浙江麗水中華大蟾蜍和黑眶蟾蜍蝌蚪對水位變化的表型響應

唐小芬, 樊曉麗, 林植華, 姚婷婷, 李 香, 金 晶, 周存通

麗水學院生態學院, 麗水 323000

浙江麗水中華大蟾蜍和黑眶蟾蜍蝌蚪對水位變化的表型響應

唐小芬, 樊曉麗, 林植華*, 姚婷婷, 李 香, 金 晶, 周存通

麗水學院生態學院, 麗水 323000

全球氣候變暖引發棲息地干涸將對生活在水中的無尾類幼體提出了挑戰。通過浙江麗水中華大蟾蜍(Bufogargarizans)和黑眶蟾蜍(Duttaphrynusmelanosticus)蝌蚪在實驗條件下對不同水位變化的表型響應，檢測表型可塑性的遺傳性和環境近因性影響。結果表明，水位變化對中華大蟾蜍蝌蚪早期發育歷期、頭寬和體重影響不顯著，對體長影響顯著，其中逐減水位最大、恒低水位最小，慢波、恒高與快波、逐增水位依次減少；水位變化對黑眶蟾蜍蝌蚪早期發育歷期、體長、頭寬和體重影響均顯著；發育歷期以恒高水位最大，恒低水位最小；體長以逐減水位最大，恒低、快波和慢波水位顯著偏小，逐增和快波水位居中；頭寬以恒低水位最小，逐增水位居中，其余較大；體重以恒低水位最小、恒高水位最大，其余居中。水位變化對中華大蟾蜍蝌蚪的變態時間、體長、頭寬和體重影響均不顯著；水位變化對黑眶蟾蜍蝌蚪的變態時間、體長和體重影響均顯著，對頭寬影響不顯著；恒低水位的變態時間最長，恒高水位的變態時間最短，其他水位變化之間差異不顯著；恒高水位的體長最大，恒低和快波水位最小，其他居中；逐增和快波水位的體重最大，恒低水位最小。研究結果表明，繁殖季節不同的中華大蟾蜍和黑眶蟾蜍蝌蚪響應水位變化的表型可塑性差異顯著，長期在容易發生干旱和水位變化的冬季繁殖的中華大蟾蜍蝌蚪的表型可塑性低，在雨水充沛的春季繁殖的黑眶蟾蜍蝌蚪的表型可塑性高，表現出表型可塑性的種間差異和遺傳性；在早期發育過程中，兩種蝌蚪體長的共同的表型變異與缺乏遺傳基礎的環境近因性影響有關；黑眶蟾蜍蝌蚪對低水位或水位下降作出減速分化的消極響應，響應程度與環境信號的強弱直接相關。

中華大蟾蜍; 黑眶蟾蜍; 蝌蚪; 水位變化; 表型可塑性

全球氣候變暖引起棲息地水溫的升高，許多地區的降雨量減少[1- 2]，濕地多樣性和可利用性不斷下降，臨時性水體可能變干或者消失，持久性池塘也可能變得短暫[3- 5]，增加了水域繁殖無尾類幼體的生存壓力[6]。如果水體干涸速度超過無尾類幼體加快生長發育的能力，那么水體干涸將是致命的[7]，水環境可利用性是影響無尾類幼體生長發育的最重要生態因子之一[8]。生活史復雜的無尾類幼體以最適的變態時間和大小完成從水生生境到陸地生境的遷移，通過在生長發育過程中產生適應性表型可塑性來提高其適合度，對于個體生存和種群動態變化至關重要[9- 11]。

不同無尾類幼體對棲息地水位的敏感性不同[6]，存在長期進化形成的遺傳特異性和特定的環境適應性。例如，二光腫肋蟾(Pleurodemadiplolister)[12]、哈蒙掘足蟾(Scaphiopushammondi)[13]、庫氏掘足蟾(Scaphiopuscouchii)[14]、斑點合跗蟾(Pelodytespunctatus)[15]、Rhinellaspinulosa[16]、短頭蛙(Sphaerothecabreviceps)[17]、強刃鋤足蟾(Pelobatescultripes)[18]蝌蚪在響應水位下降時提前變態形成較小的變態個體；歐洲大蟾蜍(Bufobufo)和黃條背蟾蜍(Bufocalamita)蝌蚪在響應自然水體干涸時形成較小的變態個體，但是變態時間卻沒有改變[19]；敘利亞鋤足蟾(Pelobatessyriacus)蝌蚪通過加快發育提早完成變態來響應棲息地的干涸，但其變態時大小與恒定水位條件下所形成的變態個體差異不顯著[20]。生活在沙漠中的庫氏掘足蟾蝌蚪在低密度的池塘中會迅速完成變態發育，而在處于干旱條件下的高密度池塘中則很少能完成幼體的變態[21]。Wilbur和Collins 認為，利用臨時性池塘進行繁殖的無尾類會比那些利用持久性水體繁殖的無尾類在蝌蚪期和變態大小這兩個方面表現出更強的可塑性[22]。歐洲林蛙(Ranatemporaria)的數據表明，北方種群相對南方種群變態時間短，但缺乏對干涸風險的適應性響應[23]。

浙江麗水中華大蟾蜍(Bufogargarizans)和黑眶蟾蜍(Duttaphrynusmelanostictus)同域分布，在持久性池塘(終年有水)繁殖，前者在冬季12—3月繁殖，后者在春季3—5月繁殖[24]，季節差異造成降雨量的差異，冬天多干旱缺水，春季多雨。本研究通過中華大蟾蜍和黑眶蟾蜍蝌蚪在實驗條件下響應不同水位變化表型可塑性的種間差異，檢測如下兩個假設：(1)若水位變化誘導無尾類幼體的表型變異與個體的適合度密切相關，則不同季節物種的表型應表現出與其所在季節相對應的響應；(2)若遺傳因素僅能部分地解釋幼體表型的變異，則水位變化實驗應能檢測到不同季節物種共同的環境近因性影響形成的表型變異。

1 材料與方法

1.1 卵帶的采集和孵化

2012年2月23日和2012年4月20日在浙江麗水學院校園(28°27′ N，119°53′ E)內的同一持久性池塘中分別采集當天產中華大蟾蜍和黑眶蟾蜍的部分卵帶(各約300枚卵)，帶回兩棲爬行動物實驗室，分別置于塑料箱(700mm×500mm×400mm，200 mm水深)中孵化(室溫(23±0.2) ℃)，待蝌蚪長至能自由游泳的26—27期[25]用于實驗。

1.2 實驗設計與管理

圖1 六種水位處理下，中華大蟾蜍和黑眶蟾蜍兩種蝌蚪的變態成活率Fig.1 Survival rate of metamorphosis of B. gargarizans and D. melanostictus tadpoles under six different water level treatments

1.3 形態測定與發育歷期鑒定

中華大蟾蜍和黑眶蟾蜍蝌蚪的水位實驗分別在2月28日—3月25日和4月24日—5月22日完成。測定第1天(N= 20)、第14天和出現前肢(42期)時蝌蚪的濕重(BM: body mass)、體長(吻端到泄殖腔的距離，SVL: snout-vent length)、頭寬(頭部最寬處距離，HW: head width)和鑒定發育歷期。用吸水紙吸干蝌蚪表面水分，用Sartorius電子天平稱取濕重(± 0.001g)；將蝌蚪和變態幼體放入底下有標尺的培養皿中，用Sony DSC-T100數碼相機記錄形態，用ImageJ 1.44p軟件讀出(± 0.01 mm)；Nikon XTS30解剖顯微鏡鑒定蝌蚪的發育階段[25]。

1.4 數據分析

用Statistica統計軟件包完成所有數據的統計分析。統計分析前，檢驗數據正態性(Kolmogorov-Smirnov test)和方差同質性(F-max test)。經檢驗，數據無需轉換符合參數統計的條件。用One-way ANOVA、One-way ANCOVA及后續的Tukey′s檢驗處理和比較相應的數據，非參數統計用x2-test。描述性統計值用Mean±SE表示，顯著性水平設置為α = 0.05。

2 結果

2.1 實驗蝌蚪初始發育歷期與形態特征的種間比較

One-way ANOVA顯示，實驗用中華大蟾蜍蝌蚪和黑眶蟾蜍蝌蚪的初始發育歷期差異不顯著，中華大蟾蜍蝌蚪的體長顯著大于黑眶蟾蜍。以體長為協變量的One-way ANCOVA顯示，特定體長的中華大蟾蜍和黑眶蟾蜍蝌蚪的頭寬差異不顯著，中華大蟾蜍蝌蚪的體重顯著大于黑眶蟾蜍蝌蚪(表1)。

表1 實驗用中華大蟾蜍和黑眶蟾蜍蝌蚪的初始發育歷期和形態特征Table 1 The initial Gosner stage and morphological characteristics of tadpoles in B. gargarizans and D. melanostictus

蝌蚪的發育歷期和體長為One-way ANOVA，其體重和頭寬均為以體長為協變量的One-way ANCOVA;BG：中華大蟾蜍(B.gargarizans)，DM：黑眶蟾蜍(D.melanostictus)

2.2 水位變化對蝌蚪早期生長發育的影響

中華大蟾蜍蝌蚪和黑眶蟾蜍蝌蚪在6種水位條件下第14天的發育歷期、個體的體長、頭寬和體重的描述性統計見圖2。種間One-way ANOVA顯示，6種水位變化處理第14天時，中華大蟾蜍蝌蚪的發育歷期顯著小于黑眶蟾蜍蝌蚪，其體長顯著大于黑眶蟾蜍蝌蚪(表2)。以體長為協變量的One-way ANCOVA顯示，特定體長的中華大蟾蜍和黑眶蟾蜍兩種蝌蚪的頭寬和體重差異均不顯著(表2)。

表2 水位變化對中華大蟾蜍和黑眶蟾蜍蝌蚪早期生長發育的影響Table 2 Effect of water levels on the early growth and development of tadpoles in B. gargarizans and D. melanostictus

發育歷期和體長為One-way ANOVA，體重和頭寬均以體長為協變量進行One-way ANCOVA;不同上標表示差異顯著(Tukey′s test,α= 0.05, a > b > c); BG：中華大蟾蜍(B.gargarizans)，DM：黑眶蟾蜍(D.melanostictus); 水位處理類型用不同數字表示，1：恒定低水位；2：恒定高水位；3：水位逐漸升高；4：水位逐漸降低；5：水位快速波動；6：水位慢速波動

以水位變化為因子One-way ANOVA顯示，水位變化對中華大蟾蜍蝌蚪早期發育歷期影響不顯著，對體長影響顯著，其中逐減水位最大、恒低水位最小，慢波、恒高與快波、逐增依次減少；以體長為協變量的One-way ANCOVA顯示，水位變化對特定體長中華大蟾蜍蝌蚪的頭寬和體重影響不顯著(表2)。

以水位變化為因子One-way ANOVA顯示，水位變化對黑眶蟾蜍蝌蚪早期發育歷期和體長影響顯著，發育歷期以恒高水位最大、恒低水位最小，蝌蚪體長以逐減水位最大、恒低、快波和慢波水位顯著偏小，逐增和快波居中。以體長為協變量的One-way ANCOVA顯示，水位變化對特定體長蝌蚪的頭寬和體重影響顯著，頭寬以恒低水位最小、逐增水位居中、其余較大，體重恒低水位最小、恒高水位最大、其余居中(表2)。

圖2 6種水位處理下對第14天中華大蟾蜍和黑眶蟾蜍兩種蝌蚪的發育歷期和形態特征的描述性統計值Fig.2 Descriptive statistics of Gosner stage and morphological traits of B. gargarizans and D. melanostictus tadpoles under six different water level treatments. The bar graphs show mean Gosner stage, SVL, HW, and BM on the 14th day (n=4 tanks/treatment); error bars represent ± SE

2.3 水位變化對蝌蚪變態時間和大小的影響

中華大蟾蜍蝌蚪和黑眶蟾蜍蝌蚪在6種水位條件下的變態時間、變態個體的體長、頭寬和體重的描述性統計見圖3。種間One-way ANOVA顯示，不同水位變化處理下中華大蟾蜍蝌蚪的變態時間和體長顯著大于黑眶蟾蜍蝌蚪，以體長為協變量的One-way ANCOVA顯示，特定體長的中華大蟾蜍和黑眶蟾蜍剛變態個體頭寬差異不顯著，中華大蟾蜍剛變態個體的體重顯著大于黑眶蟾蜍(表3)。

表3 水位變化對中華大蟾蜍和黑眶蟾蜍蝌蚪變態時間和大小的影響Table 3 Effect of water levels on the time and size at metamorphosis of tadpoles in B. gargarizans and D. melanostictus

變態時間和體長為One-way ANOVA，體重與頭寬均為以體長為協變量的One-way ANCOVA; 不同上標的平均值差異顯著(Tukey′s test,α= 0.05, a > b > c); 水位處理類型用不同數字表示，1：恒定低水位；2：恒定高水位；3：水位逐漸升高；4：水位逐漸降低；5：水位快速波動；6：水位慢速波動

以水位變化為因子One-way ANOVA顯示，水位變化對中華大蟾蜍蝌蚪的變態時間和體長影響不顯著；水位變化對黑眶蟾蜍蝌蚪的變態時間和體長影響顯著，恒低水位的變態時間最長，恒高水位的變態時間最短，其他水位變化之間的變態時間差異不顯著；恒高水位下的體長最大，恒低和快波水位最小，其他居中(表3)。以體長為協變量的One-way ANCOVA顯示，不同水位變化對特定體長的中華大蟾蜍、黑眶蟾蜍蝌蚪變態時頭寬和中華大蟾蜍蝌蚪變態時體重影響不顯著，對黑眶蟾蜍蝌蚪變態時體重影響顯著，以逐增和快波水位的體重最大，恒低水位最小(表3)。

圖3 6種水位處理下，中華大蟾蜍和黑眶蟾蜍兩種蝌蚪的變態時間和形態特征的描述性統計值Fig.3 Descriptive statistics of time to metamorphosis and morphological traits of toadlets of B. gargarizans and D. melanostictus under six different water level treatments. The bar graphs show mean time to metamorphosis, SVL, HW, and BM at metamorphosis (n=4 tanks/treatment); error bars represent ± SE

3 討論

3.1 中華大蟾蜍和黑眶蟾蜍蝌蚪對水位變化的早期表型響應

發育早期(第14天)，中華大蟾蜍和黑眶蟾蜍蝌蚪的體長均表現出對六種水位處理的生長響應(即兩種蝌蚪的體長在逐減水位體長最大，表2)，即不同季節物種應對環境變化的共同反應[23]。

兩種蝌蚪體長的這種變化與同域分布但主要在臨時性水體繁殖的虎紋蛙(Hoplobatrachuschinensis)不同[26]，反映了無尾類持久性池塘繁殖者幼體的早期生長發育比臨時性水體繁殖者對水位的敏感性高。野外觀察虎紋蛙蝌蚪喜棲息水體底部，常處于靜止或作短距離游動，故早期棲息地的充足水體不會觸發其對干涸信號的生理響應機制。而中華大蟾蜍和黑眶蟾蜍這兩種蝌蚪喜好在持久性池塘中上層群聚游動，這也許有利于感知水位的下降，觸發它們快速攝食而促進生長。與恒高水位相比，飼養在恒低水位下的中華大蟾蜍蝌蚪和黑眶蟾蜍蝌蚪的體長分別縮短了6.0%和5.6%(圖2)。由于本研究設置的密度非常低(3只/箱)，消除了密度制約效應[28]，可能原因是恒低水位限制了這兩種蝌蚪的游動范圍，繼而減少攝食，導致生長過程的抑制[13]。

與冬季繁殖的中華大蟾蜍蝌蚪相比，春季繁殖的黑眶蟾蜍蝌蚪的發育歷期、體重和頭寬還表現出不同水位處理間的顯著差異(表2)，表現出了表型可塑性的種間差異和物種特異性，即表型可塑性的遺傳性[23]。黑眶蟾蜍蝌蚪在早期階段表現出發育可塑性來響應水位變化，即恒高水位下發育最快，恒低水位下發育最慢，其他水位居中(表2)，黑眶蟾蜍蝌蚪早期生長發育對恒低水位作出消極響應[13, 29]。

3.2 中華大蟾蜍和黑眶蟾蜍蝌蚪對水位變化的變態表型響應

水位變化對中華大蟾蜍蝌蚪的變態時間和大小均不產生顯著影響(圖2，表2)，這表明該物種響應水位變化的可塑性程度低。雖然中華大蟾蜍蝌蚪在水位變化早期階段能感知到水體的減少，但后期通過補償性生長保持平衡[30]。在發育上，中華大蟾蜍蝌蚪之所以不對水位變化發生適應性的發育響應，這也可能與該物種的繁殖時間密切相關。中華大蟾蜍通常在冬季選擇持久性池塘作為繁殖地[24]，相對于黑眶蟾蜍的春季繁殖，該季節相對寒冷干燥、雨水較少，水位經常發生變化，但一般不發生干涸，長期的適應可能是中華大蟾蜍不敏感的原因。

研究表明，黑眶蟾蜍蝌蚪的變態時間受水位影響顯著(圖2，表2)。首先，恒低水位下發育最慢，變態時間最長，而恒高水位下發育最快，變態時間最短，前者比后者平均延遲了2.7 d左右，這與已有關于Hylapseudopuma[31]、二光腫肋蟾與Rhinellagranulose[16]，以及Discoglossuspictus[26]的研究結果一致，但與另外一些關于哈蒙掘足蟾[13]、虎紋蛙[26]、Ranatemporaria[32]和Scaphiopuscouchii[33]的研究結果不同，這表明了黑眶蟾蜍蝌蚪對低水位很敏感。其次，黑眶蟾蜍蝌蚪在其他水位變化下的平均變態時間分別為慢波(約24.5 d)、逐減(約24.1 d)、逐增(約23.5 d)和快波(約22.9 d)，與恒高水位相比而言，快波、逐增、逐減和慢波依次延遲0.3 d、0.9 d、1.5 d和1.9 d發生變態，這表明水位慢波變化中較長的恒低水位可能限制了該物種蝌蚪的發育，通過減慢發育延遲變態響應水位的逐減，逐增水位下黑眶蟾蜍蝌蚪的變態時間更接近于恒高水位，而非恒低水位，證實了黑眶蟾蜍蝌蚪適應于在雨后高水位水體中繁殖。

無尾類幼體的加快發育通常與變態時個體變小有關[22]，而本研究發現，6種水位處理下對黑眶蟾蜍蝌蚪變態時體長影響顯著，其中快波和恒低水位體長顯著最短，恒高水位體長顯著最長，若不考慮快波水位的情況，黑眶蟾蜍蝌蚪在恒低水位下的變態時間最長，但體長最短，而恒高水位下變態時間最短，但個體最大，這表明恒低水位同時降低黑眶蟾蜍蝌蚪生長發育速率。而恒低水位大大地限制了其游動空間影響其新陳代謝，這一結果與Székely等人[20]的研究結果一致。

因此，冬季繁殖的中華大蟾蜍和春季繁殖的黑眶蟾蜍蝌蚪面對干涸風險的表型可塑性差異顯著，長期在容易發生干旱和水位變化的冬季繁殖的中華大蟾蜍的可塑性低，在雨水充沛的春季繁殖的黑眶蟾蜍的表型可塑性高，表現出表型可塑性的種間差異和遺傳性；在早期發育過程中，蝌蚪個體大小的共同的表型變異，這種變異與缺乏遺傳基礎的環境近因性影響有關；黑眶蟾蜍蝌蚪對低水位或水位下降作出減速分化的消極響應，響應程度與環境信號的強弱直接相關。

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Phenotypic responses to water level change inBufogargarizansandDuttaphrynusmelanosticustadpoles at Lishui, Zhejiang

TANG Xiaofen, FAN Xiaoli, LIN Zhihua*, YAO Tingting, LI Xiang, JIN Jing, ZHOU Cuntong

CollegeofEcology,LishuiUniversity,Lishui323000,China

Habitat drying caused by global warming will raise a challenge for anuran larvae living in water. We investigated the phenotypic response to six different patterns of water level change inBufogargarizansandDuttaphrynusmelanosticustadpoles under laboratory conditions. The aim of this study was to examine the heritable basis and environmental proximate causes of phenotypic plasticity of these two species tadpoles. The results showed that all the six water level treatments had no significant effect on the early development Gosner stage (GS), head width (HW), or body mass (BM) ofB.gargarizanstadpoles on the 14th day, but there was a significant effect on their snout-vent length (SVL). The SVL ofB.gargarizanstadpoles raised in decreasing water levels were the longest, while the ones raised in constant low water levels had the shortest SVL than the remaining groups. Conversely, the six water level treatments had respectively significant effects on the GS, SVL, HW and BM ofD.melanosticustadpoles on the same day, respectively. Firstly, those tadpoles raised in constant high water level developed most fast, while the ones raised in constant low water level developed most slowly. Secondly, the tadpoles raised in decreasing water level had greater SVL, while the ones raised in constant low, fast fluctuation and slow fluctuation water levels had smaller SVL than the remaining groups. Thirdly, HW of the tadpoles raised in constant low water levels was the narrowest, followed by the ones raised in increasing water levels, the others raised in the remaining water level groups had the biggest HW. Lastly, BM of tadpoles in constant low water levels was the smallest, while the ones raised in constant high water level had heavier BM than the remaining groups. The water level treatments had no significant effect on the time of metamorphosis and body size at metamorphosis including SVL, HW, and BM inB.gargarizan. However, there were significant effects of the water level changes on the time of metamorphosis and body size at metamorphosis including SVL and BM, except for HW, inD.melanosticus. Tadpoles raised in the constant low water levels had protracted metamorphosis, whereas the tadpoles raised under the constant high water levels had shortened metamorphosis. SVL at metamorphosis ofD.melanosticusin the constant high water levels was the largest, while the ones raised in constant low and rapidly fluctuating water levels were the shortest. BM at metamorphosis ofD.melanosticusraised in the increasing and rapidly fluctuating water levels were the biggest, while the ones raised in the constant low water levels were the smallest. Our results suggest that there are significant interspecific differences in the phenotypic plasticity respond to desiccation risks betweenB.gargarizansandD.melanosticustadpoles: the former was weaker than the latter. Winter-breederB.gargarizanstadpoles experienced habitat drying more frequently, while spring-breederD.melanosticustadpoles experienced habitat drying rarely. This showed the interspecific differences and hereditary of the phenotypic plasticity. During the early development of the two toad tadpoles, the common phenotypic variations in their SVL were associated with lack of genetic basis of environmental proximate causes. The response to constant low or decreasing water level inD.melanosticustadpoles was negative (deceleration of differentiation), and the response degree was directly related to the strength of the environmental signals.

Bufogargarizans;Duttaphrynusmelanosticus; tadpoles; water level change; phenotypic plasticity

國家自然科學基金項目(31270443, 30970435); 浙江省大學生科技創新活動計劃(2012R429022)

2014- 04- 28;

日期:2014- 07- 07

10.5846/stxb201404280845

*通訊作者Corresponding author.E-mail: zhlin1015@126.com

唐小芬, 樊曉麗, 林植華, 姚婷婷, 李香, 金晶, 周存通.浙江麗水中華大蟾蜍和黑眶蟾蜍蝌蚪對水位變化的表型響應.生態學報,2015,35(3):911- 918.

Tang X F, Fan X L, Lin Z H, Yao T T, Li X, Jin J, Zhou C T.Phenotypic responses to water level change inBufogargarizansandDuttaphrynusmelanosticustadpoles at Lishui, Zhejiang.Acta Ecologica Sinica,2015,35(3):911- 918.