Fish sampling

For P1 and P4, the long-term database on fish distribution and environmental data was compiled during the ichthyological surveys in the Ping-Wang river-system between January 1996 and April 2009. The sampling sites were distributed among 10 sub-basins in the river-system, where a Digital Elevation Model (DEM) was used to define and divide the geographical range of the Ping-Wang river-system into sub- basins by ArcView GIS 9.2, according to the catchment area and fish sample spots. Collections of fish samples were taken at every habitat types in every selected site. Samplings were done by various methods i.e. beach seine net, cast net, multi-mesh

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gillnets as well as the electro-fishing with an AC shocker powered (Honda EM 650, DC 220 V 550BA 450VA, 1.5–2 A, 50 Hz), which was placed on the riverbank together with block nets and scoop nets. Sampling sites were chosen on the basis of accessibility, similarity in habitat types, and to maximize the diversity of habitat types (pools, cascade, falls, riffles, and stagnant water) at each sub-basin. The environmental parameters (Table 3) were measured by standard methods (APHA, 1991). All specimens were preserved in 10% formalin and then taxonomical classified, counted and measured at Maejo Aquatic Resources Natural Museum (MARNM), Chiangmai, Thailand.

Table 3 Environmental parameters and methods of measurement in this study.

No. Environmental Parameters Methods/Equipments Water qualities/Physicochemical parameters

1 Water temperature (WT; °C) YSI 556 (multi-probe system) 2 Conductivity (CON; mg/l) YSI 556 (multi-probe system) 3 Total dissolved solids (TDS; mg/l) YSI 556 (multi-probe system) 4 Dissolved oxygen (DO; mg/l) YSI 556 (multi-probe system) 5 Nitrite (NIT; mg/l) APHA (1989) protocols 6 Ammonia (AMM; mg/l) APHA (1989) protocols 7 Phosphorus (PHO; mg/l) APHA (1989) protocols 8 pH YSI 556 (multi-probe system) 9 Alkalinity (ALK; mg/l) APHA (1989) protocols 10 Hardness (HAR; mg/l) APHA (1989) protocols

11 Current velocity (CUR; m/s) Flow meter (G.O. Environmental model 1295) 12 Depth (DEP; m) Meter Tape

13 Width (WID; m) Meter Tape

14 Discharge (DIC; m3/s) Q=AV; Area of channel X Average velocity of flow

15 Altitude (ALT; m ASL) GPS GarmineTrex VISTA Geo-morphometric parameters

16 Distance from the sea (DIS; km) ArcView GIS 9.2 17 Watershed area (WSH; km2) ArcView GIS 9.2 18 Forest area (FOR; %) ArcView GIS 9.2 19 Agricultural area (AGR; %) ArcView GIS 9.2 20 Urban area (URB; %) ArcView GIS 9.2

For P2 and P5, data collection was conducted in the Mae-ngad reservoir. Fishes were sampled monthly from October 2002 to September 2003 from 10 sites in the

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tributaries and 2 stations in the reservoir (Fig. 5B). Two stations in the reservoir were a littoral zone where most of fish occupied (Prchalová et al., 2003, Brosse et al., 2007). Meanwhile, the central area of the lake is a steep shore and very deep. Therefore, very few samples are expected. For P5, data was obtained by the 12 fishermans using gill nets and the targeted species was Henicorhynchus siamensis (Fig. 6). The gill net assemblies were composed of five 30 m2 nets (10 m long X 3 m deep) with stretched mesh sizes of 10-30 mm. The nets were surface-set at twelve sites, which were equally distributed over the coastal area of the reservoir, using one gill net assembly per sampling site. All the nets were set overnight between 16h00 and 18h00 and lifted between 06h00 and 08h00. At least 120 H. siamensis were randomly sampled monthly from July 2003 to June 2004 (1,364 fish in total). Individuals were measured for total length (L, to the nearest 1 mm) and weighed (W, to the nearest 0.1 g). For P5 Data collection was focused in the tributaries connected to the reservoir. Fish samplings were conducted by using electro-fishing, i.e. a gasoline-powered electroshocker (DC, 250 V, 1.5–2 A, 50 Hz), each sampling was done with two replications for 30 to 45 minutes interval and the area cover was about 100 m2. In addition, gill net (20 x 1.2 m2, mesh size 4 cm stretched mesh) was also concurrently conducted in reservoir during the night time. The water quality parameters (Table 3) were also recorded at each sampling station by the similar protocols as in P4.

Lastly, P3, the study was conducted with Maechaem stream. Oreoglanis

siamensis (Fig. 7) were sampled monthly from October 2007 to September 2008 from

15 sites in the East part tributaries of Maechaem stream. Fish samplings were conducted by electrofishing (Honda EM 650, DC 220 V 550BA 450VA, 1.5–2 A, 50 Hz) in the upper Maechaem river system. Each tributaries sampling site was done at 45 to 60 minutes intervals or the area covered was about 100 m2, I was collected with various microhabitat, substrate type i.e. rocky, sandy, and gravel, and habitat type (riffle, pool, and run) to cover all species distributions. The skin diving was carried out to observe the abundance and behavior of the fish. Fish captured in each part were kept separate after selected O. siamensis and fixed in 10% formalin and the life specimens was released to the their habitat after measurement and weight. Then, O.

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Figure 6. The specimen of Henicorhynchus siamensis used to study in P2. (TL=215 mm)

Figure 7. The specimen of Oreoglanis siamensis study in P3. A: Top view, B: lateral view, and C: Sucking mouth O. siamensis. (TL=108 mm).

solution of anesthetic, and conditioned in ice for transportation. The process in evening at the rest room and the following data were obtained: (i) total length (TL) to the nearest 0.1 mm (ii) total weight (WT) to the nearest 0.01 g (iii) sex (iv) gonad

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weight (GW) to the nearest 0.01 g. Gonads were removed from the visceral cavity, Prior to the preservation of the ovaries/testis were classified in a macroscopic scale of gonadal development, for both sexes; for female size and colour of oocytes was also registered and, for males sperm liberation when pressing the abdomen. According to these characteristics, the following classification was considered: females – 2nd stage, immature, mature, and ripe; and males – 2nd stage, immature, mature, and ripe. Thereafter, ovaries were fixed in Bouin solution for oocytes measurements and total ripe eggs counts. The specimens were fixed in 10% formalin and preserved in 70% ethanol. Specimens were deposited at the Maejo Aquatic Resources Natural Museum.