Definiciones conceptuales

Prior to this study, N. pygmaea was known from only two sites in the Hay River in the

vicinity of the Mitchell River confluence, and one other site in the lowermost reaches of the Mitchell; a combined area of 0.06 km2 (Morgan et al. 2013). To gain a better

understanding of the extent of the distribution of this species in the Hay River system, 43 sites throughout the catchment were sampled between 2009 and 2014, with most effort focussing on non-salinised tributaries in the general vicinity of the previously known distribution (Figure 5.2). Fish sampling was initially undertaken at sites in the Mitchell River and adjacent parts of the Hay River mainstem during winter and spring of 2009, with further exploratory sampling in the Mitchell River during 2010.

As the Hay River only flows intermittently in winter/spring each year and contracts to isolated permanent pools during summer/autumn, one of the major aims of the study was to ascertain the distribution of N. pygmaea in this system during the critical

baseflow period. To achieve this, an aerial survey was conducted in March 2013 (see section 3.3.2 for a description of the methods used) to map all permanent aquatic refuge habitat in the segment of the Hay catchment that included all previously known sites of

N. pygmaea. The aerial survey covered the Hay River mainstem between Sunny Glen

gauging station (34.91° S, 117.48° E) and farmland located at the boundary of Mt Lindesay National Park (34.88° S, 117.52° E), as well as the lower reaches of the

Mitchell River (upstream to 34.85° S, 117.44° E). A number of key refuge pools that had been identified during the aerial survey were sampled in April 2013. Sampling effort was widened in winter-spring of 2013 to include several seasonally flowing tributaries of the Hay River (i.e. Mitchell River, Sheepwash Creek, Sleeman Creek), and in 2014 was further expanded into neighbouring catchments (i.e. Denmark, Kent, Bow) (Figure 5.2).

All sites where N. pygmaea occurred were mapped in ArcGIS™ Desktop 10.2

and minimum convex polygons (α-hulls) were drawn to calculate the extent of occurrence (EOO) (i.e. the area contained within the shortest continuous boundary which can be drawn to encompass all the known, inferred or projected sites of present occurrence of a taxon, excluding cases of vagrancy) following IUCN guidelines (IUCN 2014). Area of occupancy (i.e. the area within the EOO known to be occupied by a taxon, excluding cases of vagrancy) was estimated by overlaying the distribution map with a 1 km2 grid and counting the number of grid squares that contained N. pygmaea sites (IUCN 2014).

Fish were sampled using either seine nets (5 m or 10 m in length) constructed from 1 mm woven mesh and fishing to a depth of 1.8 m, or fyke nets constructed from 2 mm woven mesh and consisting of two 5 m wings, a 1.2 × 0.8 m opening, and a 5 m long pocket with two non-return funnels. Seine net sampling involved three replicate net drags at each site with the two-dimensional area of coverage of each net drag estimated in order to calculate the mean density of fishes (individuals.m-2). Fyke nets were deployed in pairs, with one net facing upstream and the other facing downstream, in order to detect the directionality of fish movements. Nets were set in the afternoon and checked the following morning. The total duration of each fyke net deployment was used to calculate catch per unit effort (CPUE) expressed as individuals.net-1.h-1. Note that at sites where the stretched wings of the fyke net did not block the entire width of the stream channel, numbers of individuals were standardised by multiplying the inverse of the proportion of stream blocked by the net by the number of individuals captured in the net as per Beatty

et al. (2014a).

All fish sampled during the study (irrespective of capture method) were identified to species level, counted, measured for total length (TL) to the nearest 1 mm, and

was ascertained by applying gentle pressure to the abdominal region of each fish and checking for exuded sperm or ova (Morgan & Beatty 2006). All fish were released alive at the site of capture, with the exception of 12 female N. pygmaea in breeding condition

that were euthanised in an ice slurry and stored in 100% ethanol for later dissection in order to elucidate life history characteristics (see section 5.2.3 below). The number of N.

pygmaea specimens that were retained for dissection in the current study was

intentionally low, so as to minimise destructive sampling of this rare species.

5.2.3 Biological characteristics

The gonads of the aforementioned female specimens were fixed in Bouin’s solution for 24 h and transferred into 100% ethanol for long term storage. Two gonads were prepared for histological assessment, firstly by dehydration in a series of alcohols before they were embedded in paraffin wax, sectioned at 6 µm and stained with Mallory’s trichrome. The sections were viewed under a compound microscope and used to describe the

composition of the ovaries of reproductively active females.

Five of the twelve remaining gonads were used to estimate fecundity by applying a modification of the methodology of Llewellyn (1979). These gonads came from

females that ranged in length between 41 and 51 mm TL. A dissecting microscope fitted with an eyepiece graticule was used to count oocytes that were assigned to arbitrary size categories. The number of large (i.e. >700 µm) oocytes were counted in full for each gonad, whereas numbers of smaller sized oocytes were estimated by excising a portion of the gonad, weighing it to the nearest 1 mg, counting oocytes within the sub-sample and applying the following formula to determine fecundity: Fecundity = N × (W1 / W2);

where, N = oocyte count; W1 = the wet weight of the whole gonad, and; W2 = the wet weight of the gonad sub-sample.

Sagittal otoliths were also removed from these 12 specimens, placed in a black dish containing glycerol, and examined under reflected light using a dissecting

microscope in order to count the number of hyaline (i.e. translucent) zones, which is often a reliable indicator of the age of a fish (see Bagenal 1973). The limited number of fish otoliths used precluded validation of annual formation of a single hyaline zone; however, it was assumed that the number of hyaline zones would be a reliable indicator of age for this species given that this aging technique has been successfully validated for all other native percichthyids in south-western Australia (e.g. Pen & Potter 1990, 1991a; Morgan

et al. 1995). The age estimates obtained from otoliths were used in conjunction with an

analysis of cohort progression in the monthly length-frequency histograms for the Hay- Mitchell sub-population in order to estimate growth rates and longevity.

Following dissection, the intestinal tract was also removed from each of the specimens used in the age and growth study. These were examined under a dissecting microscope in order to provide a qualitative evaluation of the dietary composition of this limited sample of 12 adult female N. pygmaea specimens captured in the Mitchell River

(Hay River system) during August/September 2013.

5.2.4 Environmental variables

Monthly precipitation data for Denmark and Denbarker (located centrally in the study area) were obtained from the Bureau of Meteorology website (BoM 2015).

(YSI Inc., Yellow Springs, OH, USA). Mean (±1 S.E.) estimates of three replicate measurements of water temperature (°C), pH, dissolved oxygen (%), salinity (‰ NaCl), total dissolved solids (‰), and electrical conductivity (µS.cm-1) were calculated at each sampling site on each sampling occasion.