屬魚類。同功電泳與DNA序列所得到的結果相同，二者皆支持黑塘鱧(E. melanosoma)與棕塘鱧(E. fusca)最為接近互為姊妹群，其次則為塘鱧(E. acanthopoma)。在17個同功基因座之中，ADH-1以及MDH-1可以區分三種不同的塘鱧魚類，因之可利用其作為生化鑑定的特徵。條紋塘鱧(E. faciatus)與銳頭塘鱧(E. oxycephalus)在本次研究期間均沒有在台灣以及周圍離島採獲。其中前者由於其型態描述與採自蘭嶼的棕塘鱧無法區分，因此可能是棕塘鱧的同物異名。另外採自中國大陸的銳頭塘鱧與其它三種塘鱧屬魚類的關係甚遠。它們的單系起源(monophyly)也不被DNA資料所支持。由於塘鱧屬的分布很廣，種數也多。因此其屬內的親緣關係需要進一步的分析。
塘鱧魚類在台灣的分布呈現不同種類區隔的現象。棕塘鱧主要分布在東部沿岸，黑塘鱧以及塘鱧主要分布在西部海岸。以同功電泳分析採自西部海岸五個塘鱧的族群發現，不同地區的族群分化指數極低(FST = 0.019)。顯示這些地區魚類族群遺傳結構相當均值。這種不同種間區隔分布以及種內遺傳結構均質的現象可能受到其幼體散佈以及洋流的影響。

ABSTRACT
In this study, I used genetic marker to analyse the phylogenetic relationships between eleotrids and gobiid within gobioids. The main achivement can be described as the following five sections:
(1) The conserved regions of tRNAPHE and 16S rRNA in the vertebrate mitochondrial genome were compared in order to design the primers, 12SR and 12SL. These universal primers can be broadly used to amplify a 1.2-kb DNA fragment by polymerase chain reaction (PCR) over a wide range of major vertebrate lineages represented by the species listed in the text. There is little length variation of the PCR product among different taxa. Further sequence analysis revealed that the fragment contains complete lengths of 12S rRNA and tRNAVAL, and that the length of 16S rRNA is 200 bp. In tests through all representative taxa investigated, the above 2 primers could amplify the complete 12S rRNA gene from all representative taxa investigated. As the 12S rRNA gene is widely used for phylogenetic analyses among different hierarchies, these primer sets are useful for vertebrate phylogeny.
(2) These primer sets were further used to investigate the phylogenetic relationships of Gobioidei. The molecular phylogeny of gobioid fishes studied comprising 33 genera and 43 valid species were examined by using complete mitochondrial 12S rRNA and tRNAVAL genes. Both parsimony and neighbor-joining analyses reveal comparable results and are congruent with that of morphological studies. The Odontobutis, which always formed at the root of the phylogenetic trees, can be treated as sister group of all other non-rhyacichthyid gobioids. Within eleotrid fishes, the monophyly of Eleotrinae (Hoese and Gill 1993) is strongly supported by molecular data. The Butinae is closer and should be treated as sister group of five-branchiostegal rays group. Of the five-branchiostegal rays, except of sicydine, can be divided into two groups according to their epural counts. The fish with one epural, Gobiinae of Pezold (1993) plus microdesmid, is resolved as a monophyletic group and sister to that with two epurals, Oxudercinae and Gobionellinae of Pezold (1993). However, with one epural, sicydine is more close to Oxudercinae and Gobionellinae rather than Gobiinae. Since the progressed reduction of epural number has been observed along this lineage. The sicydine should be treated as derived group within the group with two epural.
(3) The 12S rRNA sequences of 43 gobioid species and nine diverse assortments of fishes were further analyzed and employed to establish a "core" secondary structure model for fish 12S rRNA. Of 43 stems recognized, 41 of which were supported by at least some compensatory evidence among vertebrates. An analysis of compensatory substitution shows that the percentage of co-variation is 68% and the weighting factor for phylogenetic analyses to account for the dependence of mutations should be 0.66. Different stem/loop weighting schemes were applied to the analyses of phylogenetic relationships of Gobioidei which show that down-weighting paired region due to non-independence is irrelevant in the present phylogenetic analysis. The biased nucleotide composition (A% > T%, C% > G%) in loop regions was also observed on its mammalian counterpart. The exceed of A and C in loop region may be due to asymmetric mechanism of mtDNA replication which leads to the spontaneous deamination of C and A. This process may also contribute to the preference for transitions over transversions in both loop and stem regions.
(4) For further understanding the relationship between Eleotris dispersal and sea current, the reproductive cycle of Eleotris acanthopoma was investigated. For over 1 year, gonad gross morphologies of fish were examined by using light microscopy and the plasma 17 beta-estradiol (E2) and testosterone (T) were analyzed by using ELISA. In male E. acanthopoma, plasma T concentrations show a single seasonal cycle, with a peak in summer (June to August) following the profile of gonadosomatic index. In females, plasma E2 concentrations are significantly elevated during summer in accordance with the GSI. Low temperatures and a short photoperiod in winter correlate well with the arrest of gonad maturation. Annual patterns of plasma E2 and T levels are similar to those of GSI changes existing in Eleotris acanthopoma of either sexes. These findings indicate that both temperature and photoperiod dominantly affect the reproductive cycle of Eleotris acanthopoma.
(5) The systematic of genus Eleotris was carried out by isozyme electrophoresis and DNA sequences analysis. Both data sets achieve the similar conclusion, which indicates that the E. acanthopoma is sister group to E. melanosoma and E. fuscus. Among 17 loci analyzed, ADH-1 and MDH-2 can be used as biochemical key to identify these three species in Taiwan. E. faciatus were not found during this study period. Since the morphological characters of E. faciatus described by Chen (1968) can not distinguish from E. fuscus collected from Orchid Island. I suppose that the E. faciatus is synonymous to E. fuscus. E. oxycephalus was not collected either due to its rareness in Taiwan. The E. oxycephalus collected from Mainland China show distantly related with other three species. The phylogeny of this genus may need further investigation.
The distributions of Eleotris species around Taiwan are different: E. fuscus is confined to the east coast whereas the E. acanthopoma and E. melanosoma to the west. Samples of E. acanthopoma from different localities show extremely low level of differentiation (FST = 0.019), indicates the genetic structures of this species among different collecting site are homogenous. Both differentiated distributions between species and homogenous genetic structures within species are due to the association of reproductive cycle with seasonal changed water current.

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