August 2008 School of Environmental and Life Sciences University of Newcastle

Status of populations of threatened stream species in the upper catchment of the Styx River on the New England Tablelands, near sites where trout releases occur.

Year 2: establishing sites for the study of trout impacts on population demographics.

Simon Clulow, John Clulow & Michael Mahony

School of Environmental and Life Sciences, University of Newcastle

Prepared For

Recreational Freshwater Fishing Trust New South Wales Department of Primary Industries

August 2008

Status of Populations of Threatened Stream in the New England Tablelands. August 2008 School of Environmental and Life Sciences University of Newcastle

EXECUTIVE SUMMARY ...... 1 1.0 INTRODUCTION ...... 1 1.1 Scope ...... 1 1.2 Local Context ...... 3 1.3 Legislation and Licensing Requirements ...... 3 1.4 Background to the Problem and Proposed Study ...... 4 1.4.1 The Recent Decline of in Australia ...... 4 1.4.2 The Implication of Introduced Trout in the Decline of Stream Frogs ...... 5 1.4.3 Addressing the Fisheries Management Strategy ...... 6 1.5 Project Objectives...... 6 1.6 Target Species Profiles ...... 7 1.6.1 Glandular frog (Litoria subglandulosa) ...... 7 1.6.2 Stuttering frog (Mixophyes balbus)...... 8 2.0 METHODS ...... 9 3.0 RESULTS ...... 15 3.1 Frog distributions along transects and a comparison of frog population densities between trout and control transects...... 15 3.2 Effects of trout on various measures of body condition and environmental stress . 19 3.3 The relationship between stream and riparian variables and frog distributions along stream transects...... 21 4.0 DISCUSSION...... 26 5.0 CONCLUSIONS...... 27 6.0 RECOMMENDATIONS...... 27 7.0 REFERENCES ...... 28

Status of Populations of Threatened Stream Frogs in the New England Tablelands.

August 2008 School of Environmental and Life Sciences University of Newcastle

EXECUTIVE SUMMARY

The authors of this report were engaged to assess the status of populations of threatened stream frogs in and around the upper catchment of the Styx River on the New England Tablelands in areas where trout releases occur over the spring/summer periods of 2005/2006 and 2006/2007. The brief for this study required an assessment of the impact of introduced trout on these threatened frog populations in streams where trout have been released. The rationale for this study was the implication of trout in the decline of several Australian specialist stream breeding species in 1999 (Gillespie & Hero, 1999). The initial surveys of 2006 involved broad landscape scale surveys of the presence/absence of a number of threatened species that were known to be present in the New England Tablelands historically. In 2007, the studies were focussed on a smaller number of permanent transects that were established at 11 sites in the Styx River area to investigate more intensely potential impacts of trout on two endangered frogs: the Glandular Frog, Litoria subglandulosa and the Stuttering Frog, Mixophyes balbus. One year of demographic data from surveys in October and December 2007 was used to assess effects of trout on population densities and demographics, and the condition of adult frogs of these species as a measure of environmental stress. No effects of the presence of trout were demonstrated on any of the parameters used as potential measures of impact. Nevertheless, it is recognised that the power of the analysis was limited, since it was based on surveys during just one season. Consequently it is recommended that study sites continue to be surveyed during further years. The study also identified various characteristics of the streams and riparian zones that may help identify stream systems where these species are present, and stream systems that may offer favourable habitat to be managed in the long term for the conservation of these species.

1.0 INTRODUCTION

1.1 Scope The authors of this report were engaged by the New South Wales Department of Primary Industries (Recreational Freshwater Fishing Trust) to undertake an assessment of the status of populations of threatened stream frogs in and around the upper catchment of the Styx River on the New England Tablelands in areas where trout releases occur. The purpose of this assessment was to provide an insight into the impact of the release of introduced trout species on the distribution and abundance of threatened stream dependent frogs.

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The first year of the study of the involved a wide ranging survey of streams in the Styx River Catchment area that were either trout release streams or streams in which trout were not released. Those surveys showed that two endangered species, the Glandular Frog (Litoria subglandulosa) and the Stuttering Frog (Mixophyes balbus) were distributed along streams where trout release occurs, as well as along streams where trout were not known to be released. Analysis of the data did not show a significant difference in the proportions of streams with populations of these frogs between trout release and non-trout release streams. Nevertheless, it was concluded in the report on that initial surveys (Clulow et al, August, 2006) that the presence of populations of these frog species in streams co-habited by trout did not necessarily indicate that trout had no negative impact on those threatened frog species. Rather, the study only indicated that trout were not associated with the elimination (local extinction) of the frogs from those streams. This was because the study was only able to determine the presence or absence of frog populations (due to the objective of surveying a large number of stream sites), but did not determine whether frog population size and density was reduced in the presence of trout. The report recommended that some longer term study sites be established to determine whether trout are associated with a decline in the population status of these frog species by comparing the population demographics of frog populations over time in streams with and without trout. Support from the Freshwater Fishing Trust was provided to continue that work into a second year, with the focus on assessing the demographics and status of these species in a smaller number of more intensively studied streams. The design of the field project is described in more detail in the Methods section. The benefit of continuing this study is the capacity to determine whether trout have a negative effect on populations of frogs they share stream with. Knowing this will indicate whether there might be long term, incremental effects leading possibly to loss of threatened stream frogs in the long term, or whether alternatively trout and threatened stream frogs may persist indefinitely together in the same stream systems. The outcome should be improved management of both the endangered frog species on the New England Tablelands and the trout fisheries of the region.

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1.2 Local Context The study area is located in north-eastern NSW on the New England Tablelands and is situated between Coffs Harbour to the east, Armidale to the west, Grafton to the north and Kempsey to the south (see Figure 1). The region is associated with large areas of National Park, State Forest and freehold rural lands. The landscape is characterised by numerous creeks, streams and rivers, numbers of which are utilised for stocking with introduced trout. The vegetation and landscape throughout the study area varies greatly with rural townships, cleared land (generally utilised for farming), and natural forest areas.

Figure 1 Regional context showing the location of the study area in the Styx River catchment system on the New England Tablelands.

1.3 Legislation and Licensing Requirements This project was undertaken in accordance with the following Policies and Approvals:

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• Commonwealth Environment Protection and Biodiversity Conservation Act 1999 (EPBC Act); • NSW Threatened Species Conservation Act 1995 (TSC Act); • NSW Threatened Species Conservation Amendment Act 2002; • National Parks and Wildlife Act 1974 (NP&W Act); • Fisheries Management Strategy – Freshwater Fish Stocking in NSW Environmental Impact Statement (NSW Fisheries, 2003) • NSW NPWS Scientific License S10382 • University of Newcastle Ethics Approval 6771008

Schedules 1 and 2 of the TSC Act contain lists of flora and fauna species, populations and communities, which have been determined by the NSW Scientific Committee as being under threat of serious decline that could ultimately lead to extinction. Schedule 3 of the TSC Act contains a list of ‘key threatening processes’ deemed to be processes that have a negative impact on threatened species, populations or communities.

NSW National Parks and Wildlife Service and the University of Newcastle Animal Ethics Committee have approved these animal surveys.

1.4 Background to the Problem and Proposed Study

1.4.1 The Recent Decline of Amphibians in Australia In the early 1980’s the decline of an Australian amphibian was observed for the first time with the disappearance of the Gastric Brooding (Rheobatrachus silus) frog in south- eastern Queensland. Since then, there has been a recorded decline in the distribution and abundance of numerous native frog species throughout Australia (see papers in Campbell, 1999). Stream dependent frogs (species that rely on streams for breeding and larval support) make up the majority of Australian amphibians undergoing decline (Morrison & Hero, 2004). The cause of this decline is a matter of considerable debate and current research among scientists today. Several hypotheses have been put forward to explain this

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August 2008 School of Environmental and Life Sciences University of Newcastle decline including disease, habitat loss and degradation, climate change, pollution, increased levels of UV-B radiation, and predation by introduced species (Alford & Richards, 1999). Today it is estimated that a possible 32% of amphibian species are at risk of extinction globally (Stuart et al, 2004). These declines have resulted in numerous species being listed as threatened in Australia under both state and federal legislation. Accordingly, activities that may impact on populations of these frogs or their habitats are required to be subject to impact assessment.

Five stream dependent frogs that are listed as threatened occur in the New England region. Two that have population records near to the sites of fish releases on the Styx River on the New England Tablelands are the Stuttering frog (Mixophyes balbus) and Glandular frog (Litoria subglandulosa). Populations of three other threatened frog species are also likely to occur in the area, the Peppered frog (Litoria piperata), the Booroolong frog (Litoria booroolongensis) and the Tusked frog (Adelotus brevis). Clulow et al (2006) in the first year of this study confirmed the presence of significant populations of the first two of these endangered species throughout the study area.

1.4.2 The Implication of Introduced Trout in the Decline of Stream Frogs Considerable research has gone into investigating the affect that both introduced fish, and native fish translocated into areas where they previously did not occur, have had on amphibian larvae. Studies of the effects of presence of species such as the introduced Mosquito Fish (Gambusia spp.) have suggested that they may play a role in the decline of several amphibian species through predation on tadpoles (for example Morgan & Buttemer, 1996; Pyke & White, 1996; Webb & Joss, 1997). Introduced trout have been linked to the decline of macroinvertebrates in Utah (Luecke, 1990) and several overseas studies have implicated various salmonids in the decline of endemic amphibian populations (for example Braña et al, 1996). Gillespie and Hero (1999) suggested that various trout species, including the Brown Trout (Salmo trutta) and Rainbow Trout (Oncorhynchus mykiss), were playing a significant role in the decline of several species of stream dwelling frogs in Australia. In particular, the species considered to be at threat from trout were those that breed exclusively in streams throughout south-eastern

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Australia (Gillespie & Hero, 1999). Such species include the Spotted Tree frog (Litoria speceri), Leaf-green Tree frog (Litoria phyllochroa) and the Booroolong frog (Litoria booroolongensis) (Gillespie & Hero, 1999; Gillespie 2001).

1.4.3 Addressing the Fisheries Management Strategy The first goal of the Fisheries Management Strategy (FMS) is to “manage the activity in a manner that minimises impacts on aquatic biodiversity including threatened species and genetic resources” and it sets out to achieve this by a set of objectives, the second of which states, “to minimise or eliminate any negative impact from the activity on threatened species, populations or ecological communities, and where possible promote their recovery.” (NSW Fisheries, 2003).

This translates to the need to appropriately manage trout stocking areas where the activity may adversely affect threatened species. The FMS notes that any stocking event that has the potential to affect threatened species will be thoroughly reviewed with a view to preventing or minimising any potential impacts (objective 1.2a). Accordingly it is a high priority to assess the potential for fish stocking of the Styx River on the New England Tableland to impact on threatened frog fauna.

1.5 Project Objectives The goals and objectives of the current year of this study (Year 2), follow on from the previous year which located and mapped extensively the local distribution of the two endangered frogs, L. subglandulosa and M. balbus. The objective of Year 2 of this project was to establish appropriate population monitoring transects for the Glandular and Stuttering Frogs along streams in the Styx River catchment that may be used to obtain baseline data for comparing frog population demographics between streams that are sites of trout release and streams that are trout free. The intention was to obtain initial population data in 2007, but to establish these as permanent transect sites so that they can be used as long term monitoring sites to acquire data into the future with high levels of statistical and scientific confidence.

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1.6 Target Species Profiles

1.6.1 Glandular frog (Litoria subglandulosa) (Figure 2) This species is similar to other members of the Litoria citropa complex and for many years was considered to be a form of L. citropa. It is now clear that it is a distinct species and has been further divided into two sibling species (L. subglandulosa, L. daviesae) in recent years (Mahony et al, 2001). The dorsum ranges from green to olive brown above with the sides almost always remaining green (Robinson, 1998). The head stripe is similar to L. citropa, containing a broad dark stripe running from the nostrils, through the eyes and above the indistinct tympanum, with a thin gold stripe running above it. The distinct white stripe on the upper jaw however is much reduced or absent in L. citropa (Barker et al, 1995). The backs of the thighs are reddish-brown and the toes are almost fully webbed while the fingers remain free (Barker et al, 1995). There are vomerine teeth behind the choanae, and maxillary teeth (Robinson, 1998).

The Glandular frog has a relatively small distribution, occurring along the ranges from the Southern New England Tablelands to the northern most forests of NSW, extending just into south-eastern QLD (Figure 7) (Barker et al, 1995; Anstis, 2002). A large portion of its range, and indeed its type locality, falls in the New England Tablelands and it is sometimes referred to as the New England Tree frog (Tyler & Anstis, 1975). To the south of this distribution extending from the Barrington Range to the southern border of the New England Range is a closely related sibling species Litoria daviesae (Mahony et al, 2001). Both species are associated with small permanent creeks, streams and rivers in rainforest, montane areas or wet sclerophyll forest above 300m (Anstis, 2002) although they may also be found along streams running through disturbed grazing lands (Barker et al, 1995). Males call during summer and early spring in a variety of weather conditions from low vegetation surrounding the stream. Calling intensity greatly increases during and after rain events (Anstis & Littlejohn, 1996) and breeding generally occurs in shaded pools where the flow rate is slower (Anstis, 2002). Larvae in L. subglandulosa and L.

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August 2008 School of Environmental and Life Sciences University of Newcastle daviesae are unique in that they lack tooth rows and jaw sheaths. Instead they contain a large suctorial oral disk with numerous finely pointed papillae that increase in size towards the mouth opening (Anstis, 2002). Tadpoles may be found frequenting the bottom of shallow, slowly flowing sections of the stream where they are well camouflaged and can be observed using a unique ‘oral locomotion’ not observed in other species when feeding (Anstis, 2002).

Figure 2. The Glandular frog (Litoria subglandulosa)

1.6.2 Stuttering frog (Mixophyes balbus) (Figure 3) The Stuttering Frog Mixophyes balbus, is among the largest amphibians in Australia and has appeared to reduce greatly in number in recent times (Daly, 1998). The species is yellow-grey above and finely granular with a dark irregular mid-dorsal band extending from between the eyes to the rear of the back. The limbs have narrow dark cross-bars that

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August 2008 School of Environmental and Life Sciences University of Newcastle may be incomplete or indistinct (not to be confused with Lechriodus fletcheri) (Cogger 2000). Individuals grow to a length of approximately 80mm. The historical distribution of the Stuttering Frog was from east of the Great Divide and extended from south-eastern Queensland through NSW to Victoria (Figure 8) (Barker et al, 1998). The preferred habitat of the Stuttering Frog is along streams in rainforest, Antarctic beech and wet sclerophyll forests (Cogger 2000). The species depends on freshwater streams and riparian vegetation for breeding and habitation. It makes use of riffle zones along creeks and streams where it makes hollowed out nests in the gravel in which it deposits its eggs at very precise times when the water flow is correct (M Mahony unpublished data). The tadpoles are then washed into deeper pools where they grow and metamorphose. The best time to detect naturally calling males therefore is after heavy rains when the creeks and streams have filled and are beginning to recede, forming ideal riffle zones. The males will however respond readily to call playback most of the time through spring and summer, the season in which it is known to breed.

Figure 3. The Stuttering frog (Mixophyes balbus)

2.0 METHODS

Permanent 100m transects were established along 11 streams in 2007 to allow monitoring and assessment of the status of the two threatened stream frogs, the Stuttering Frog

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(Mixophyes balbus) and the Glandular Frog (Litoria subglandulosa) in the presence and absence of trout. These streams provide 5 trout inhabited sites and 4 trout free control sites for the Glandular Frog and 4 trout inhabited sites and 5 trout free control sites for the Stuttering Frog (Mixophyes balbus). Some sites selected contain both target species and as such are used to assess both species at those sites.

Stream and riparian characteristics were mapped at all sites and included features such as flow rate, stream width and depth, substrate under water, exposed substrate, pools and riffle zones, riparian vegetation cover, ground cover and amount of vegetation overhanging the banks. The location of each site established is shown below:

Litoria subglandulosa

Control Trout Inhabited

Sunday Ck Lower (420500E, 6608000N) Little Styx River [Thungutty] (441200E, 6625550N) Little Sunday Ck (421900E, 6608750N) Styx River [Hyatt’s Flat] (435700E, 6623400N) Sunday Ck Upper (421000E, 6610050N) Eely Ck [Loop Rd] (425350E, 6615500N) George’s Ck (439500E, 6623750N) Eely Ck [Jack’s Rd] (427650E, 6616950) Backwater Ck (435100E, 6618600N)

Mixophyes balbus

Control Trout Inhabited

George’s Ck (439500E, 6623750N) Eely Ck [Loop Rd] (425350E, 6615500N) Sunday Ck Lower (420500E, 6608000N) Eely Ck [Jack’s Rd] (427650E, 6616950N) Little Sunday Ck (421900E, 6608750N) Spankers Gully (422650E, 6613650N) Sunday Ck Upper (421000E, 6610050N) Backwater Ck (435100E, 6618600N) Boggy Ck (417450E, 6609600N)

The study sites and transect locations are also shown in Figs 4-6.

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Two surveys were conducted along each transect in 2007, one in October and one in December. Both diurnal and nocturnal surveys were undertaken and included surveying for tadpoles in the day and surveying for adult at night. Standardised survey techniques were employed throughout the survey periods and included call playback, dip netting for tadpoles, and searching along transects using a head torch to locate animals by eyeshine. The chainage of frogs caught was recorded (location along transect, distance from water, and height off the ground) and the frogs were weighed, sexed and measured before being released at the position where they were found. Any tadpoles caught were identified according to the key by Anstis (2004) and tadpole data was recorded by way of presence/absence as well as an approximate estimation of number of tadpoles observed.

The morphometrics and condition of frogs along transects were established by measuring parameters of body condition including body weight, head width, snout-vent length, body condition index (body weight/snout-vent length) and fluctuating asymmetry (based on a comparison of left and right leg tibia length). Fluctuating asymmetry is a measure of environmental stress in which animals subjected to increased levels of environmental stress are predicted to show greater degrees of asymmetry between left and right sides of the body (induced by stress during early development) than animals in benign environments.

Statistical Analysis of the data

Analysis of the first year of transect monitoring data collected during the 2007 surveys has been completed using parametric and non-parametric approaches. Multi-variate regression analysis was used to determine correlations between stream and riparian variables and the distribution of frogs along transects.

Analysis of variance and t-tests were used compare the frog population densities on transects between trout and non-trout sites. Analysis of variance was also used to analyse body condition variables and fluctuating asymmetry between trout and control (non-trout) transects.

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Figure 4 Location of the study sites in relation to the location of the Dutton Trout Hatchery. See Insets 1 and 2 in Figs 5 and 6 respectively.

Status of Populations of Threatened Stream Frogs in the New England Tablelands. 12 August 2008 School of Environmental and Life Sciences University of Newcastle

Figure 5 Location of the trout and control study sites in Inset 1 (Figure 4)

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August 2008 School of Environmental and Life Sciences University of Newcastle

Figure 6 Location of the trout and control study sites in Inset 2 (Figure 4)

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3.0 RESULTS

3.1 Frog distributions along transects and a comparison of frog population densities between trout and control transects.

Calling adult males of both species were located along trout and control transects in both survey periods (October and December 2007). However, fewer L. subglandulosa were located in the December surveys (Table 1). since it is primarily a species that calls in spring and early summer. Both species were located along transects in trout and control sites, with the patterns of distribution varying between transects, but showing no evidence of being related to the occurrence of trout (Fig. 7). There was overlap in the occurrence species along transects where conditions were appropriate, and some evidence of clustering of animals together along transects (analysis of clustering effects is ongoing). However, any clustering did not appear on initial inspection of the data (eg Fig. 7) to be related to their occurrence on streams with trout.

Mean numbers of animals for both species recorded along transects were higher in the October than in the December survey periods (Table 1). There was no relationship however, in either survey period, or in analyses of the data pooled between both survey periods, between mean population counts (based on calling males captured) and the occurrence of trout (Tables 1 and 2). This was despite mean counts being higher in a number of transects on control streams. When error values were taken into account, no significant statistical differences were found. Mean values were generally in the order of 1-4 frogs per transect per survey period with the exception of low count rates for L. subglandulosa in the December surveys. Obviously, with the magnitude of the errors (standard deviations), there were much higher counts on a number of individual transects.

Status of Populations of Threatened Stream Frogs in the New England Tablelands. August 2008 School of Environmental and Life Sciences University of Newcastle

Control Sites Trout Sites

George's Ck Backwater Creek

M. balbus M L. subglandulosa M M. balbus M M. balbus F L. subglandulosa M

10 10

0 0

-10 -10 0 102030405060708090100 0 102030405060708090100 Chainage Chainage Upper Sunday Ck Eely Ck (Loop Rd)

M. balbus M L. subglandulosa M M. balbus M L. subglandulosa F

10 10

0 0

-10 -10 0 102030405060708090100 0 102030405060708090100 Chainage Chainage

Figure 7 Distributions of L. subglandulosa and M. balbus along four transects in the Styx River study area. Each panel shows a separate transect using pooled data from the October and December 2007 surveys. The x-axis represents the distance along the transect from 0-100m, and the y-axis represents the distance frogs were located from the stream edge. Most frogs were located by calls close to the water’s edge in both trout and control sites. Both frog species may occur together.

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Group N Mean Std. Deviation Std. Error Mean

M. balb cont 5 3.60 2.074 .927 Oct 2007 trout 6 2.67 3.615 1.476

M. balb cont 5 1.80 1.095 .490

Dec 2007 trout 6 1.17 1.472 .601

M. balb cont 5 5.40 1.817 .812

Oct + Dec 2007 trout 6 3.83 4.997 2.040

L. subgland cont 5 3.80 4.970 2.223

Oct 2007 trout 6 2.67 2.875 1.174

L. subgland cont 5 .00 .000 .000

Dec 2007 trout 6 .83 1.169 .477

L. subgland_ cont 5 3.80 4.970 2.223

Oct + Dec 2007 trout 6 3.50 3.834 1.565 Table 1 Mean number (and standard deviation, standard error of the mean) of adult M. balbus and L. subglandulosa recorded per transect during surveys in October and December 2007. N = number of transects.

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Levene's Test for Equality of Variances t-test for Equality of Means

95% Confidence Interval of the Sig. (2- Mean Std. Error Difference

F Sig. t df tailed) Difference Difference Lower Upper

M. balb Equal variances 1.955 .196 .509 9 .623 .933 1.834 -3.215 5.081 Oct 2007 assumed

Equal variances .535 8.142 .607 .933 1.743 -3.074 4.940 not assumed

M. balb Equal variances .161 .698 .794 9 .448 .633 .798 -1.172 2.439 Dec 2007 assumed

Equal variances .817 8.926 .435 .633 .775 -1.123 2.389 not assumed

M. balb Equal variances 3.063 .114 .661 9 .525 1.567 2.371 -3.798 6.931 Oct + Dec assumed

2007 Equal variances .714 6.507 .500 1.567 2.196 -3.706 6.839 not assumed

L. subgland Equal variances 1.358 .274 .474 9 .647 1.133 2.389 -4.272 6.538 Oct 2007 assumed

Equal variances .451 6.159 .668 1.133 2.514 -4.979 7.245 not assumed

L. subgland Equal variances 6.392 .032 -1.579 9 .149 -.833 .528 -2.027 .360 Dec 2007 assumed

Equal variances -1.746 5.000 .141 -.833 .477 -2.060 .394 not assumed

L. Equal variances .298 .599 .113 9 .912 .300 2.649 -5.693 6.293 subgland_ assumed

Oct + Dec Equal variances .110 7.480 .915 .300 2.718 -6.045 6.645 2007 not assumed Table 2 Statistical tests of the significance of differences in the mean numbers of adult M. balbus and L. subglandulosa recorded per transect during surveys in October and December 2007. There are no significant differences in the mean numbers of frogs for either species in any survey period between trout release and control streams (determined from t-tests either assuming or not assuming equal variances).

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3.2 Effects of trout on various measures of body condition and environmental stress

A total of 26 L. subglandulosa and 43 M. balbus adult males were captured and measured during the surveys in October and December, 2007.

There was no significant difference in any of the morphometric characters measured (body weight, head width, snout-vent length, body condition index) for either species between trout and control streams (Tables 3 and 4).

Right and left tibia lengths were measured individually (each measurement replicated 3 times to achieve a high level of accuracy in measurements) for adult frogs of both species (Tables 5 and 6). The difference between the left and right tibia length in individual animals was used as a measure of asymmetry within each animal and the mean asymmetries for both species in trout and control animals determined (Tables 5 and 6). Statistical analysis showed that there was a significant asymmetry between tibias within individual animals. However, there was no significant difference in the mean level of asymmetry between animals in trout and control streams for either species (Tables 5 and 6). This indicated that there was no evidence of increased environmental stress in frogs from streams containing trout that was reflected in developmental differences relating to asymmetry.

Mean ± 1 standard deviation Trout Control Combined p Body weight (g) 4.90 ± 0.68 5.17 ± 0.54 4.99 ± 0.64 ns Head width (mm) 16.14 ± 5.86 14.49 ± 0.78 15.57 ± 4.77 ns Snout-vent Length (mm) 37.64 ± 6.52 38.59 ± 1.98 37.97 ± 5.36 ns Body Condition Index # 0.137 ± 0.048 0.134 ± 0.012 0.135 ± 0.039 ns N 17 9 26 Table 3 Comparative morphometrics of adult male L. subglandulosa from trout release and control streams during October and December 2007 surveys. # Body Condition Index (BCI) = Body weight (g)/Snout-vent length (mm). p = level of significance of mean difference between trout release and control stream frogs; ns = no significant difference

Mean ± 1 standard deviation Trout Control Combined p Body Weight (g) 26.02 ± 3.79 25.47 ± 4.44 25.70 ± 4.15 ns Head Width (mm) 35.96 ± 15.83 37.90 ± 16.34 37.09 ± 15.97 ns

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Snout-Vent Length (mm) 51.55 ± 16.33 48.52 ± 17.54 49.78 ± 16.91 ns Body Condition Index # 0.575 ± 0.249 0.619 ± 0.298 0.601 ± 0.276 ns N 18 25 43 Table 4 Comparative morphometrics of adult male M. balbus from trout release and control streams during October and December 2007 surveys. # Body Condition Index (BCI) = Body weight (g)/Snout-vent length (mm). p = level of significance of mean difference between trout release and control stream frogs; ns = no significant difference

Mean ± 1 standard deviation Trout Control Combined p Left tibia (mm) 20.77 ± 0.82 21.31 ± 1.27 20.96 ± 1.01 ns Right tibia (mm) 20.74 ± 0.83 21.30 ± 1.21 20.93 ± 0.99 ns Difference (asymmetry) between 0.14 ± 0.11 0.09 ± 0.06 0.13 ± 0.95 ns left and right tibias (mm) Significance of asymmetry 0.001* N 17 9 26 Table 5 Mean tibia length of adult male L. subglandulosa from trout release and control streams during October and December 2007 surveys, and levels of asymmetry between left and right tibias (as determined by difference in length between left and right tibias). * indicates significant asymmetry between left and right tibias; there was no significant difference (ns) between the level of asymmetry in trout and control stream frogs.

Mean ± 1 standard deviation Trout Control Combined p Left tibia (mm) 38.99 ± 1.73 38.23 ± 2.03 38.56 ± 1.92 ns Right tibia (mm) 39.19 ± 1.77 38.33 ± 1.95 38.69 ± 1.90 ns Difference (asymmetry) between 0.26 ± 0.24 0.28 ± 0.39 0.27 ± 0.33 ns left and right tibias (mm) Significance of asymmetry 0.001* N 18 25 43 Table 6 Mean tibia length of adult male M. balbus from trout release and control streams during October and December 2007 surveys, and levels of asymmetry between left and right tibias (as determined by difference in length between left and right tibias). * indicates significant asymmetry between left and right tibias; there was no significant difference (ns) between the level of asymmetry in trout and control stream frogs.

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3.3 The relationship between stream and riparian variables and frog distributions along stream transects.

A multivariate analysis of stream and riparian physical and vegetation characteristics was undertaken to identify variables or groups of variables that would explain the distribution of frogs along transects, independent of the occurrence of trout. The results of those analyses are summarised in Tables 6 and 7, and some key variables identified are plotted in Figures 8-11.

In terms of stream related variables, the occurrence of L. subglandulosa was less predictable in relation to particular variables than was M. balbus. L. subglandulosa showed some correlation with the proportion of stream bottom consisting of sand (Table 7), but it is not clear why such a relationship existed. The occurrence of M. balbus, on the other hand, in quadrats along transects was strongly correlated with particular stream variables including the total length of riffle zones, the presence of mud on the bottom, and the shallowness (positive relationship) and area of completely exposed creek bed (Table 7, Fig.s 9-11). These statistically significant associations with stream variables may be explained by the biology of M. balbus which breeds (lays eggs) in riffle zones (areas of shallow pebble substrate in streams with gentle flow), around which males sit in the open at night on areas of exposed creek bed or bank to call before spawning with females directly into the riffle zones.

In terms of riparian vegetation characteristics (Table 8), L. subglandulosa showed some correlation with the occurrence of grasses along stream banks (Fig 8, although the reason for this is not clear), but no significant correlation with the amount of overhanging vegetation along banks. This latter non-correlation is interesting, since L. subglandulosa calls from overhanging vegetation, and this non-correlation may indicate that the distribution of L. subglandulosa along the streams in the study area is not limited by an availability of overhanging vegetation ie there is more than enough for to meet the requirements of calling males, and other environmental factors may be determining its abundance. The only riparian variable that was correlated with the occurrence of M.

Status of Populations of Threatened Stream Frogs in the New England Tablelands. 21

August 2008 School of Environmental and Life Sciences University of Newcastle balbus was the abundance of and Gahnia (sword grass) cover, and this relationship was negatively correlated (Table 8). M. balbus appears to be more associated with streams with large areas of riffle zones and significant amounts of exposed, silted stream edges that provide open sites for occupation.

M. balbus L. subglandulosa r p r p Turbulence 0.171 0.073 -0.029 0.764 Shade % 0.011 0.908 -0.040 0.681 Depth (m) -0.231 0.015* -0.025 0.796 Width under water (m) -0.051 0.593 0.048 0.620 % stream area under water -0.206 0.031* 0.071 0.463 Width exposed (m) 0.084 0.383 0.003 0.974 % stream area exposed 0.206 0.031* -0.071 0.463

Substrate Under Water mud % 0.026 0.787 0.053 0.580 sand % 0.088 0.362 -0.209 0.028* gravel % 0.020 0.834 -0.090 0.350 stone & rock % 0.033 0.729 0.231 0.015* bare rock % -0.054 0.574 -0.073 0.450 log & wood % -0.053 0.580 -0.120 0.211

Substrate exposed mud% 0.219 0.022* -0.046 0.630 sand% -0.006 0.946 -0.079 0.410 gravel% 0.160 0.095 0.118 0.219 stone & rock % 0.239 0.012* 0.135 0.159 bare rock% -0.116 0.229 0.074 0.443 log & wood % 0.077 0.425 -0.042 0.659

Riffles - length (m) 0.497 0.0001**** -0.068 0.482

Pools Length (m) -0.028 0.773 -0.077 0.426 Aver. width (m) -0.080 0.406 -0.085 0.380 Aver. Depth (m) -0.012 0.899 -0.044 0.645 Aver. Area (m2) -0.069 0.474 -0.091 0.346 Aver. Volume (m3) -0.052 0.590 -0.073 0.449 Stream logs (m) -0.168 0.080 -0.090 0.350

Table 7 Spearman’s non-parametric correlation co-efficients for correlation of M. balbus and L. subglandulosa numbers with various stream riparian characteristics. Data from multi-variate regression analysis of 110 quadrats in 11 transects using total frog counts recorded in quadrats in October and December, 2007. r = Spearman’s correlation coefficient; p = statistical significance of r; * p ≤ 0.05.

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M. balbus L. subglandulosa Left Bank Right Bank Left Bank Right Bank r P r p r p r p % canopy cover 0.007 0.941 0.092 0.341 -0.062 0.523 -0.038 0.691 Length bank overhang (m) -0.052 0.591 -0.117 0.225 0.165 0.086 0.113 0.238 Bank Lomandra cover % -0.054 0.575 -0.087 0.368 0.098 0.306 0.112 0.245 Bank Gahnia cover % -0.157 0.102 -0.107 0.267 -0.113 0.240 -0.045 0.644 Bank Lom & Gah cover % -0.188 0.050* -0.182 0.057* -0.050 0.604 0.032 0.739 Bank shrub cover % -0.177 0.065 -0.088 0.360 0.138 0.150 -0.080 0.408 Bank fern cover % -0.025 0.793 0.085 0.377 -0.192 0.044* -0.089 0.356 Bank short grass cover % 0.087 0.366 0.108 0.259 0.259 0.006* 0.102 0.289 Bank long grass cover % 0.051 0.599 -0.114 0.235 0.237 0.013* 0.016 0.870 Bank short + long gras cov % 0.067 0.485 0.038 0.697 0.272 0.004 0.073 0.446 Bank litter & dirt cover % -0.043 0.654 -0.018 0.849 -0.178 0.062 -0.042 0.666 Bank rock & stone cover % -0.103 0.285 -0.075 0.438 0.153 0.112 0.118 0.222 Bank log ground cover % -0.004 0.964 -0.090 0.350 -0.027 0.779 0.040 0.682 Bank logs lngth of bank (m) -0.053 0.584 -0.124 0.199 -0.045 0.642 0.056 0.560

Table 8 Spearman’s non-parametric correlation co-efficients for correlation of M. balbus and L. subglandulosa numbers with various stream bank characteristics. Data from multi-variate regression analysis of 110 quadrats in 11 transects using total frog counts recorded in quadrats in October and December, 2007. r = Spearman’s correlation coefficient; p = statistical significance of r; * p ≤ 0.05.

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Figure 8 L. subglandulosa counts along transects (y-axis) against proportion of bank covered by grass (x-axis).

Figure 9 M. balbus counts along transects (y-axis) against average riffle length (x-axis) in transects

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Figure 10 M. balbus counts along transects (y-axis) against the average depth of pools along transects (x-axis)

Figure 11 M. balbus counts along transects (y-axis) against the proportion of stream bottom substrate consisting of mud (x-axis) along transects.

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4.0 DISCUSSION

The work in the field during 2007 successfully established 11 permanent, marked transects on trout release and trout free streams in the Styx River area of the New England Tablelands. These transects are now established and are amenable to repeated regular surveys into the future to investigate interactions between stream frogs and introduced trout. They also have the potential in the future to be sites of experimental field manipulations where the level of trout release might be varied under controlled conditions to determine effects of trout densities on stream fauna complexes.

The results presented in this report cover one year of survey work aimed at establishing permanent survey transects and initiating studies on the population demographics of L. subglandulosa and M. balbus along transects of streams that contain trout or have no recent history of trout release. Although the data lacks a certain level of power because there has only been one year of survey effort, the data have not produced any results that indicate a significant effect of the presence of trout in streams on the distribution and abundance (population densities along transects) of either frog species along streams in the Styx River study area.

Analysis of the data also failed to show any association of the presence of trout in streams on parameters of body condition (such as body weight, length, head size, body condition index or level of asymmetry in leg length).

Nevertheless, despite the absence of trout effects in the study to date, there is a strong argument for continuing the study beyond the 2007 year to increase the power of the analysis and the confidence in future conclusions based on these data.

In broad terms, the authors of this study continue to be of the view that the main cause of the decline of New England Tablelands stream frog species has been due to the impacts of the introduced chytrid fungus that is now widely recognised as the primary cause of most amphibian species declines after a scientific debate that has continued over more than two decades (Pyke & White, 1996; Mahony, 1997, 1999; Alford & Richards, 1999; Hamer et al, 2002; Morrison et al, 2004; Stuart et al, 2004). Nevertheless, even though chytridiomycosis is likely to have been the primary and initial cause of the decline of endangered species in the New England Tablelands, other environmental factors (such as trout predation) may subsequently contribute as secondary causes of species decline. Under that scenario, it is important to study the broader ecology of the endangered frogs to understand factors that might further drive declines, and hence, understanding the impacts of trout is an important component of managing these stream frogs.

Besides investigating the effects of trout on the population demographics of the subject frog species, the study has contributed some fundamental ecological knowledge relating to the ecology of these species that may be of assistance in their management in trout or trout free systems. The results suggest that the occurrence of L. subglandulosa is not related to clearly defined stream or riparian characteristics, but that the species may occur locally along any stream with appropriate flow rates and a range of riparian

Status of Populations of Threatened Stream Frogs in the New England Tablelands. 26

August 2008 School of Environmental and Life Sciences University of Newcastle characteristics. On the other hand, the occurrence of M. balbus would appear to be strongly biased towards streams with distinct stream characteristics (shallow, extensive exposed substrate, bare banks derived from muddy sediments, and extensive ripple zones). These characteristics may be predictors of the presence of M balbus, and streams with these characteristics may be targeted to manage and conserve the species.

5.0 CONCLUSIONS

(1) The occurrence of L. subglandulosa and M. balbus along New England Tablelands streams in the Styx River area is related to stream and vegetation characteristics, but the relationship between particular stream variables and occurrence of the frogs is stronger for M. balbus than L. subglandulosa. (2) Permanent transects suitable for long term monitoring of these species in the Styx River area to determine the effects of trout on population demographics and fitness have been established successfully. (3) Notwithstanding the limited power of the analysis based on one season’s data, there is no evidence that the presence of trout in streams is suppressing population densities or having measurable effects on population age structure or individual fitness levels in either species. (4) Given the absence of evidence for a direct effect of trout on L. subglandulosa and M. balbus, more parsimonious explanations of the causes of their decline (such as the effects of chytridiomycosis) seem reasonable. (5) Continuing the study over further seasons is warranted to reduce error values in the statistical analyses, and increase confidence in the conclusions of the study.

6.0 RECOMMENDATIONS

(1) It is recommended that the current permanently established transects be maintained and the populations of L. subglandulosa and M. balbus be monitored for at least two more seasons (bringing the total to three) to increase the level of confidence in the outcomes of the study to date.

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7.0 REFERENCES

Alford, R. A. & Richards, S. J. (1999). Global amphibian declines: a problem in applied ecology. Annual Review of Ecology and Systematics. 30; 133-165.

Anstis, M. (2002). Tadpoles of South-eastern Australia: A Guide With Keys. New Holland: Australia.

Anstis, M. & Littlejohn, M. J. (1996). The breeding biology of Litoria subglandulosa and L. citropa (Anura: Hylidae), and a re-evaluation of their geographic distribution. Transactions of the Royal Society of South Australia 120(3): 83-99.

Anstis, M., Alford, R. A. & Gillespie, G. R. (1998). Breeding biology of Litoria booroolongensis (Moore, 1961) and Litoria lesueuri (Dumeril and Bibron, 1841) (Anura: Hylidae) and comments on population declines of L. booroolongensis. Transactions of the Royal Society of South Australia 122(1): 33-43.

Australian Museum. New England Frogs Database.

Barker, J., Grigg, C. G. and Tyler, M. J. (1995). A Field Guide to Australian Frogs. Surrey Beatty & Sons: Norton.

Braña, F., Frechilla, L. and Orizaola, G. (1996). Effect of introduced fish on amphibian assemblages in mountain lakes of northern Spain. Herpetological Journal, 6: 145-148.

Campbell, A. (1999). Declines and Disappearances of Australian Frogs. Environment Australia: Canberra.

Cogger, H. (2000) Reptiles and Amphibians of Australia - 6th edn. Reed New Holland.

Daly, G. (1998). Review of the status and assessment of the habitat of the Stuttering frog, Mixophyes balbus, (Anura: Myobatrachidae) on the south coast of New South Wales. Herpetofauna. 28(1), 2- 11.

Daly, G. (1995). Observations on the Tusked Frog Adelotus brevis (Anura: Myobatrachidae). Herpetofauna 25(2): 32-35.

Gillespie, G. R. (2001). The role of introduced trout in the decline of the spotted tree frog (Litoria spenceri) in south-eastern Australia. Biological Conservation. 100(2), pp. 187-198.

Gillespie, G. R. & Hero, J. M. (1999). Potential impacts of introduced fish and fish translocations on Australian amphibians. In Declines and Disappearances of Australian Frogs, ed. A. Campbell. Environment Australia, Canberra, pp. 131-144.

Gillespie, G. R. & Hines, H. B. (1999). Status of temperate riverine frogs in south- eastern Australia. In Declines and Disappearances of Australian Frogs, ed. A. Campbell. Environment Australia, Canberra, pp. 109-130.

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Hamer, A.J., Lane, S.J., Mahony, M.J. (2002). Management of freshwater wetlands for the endangered green and golden bell frog (Litoria aurea): roles of habitat determinants and space. Biological Conservation 106(3): 413-424.

Luecke, C. (1990). Changes in abundance and distribution of benthic macroinvertebrates after introduction of cutthroat trout into a previously fishless lake. Transactions of the American Fisheries Society. 119(6), pp. 1010-1021.

Mahony, M.J. (1999). Review of the declines and disappearances within the bell frog species group (Litoria aurea species group) in Australia. In Declines and Disappearances of Australian Frogs, ed. A. Campbell. Environment Australia, Canberra, pp. 81-93.

Mahony, M. (1997). Survey of the distribution and abundance of declining frog species in upper north east New South Wales. Prepared for the Threatened Frogs of Queensland and north east New South Wales Recovery Team.

Clulow, S, Price, L, Clulow J & Mahony, MJ (2006) Status of Populations of Threatened Stream Frog Species in the Upper Catchment of the Styx River on the New England Tablelands Near Sites Where Trout Releases Occur. Report prepared for the Recreational Freshwater Fishing Trust, NSW Dept of Primary Industries.

Mahony, M., Knowles, R., Foster, F. & Donnellan, S. (2001). Systematics of the Litoria citropa (Anura: Hylidae) complex in northern New South Wales and southern Queensland, Australia, with the description of a new species. Records of the Australian Museum 53(1): 37-48.

Morgan, L. A. and Buttemer, W. A. (1996). Predation by the non-native fish Gambusia holbrooki on small Litoria aurea and L. dentata tadpoles. In G. H. Pyke and W. S. Osborne (eds), The Green and Golden Bell Frog (Litoria aurea): Biology and Conservation. Royal Zoological Society of NSW, pp. 143-149.

Morrison, C., Hero, J. M. & Browning, J. (2004). Altitudinal variation at the age of maturity, longevity, and reproductive lifespan of anurans in subtropical Queensland. Herpetologica. 60(1), p.34-44.

NSW Fisheries (2003). Freshwater Fish Stocking In NSW Environmental Impact Statement: Public Consultation Document. Volume 1. Published in November 2003 by NSW Fisheries, Cronulla, NSW.

NSW Fisheries (2003). Freshwater Fish Stocking In NSW Environmental Impact Statement: Public Consultation Document. Volume 2. Published in November 2003 by NSW Fisheries, Cronulla, NSW.

NSW Fisheries (2003). Freshwater Fish Stocking In NSW Environmental Impact Statement: Public Consultation Document. Volume 3. Published in November 2003 by NSW Fisheries, Cronulla, NSW.

Pyke, G. H. and White, A. W. (1996). Habitat requirements of the green and golden bell frog Litoria aurea (Anura: Hylidae). In G. H. Pyke and W. S. Osborne (eds), The Green and Golden Bell Frog (Litoria aurea): Biology and Conservation. Royal Zoological Society of NSW, pp. 224-232.

Reader’s Digest (2005). Encyclopedia of Australian Wildlife. Reader’s Digest: Australia.

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Robinson, M. (1998). A Field Guide to Frogs of Australia. Reed New Holland: Sydney.

Stuart, S. N., Chanson, J. S., Cox, N. A., Young, B. E., Rodrigues, A. S. L., Fischman, D. L. and Walter, R. L. (2004). Status and trends of amphibian declines and extinctions worldwide. Science. 306(5702), pp. 1783-1786.

Tyler, M. J. & Anstis, M. (1975). and biology of frogs of the Litoria citropa Complex (Anura: Hylidae). Records of the South Australia Museum 17: 41-50.

Webb, C. and Joss, J. (1997). Does predation by the fish Gambusia holbrooki (Atheriniformes: Poeciliidaae) contribute to declining frog populations? Australian Zoologist, 30 (3): 316-324.

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