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Greater Paraburdoo Subterranean Fauna Survey

7.3 Types of impacts to stygofauna

Direct impacts to stygofauna assemblages and habitats comprise the removal of porous/ fractured hydrogeological strata from BWT mining, and depletion of groundwater by abstraction for mine dewatering and operational uses. The propagation of groundwater drawdown throughout all suitable, connected hydrogeological habitats is considered to be a direct impact, regardless of distance from the mine or whether the drawdown propagates through strata with different porosities.

Nevertheless, the direct impact from groundwater drawdown is more complex to measure and assess than removal of porous hydrogeological habitat, as the propagation of drawdown must be modelled in three spatial dimensions, and a temporal dimension, taking into consideration natural seasonal fluctuations. Drawdown of a given hydrogeological habitat within the range of variability/ seasonal fluctuations of the natural water table would not be considered to have any direct impact. Hydrogeological complexity created by variable permeability, geological disconformity, the presence of intrusive barriers, or compartmentalisation of hydrogeological units can further complicate the assessment, as can the occurrence of multiple layered aquifers with different habitat characteristics, and interactions with surface drainage features.

As the hydrogeological setting at Greater Paraburdoo shows many of these complexities, detailed 3D modelling has been undertaken of pre-mining groundwater habitats, ‘current’ (CUR) groundwater habitats (i.e. including current drawdown from existing approved mining), and ‘worst- case scenario’ (WCS) habitats following the maximum mining and drawdown associated with the proposed development. The risk assessment for stygofauna was based upon the best available taxonomic and ecological information regarding the stygofauna species occurring only within the predicted drawdown, as well as the best available understanding of hydrogeological habitats and habitat connectivity, and 3D modelling of potential impacts and habitat remaining under the WCS model (Appendix 6).

7.4 Risks to stygofauna species

Many of the stygofauna taxa recorded throughout the Study Area and surrounding hydrogeological units are considered locally or regionally widespread, and almost all of the remaining taxa were found both inside and outside of the estimated drawdown at Paraburdoo. As such, most stygofauna taxa are not considered to be at risk from impacts associated with the proposed development. Nevertheless, one stygal taxon was recorded only from within the proposed groundwater drawdown area in Seven-Mile Creek; the syncarid Bathynella ‘WAM-BATH001’.

A summary of taxonomic, distribution and habitat factors affecting the risk assessment for this species is presented in Table 7.1, while Figures 7.3a and 7.3b map the known locations of this taxon against the CUR and WCS extent of suitable groundwater habitats beneath Seven-Mile Creek. Appendix 6 (BWT) shows 3D habitat modelling (long sections and cross sections) of CUR and WCS extent of suitable groundwater habitats beneath Seven-Mile Creek.

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Bathynella ‘WAM-BATH001’ belongs to an obligate stygobitic group (Bathynellidae) that is currently undergoing a taxonomic study to describe previously unrecognised species and genera found in Western Australia (G. Perina pers. comm. 2018). Current patterns of diversity across the region suggest the occurrence of many SRE taxa, with turnover at the species-level and generic level between different catchments and discrete hydrogeological habitat units (Perina et al. 2018).

Bathynellids are known to have limited dispersal abilities due to their habit of crawling over the sub-strata as opposed to free swimming within water-filled subterranean cavities (Coineau & Camacho 2013). This would suggest that the distributions of bathynellids may be more limited, and species turnover potentially higher per unit of distance, than the more vagile, free-swimming stygofauna such as cyclopoid copepods, ostracods, or amphipods (i.e. groups that contain more well-known, widespread species in the Pilbara).

Despite the specimens from Paraburdoo aligning morphologically to the genus Bathynella (based on a key published by Schminke 1973 that described one female specimen only), more recent work suggests this genus is probably not represented in Australia (Perina et al. 2019). More recent studies revealed the genus Pilbaranella from the upper Fortescue River (Perina et al. 2018), while specimens collected north of the De Grey River (Perina et al. 2019) and at West Angelas (G. Perina pers. comm. 2018) were considered to represent new, undescribed genera. No Paraburdoo specimens were available at the time of these studies but owing to the considerable distances it is suspected that Bathynella ‘WAM-BATH001’ could represent another unique genus (G. Perina pers. comm. 2018). Based on this assessment, Bathynella ‘WAM-BATH001’ is a Potential SRE which may be restricted to the Greater Paraburdoo area.

Bathynella ‘WAM-BATH001’ was detected from 72 individuals collected at bores MB15NLC001 (3 specimens April 2018, 10 specimens June 2018), MB15NLC005 (57 specimens June 2018) and WB17NLC001 (2 specimens June 2018). Sampling in the nearby bores and holes in Seven-Mile Creek (5 sites to the north and 5 sites to the south of the central area where Bathynella ‘WAM- BATH001’ was collected) did not detect further records of the species despite the detection of other stygofauna (Table 7.2). However, only 14 bores were sampled in this area, and the majority of bores were only sampled on one occasion during the April or June/ July surveys in 2018 (excluding MB15NLC001, which was sampled during both surveys). The likelihood of detecting additional specimens of Bathynella ‘WAM-BATH001’ would be increased by additional sampling, particularly in hydrogeological habitats likely to be temporarily connected to the current locations of the species following flooding in Seven-Mile Creek.

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Table 7.2: Groundwater characteristics of bores sampled in Seven-Mile Creek. Red font indicates bores where Bathynella ‘WAM-BATH001’ was detected.

DTW Temp EC Salinity Acidity Redox DO Slot Bore Stygo Comments (m) (°C) (μS/cm) (ppm) (pH) (mV) (ppm) depth NORTH MB15PAFL002 Y 12 30.4 1220 0.54 8.25 27.4 2.87 13-25m Superficial MB16PAFL0003 N 7 20.5 3015 1.46 7.38 -14.6 1.03 15-21m Superficial MB16PAFL0002 Y 7 31.1 3223 1.48 7.48 -90.7 0.75 18-24m Superficial MB16PAFL0001 Y 9 30.7 3122 1.45 7.82 -58.9 1.21 20-26m Superficial 60m MB17NLC0005 N 4 27.1 2359 1.16 7.59 -32.8 1.36 Superficial EOH Average (North) 7.80 27.9 2587.8 1.2 7.7 -33.9 1.4 CENTRAL PMO1(PVC01) Y 6 26.6 2453 1.22 7.81 21.5 1.91 18-46m Orebody MB15NLC001 Y 8 31.15 2103 0.945 8.97 -62.25 0.88 26-50m Orebody MB15NLC005 Y 16 30.3 2146 0.98 8.85 -97.9 0.93 24-30m Orebody 65m Orebody, WB17NLC001 Y 13 29.2 1302 0.59 9.82 -128.9 1.24 EOH rusty Average (Central) 10.7 29.3 2001.0 0.9 8.8 -66.8 1.2 SOUTH 4ED05 N n/a 30.2 1702 0.77 7.96 45.7 3.7 46-113m DW pump PMO8 Y 3.5 29.5 215.2 0.09 8.86 -124.9 0.57 6m EOH Hyporheic 37m HM17NLC0001 N 36 30.4 1025 0.45 11.32 19.7 1.05 Orebody EOH PMO7 N 35 30.9 1453 0.64 7.8 44.6 3 21-63m Orebody Average (South) 30.2 1098.8 0.4 8.9 -3.7 2.0 SOUTHERN

BOREFIELD PMP02 Y 19 30.45 1619 0.73 7.48 117.1 4.71 10-40m Dolomite

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7427000 Morphospecies, Risk Low : 1m GF Bathynella `WAM-BATH001`, High risk Rio Tinto Iron Ore Other fauna detected Drawdown (mBWT) !( Stygofauna 2 - 30 Greater Paraburdoo Subterranean Fauna Survey )" Troglofauna 31 - 70 Fig. 7.3a: CUR (modelled) extent of BWT habitat 71 - 120 for stygofauna known only from Paraburdoo 1:25,000 121 - 170 Coordinate System: GDA 1994 MGA Zone 50 0 0.325 0.65 1.3 Projection: Transverse Mercator 171 - 228 ¯ km Datum: GDA 1994 Size A3. Created 01/05/2019 556000 557000 558000 559000 560000 561000 562000 563000 564000 565000

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7427000 Morphospecies, Risk Low : 1m GF Bathynella `WAM-BATH001`, High risk Rio Tinto Iron Ore Other fauna detected Drawdown (mBWT) !( Stygofauna 2 - 30 Greater Paraburdoo Subterranean Fauna Survey )" Troglofauna 31 - 70 Fig. 7.3b: WCS (modelled) extent of BWT habitat 71 - 120 for stygofauna known only from Paraburdoo 1:25,000 121 - 170 Coordinate System: GDA 1994 MGA Zone 50 0 0.325 0.65 1.3 Projection: Transverse Mercator 171 - 228 ¯ km Datum: GDA 1994 Size A3. Created 01/05/2019

Greater Paraburdoo Subterranean Fauna Survey

Hydrogeological investigations in Seven-Mile Creek revealed compartmentalisation within the Orebody aquifers beneath the Detrital aquifer. The aquifer compartments appear to be separated by impermeable geologies (e.g. Mt McRae Shales and dolerite intrusives) that have been shifted through the profile by a series of faults (RTIO 2018) (Figure 7.4). These impermeable layers create hydraulic barriers restricting the downstream flow of groundwater and creating a ‘bath-tub’ like aquifer compartment in the central area (RTIO 2018), where Bathynella ‘WAM-BATH001’ was detected (bores MB15NLC001, MB15NLC005, and WB17NLC001 – approximately at the same location as MB15NLC002 shown in Figure 7.4).

The groundwater levels measured during sampling broadly concurred with the water table differences shown in the hydrogeological cross section. Table 7.2 shows a high water table in the north of Seven-Mile Creek (DTW average 7.8 m at the time of sampling, also indicated by photos in Figure 7.4 showing dense riparian vegetation), moderate depths to water in the central compartment (DTW average 10.7 m), and greater depths in the south (DTW in bores HM17NLC0001 and PMO7 >35m). This suggests that the groundwater in the southern section readily drains away through permeable strata to the south, while in the central compartment and in the north throughflows are blocked by impermeable barriers. Habitat modelling in Appendix 6 (BWT) and Figure 7.3a shows that the central and southern hydrogeological units are currently affected, but not totally depleted, by drawdown from existing pits at 4W/ 4E, while the WCS drawdown for the 4EE pit (Figure 7.3b) indicates complete dewatering in the central and southern aquifers beneath Seven-Mile Creek. Based on current modelling, the risk of impacts from groundwater drawdown on the known extent of habitat for Bathynella ‘WAM-BATH001’ are high.

There is a reasonable likelihood that Bathynella ‘WAM-BATH001’ may occur elsewhere beyond the central aquifer compartment beneath Seven-Mile Creek, despite the absence of other records from current sampling. The current survey only sampled bores in Seven-Mile Creek aquifers once or at most twice during 2018 and one of these events was during the dry season. Hydrogeological studies suggest that flood events may periodically raise groundwater levels above the impermeable barriers beneath Seven-Mile Creek, potentially facilitating wider groundwater connectivity upstream and downstream of the aquifer compartments. Based on current sampling, it is not possible to rule out the likelihood that Bathynella ‘WAM-BATH001’ may occur within the superficial aquifers north of the Paraburdoo Range and/ or the Duck Creek Dolomite aquifers of the Southern Borefield, both of which are predicted to retain sufficient High and Medium (certain) BWT habitat under the WCS habitat modelling (Appendix 6 BWT). Nevertheless, the potential risks to Bathynella ‘WAM-BATH001’ from the proposed development are considered high based on its current occurrence only within habitats predicted to be completely dewatered by groundwater drawdown in the WCS modelling.

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Figure 7.4: Hydrogeological cross section of Seven-Mile Creek showing hydraulic barriers (compartmentalisation by impervious Mt McRae shales and dolerite dykes). Figure from RTIO (2018)

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8. CONCLUDING REMARKS

The key findings of the Greater Paraburdoo Subterranean Fauna Survey are based on:

• results of all sampling to date within the Study Area and surrounds, • available habitat information and 3D modelling, and • impact information regarding proposed mining and dewatering.

These findings may be subject to change following the receipt of additional or revised information regarding fauna taxonomy, subterranean habitats, and/ or proposed impact areas.

Eight (8) troglofauna taxa are currently known only from the direct impact areas of the proposed development and were therefore considered to be potentially at risk. The potential risks to these taxa were characterised as follows, using a three-point system (i.e. high, moderate, or low risk):

• Low risk (3 taxa): Eukoenenia `WAM-PALE002`, Decapauropus `WAM-PAUD004`, and Symphyella `WAM-SYMPH002`.

These taxa were regarded as low risk because their known records were located near to the boundaries of proposed pits. Three-dimensional habitat modelling indicated that suitable, continuous habitat will remain intact under the ‘worst-case scenario’ below their known locations, and extensively beyond the impact area.

• Moderate risk (5 taxa): Paraplatyarthrus `WAM-PARA001`, Decapauropus `WAM- PAUD003`, Scutigellera `WAM-SCUTI004`, Symphyella sp. indet., and Trinemura `WAM- ZYGS001`.

These taxa were regarded as moderate risk because they were known only from the central areas of their respective pits, and under the ‘worst-case scenario’ habitat modelling, no suitable habitat was expected to remain below their recorded locations. Nevertheless, the same habitat modelling showed extensive areas of suitable habitat beyond the impact areas in the nearby vicinity. Despite being recorded as singletons, most of these species can be reasonably expected to occur elsewhere within the modelled extent of suitable, connected habitat beyond the impact areas. This inference is supported by the known distribution of Trinemura `WAM-ZYGS001`, which has already been recorded from several sites in multiple proposed pits connected by the modelled extent of continuous habitat in Western Range.

One stygofauna taxon is currently known only from sites within the proposed groundwater drawdown at Paraburdoo, within the Orebody aquifer/ Detrital aquifer beneath Seven-Mile Creek. Using a three-point classification, the risk to this taxon from the proposed mining/ drawdown was characterised as:

• High risk (1 taxon): Bathynella ‘WAM-BATH001’

This taxon is regarded as a potential SRE stygobite due to the known patterns of occurrence throughout the family, and its limited dispersal capabilities. The only known records of this taxon

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Greater Paraburdoo Subterranean Fauna Survey are in a highly compartmentalised aquifer beneath Seven-Mile Creek. Hydrogeological connectivity with other nearby aquifers is limited by dolerite intrusives and impermeable layers Mt McRae Shales in this area. This aquifer is also affected by approved groundwater drawdown from the dewatering of the nearby 4W and 4E pits. In addition, the proposed ‘worst-case scenario’ drawdown modelling for the 4EE pit appears to completely dewater all aquifers beneath Seven- Mile Creek, including the known location of Bathynella ‘WAM-BATH001’.

It is possible that Bathynella ‘WAM-BATH001’ could occur elsewhere beyond the central aquifer compartment beneath Seven-Mile Creek. Prior to the approved groundwater drawdown, groundwater levels throughout Seven-Mile Creek were historically higher, and flood events would have facilitated wider connectivity between other hydrogeological habitats upstream and downstream. Major floods of Seven-Mile Creek may still periodically raise groundwater levels above the impermeable barriers constraining the central compartmentalised aquifer.

Suitable groundwater habitats appear to persist despite the proposed drawdown under the ‘worst- case scenario’ modelling, including detrital aquifers north of the Paraburdoo Ranges, and Duck Creek Dolomite aquifers at the Southern Borefield. While the current survey detected Bathynella ‘WAM-BATH001’ only within the central aquifer compartment beneath Seven-Mile Creek, additional sampling could detect the species further afield, particularly in adjacent aquifers that may become temporarily connected to the central aquifer compartment during flooding.

Nevertheless, based on current taxonomic and ecological information, the extent of ‘worst-case scenario’ drawdown modelling, and the current records of the species only within the central aquifer compartment beneath Seven-Mile Creek, the potential risk to Bathynella ‘WAM-BATH001’ is considered high.

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Perina, G., A. I. Camacho, J. Huey, P. Horwitz, and A. Koenders. 2019. New Bathynellidae (Crustacea) taxa and their relationships in the Fortescue catchment aquifers of the Pilbara region, Western Australia. Systematics and Biodiversity DOI:10.1080/14772000.2018.1559892.

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Rathbone, S. 2005. A groundwater model of the 4West and 4East pits, incorporating the Seven Mile, Pirraburdoo Creeks and Ratty Springs. Paraburdoo - Western Australia. RTIO-PDE- 0146403.

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———. 2015. Paraburdoo Operations. Groundwater Operating Strategy. Unpublished report for Rio Tinto Iron Ore. April 2015.

———. 2016a. Greater Paraburdoo. 2016 Hydrogeological Conceptual Report. Southern Borefield Assessment. December 2016.

———. 2016b. Greater Paraburdoo. 2016 Hydrogeological Conceptual Report. Ratty Springs Hydrogeological Conceptualisation. October 2016.

———. 2017. H2 Basic Hydrogeological Assessment. Channar Wellfield and 64E5 Supply Borefield. June 2017.

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Schminke, H. K. 1973. Evolution, System und Verbreitungsgeschichte der Familie Parabathynellidae (Bathynellacea, Malacostraca). Akademie der Wissenschaften und der Literatur Mainz, Mathematisch-Naturwissenschaftliche Klasse, Mikrofauna des Merresbodens 24:1–192.

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Watts, C. H. S., and W. F. Humphreys. 2004. Thirteen new Dytiscidae (Coleoptera) of the genera Boongurrus Larson, Tjirtudessus Watts & Humphreys and Nirripirti Watts and Humphreys, from underground waters in Australia. Transactions of the Royal Society of South Australia 128:99– 129.

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10. APPENDICES

Appendix 1: Regulation 17 – Licence to take fauna for scientific purposes

Appendix 2: Conceptual methodology for subterranean fauna habitat modelling

Appendix 3: DNA report

Appendix 4: Western Range 3D Habitat Modelling (AWT) – Long and cross sections

Appendix 5: Eastern Range 3D Habitat Modelling (AWT) – Long and cross sections

Appendix 6: Paraburdoo 3D Habitat Modelling (AWT/ BWT) – Long and cross sections

Appendix 7a: Bores and drill holes visited and sampled during the survey

Appendix 7b: Database search data (WAM & PSS)

Appendix 7c: Specimen data – Current survey

Appendix 7d: Water physicochemical measurements observed during the current survey

Page | 128

Greater Paraburdoo Subterranean Fauna Survey

Appendices

Appendix 1: Regulation 17 – Licences ...... 2 Appendix 2: Conceptual methodology for subterranean fauna habitat modelling ...... 7 Appendix 3: DNA Report (WA Museum) ...... 11 Appendix 4: Western Range 3D Habitat Modelling (AWT) – Long and cross sections ...... 57 Appendix 5: Eastern Range 3D Habitat Modelling (AWT) – Long and cross sections ...... 74 Appendix 6: Paraburdoo 3D Habitat Modelling (AWT/ BWT) – Long and cross sections ...... 87 Appendix 7a: Bores and drill holes visited and sampled during the survey ...... 106 Appendix 7b: Database search data (WAM & PSS) ...... 106 Appendix 7c: Specimen data – Current survey ...... 106 Appendix 7d: Water physicochemical measurements observed during the current survey ...... 106

Appendix Page | 1

Greater Paraburdoo Subterranean Fauna Survey

APPENDIX 1: Regulation 17 – Licences

Appendix Page | 2

DEPARTMENT OF PARKS AND WILDLIFE

Enquiries: 17 DICK PERRY AVE, KENSINGTON, WESTERN AUSTRALIA Telephone: 08 9219 9000 Facsimile: 08 9219 8242 Web Site: https://wildlifelicensing.dpaw.wa.gov.au Correspondance: Locked Bag 30 PAGE 1 Bentley Delivery Centre WA 6983 NO. 08-000722-1

Wildlife Conservation Act 1950 REGULATION 17 Regulation 17 – Licence to take fauna for scientific purposes (Regulation 17 - Standard) The undermentioned person may take fauna for research or other scientific purposes and where authorised, keep it in captivity, subject to the following and attached conditions, which may be added to, suspended or otherwise varied as considered fit. Director General

Conditions

1 The licensee shall comply with the provisions of the Wildlife Conservation Act 1950, Wildlife Conservation Regulations 1970 and any Notices in force under this legislation. 2 The licensee shall take fauna only in the manner stated on the endorsed Regulation 17 licence application form and endorsed related correspondence. 3 Unless specifically authorised in the conditions of this Licence or otherwise in writing by the Director General, species of fauna declared as likely to become extinct, rare or otherwise in need of special protection shall not be taken. 4 Any by-catch of fauna, which is declared to be rare, likely to become extinct, or otherwise in need of special protection shall be released immediately at the point of capture. Where such fauna taken under this licence is injured or deceased, the licensee shall contact the Department’s Wildlife Licensing Section for advice on disposal. Records must be kept of any such fauna so captured and details are to be included in the report required under further condition below. 5 Any interaction involving Gazetted Threatened Fauna that may be harmful to the fauna and/or invasive may require approval from the Commonwealth Department of the Environment ph 02 6274 1111. Interaction with such species is controlled by the Commonwealth Environment Protection and Biodiversity Conservation Act 1999 and Environment Protection and Biodiversity Conservation Regulations 2000 as well as the Wildlife Conservation Act 1950 and Wildlife Conservation Regulations 1970. 6 No fauna shall be taken in areas where it would impinge on pre-existing scientific research programs. 7 Except in the case of approved lethal traps, the licensee shall ensure that measures are taken in the capture and handling of fauna to prevent injury or mortality resulting from that capture or handling. Where traps or other mechanical means or devices are used to capture fauna these shall be deployed so as to prevent exposure of trapped animals to ants and debilitating weather conditions and inspected at regular intervals throughout each day of their use. At the conclusion of research all markers used, and signs and structures erected by the licensee shall be removed and the environment returned to its original condition. 8 Not more than ten specimens of any one protected species of fauna shall be taken and removed from any location less than 20km apart. Where exceptional circumstances make it necessary to take a larger number of specimens from a particular location in order to obtain adequate statistical data, the collector must proceed with circumspection and justify their actions to the Director General in advance. 9 The licensee shall not release any fauna or their progeny in any area where it does not naturally occur, nor hand such fauna over to any other person or authority unless approved by the Director General, nor dispose of the remains of such fauna in any manner likely to confuse the natural or present day distribution of the species. 10 Bioprospecting involving the removal of sample aquatic and terrestrial organisms for chemical extraction and bioactivity screening shall not be conducted without specific written approval by the Director General. 11 No fauna shall be taken from any CALM land, as defined in the Conservation and Land Management Regulations 2002, without prior written approval of the Director General. No fauna shall be taken from any public land without the prior written approval of the Government Authority managing that land. 12 The licensee shall not enter upon any private property or pastoral lease for the purposes of this licence, nor take any fauna from any private land or pastoral lease without the prior consent in writing of the owner or occupier. Similarly, in the case of Aboriginal lands, the licensee must not enter upon or take fauna from such lands without the written approval of the Department of Aboriginal Affairs and/or the relevant native title holders or applicants. 13 Copies of this licence and any written approval or consent required by conditions of this licence must be carried by the licensee and any person/s authorised under the licence at all times when conducting activities relevant to the licence DEPARTMENT OF PARKS AND WILDLIFE

Enquiries: 17 DICK PERRY AVE, KENSINGTON, WESTERN AUSTRALIA Telephone: 08 9219 9000 Facsimile: 08 9219 8242 Web Site: https://wildlifelicensing.dpaw.wa.gov.au Correspondance: Locked Bag 30 PAGE 2 Bentley Delivery Centre WA 6983 NO. 08-000722-1

and must be presented to an authorised officer of the Department upon request. 14 All holotypes and syntypes and a half share of paratypes of species or subspecies permitted to be permanently taken under this licence shall be donated to the Western Australian Museum. Duplicates (one pair in each case) of any species collected, which represents a significant extension of geographic range shall upon request be donated to the Western Australian Museum. 15 To prevent any unnecessary collecting in this State, all specimens and material taken and retained under the authority of this license shall, upon request, be loaned to the Western Australian Museum. Any unused portion or portions of any specimen collected under the authority of this license shall be offered to the Western Australian Museum for inclusion in its collection or made available to other scientific workers if so required. 16 Within one month of the expiration of this licence, the holder shall submit an electronic return into the department’s Wildlife Licensing System, detailing the locality, site, geocode, date and number of each species of fauna captured, sighted or vouchered during the currency of the licence. A copy of any paper, report or thesis resulting from the research shall upon completion be lodged with the Director General.

Purpose

Subterranean fauna survey sampling of troglofauna will be via PVC leaf-litter traps, net haul scrapings and stygofauna haul nets for Rio Tinto Iron Ore Paraburdoo in accordance with EPA guidance statement 54a.

Locations

The Greater Paraburdoo Study Area is located in the central Pilbara region, approximately 9 km south west of the town of Paraburdoo

Authorised Person Surname Given name(s) Durrant Bradley Volschenk Erich Main Dean

Original Date of Issue 30/05/2017 Date of Issue 30/05/2017 Valid From 08/06/2017 Date of Expiry 07/06/2018

Licensee: Mr Shae Callan Address Office G 162 Rokeby Road Subiaco WA 6008 Australia

Issued by a Wildlife Licensing Officer of the Department of Parks and Wildlife under delegation from the Minister for Environment pursuant to section 133(1) of the Conservation and Land Management Act 1984. DEPARTMENT OF PARKS AND WILDLIFE

Enquiries: 17 DICK PERRY AVE, KENSINGTON, WESTERN AUSTRALIA Telephone: 08 9219 9000 Facsimile: 08 9219 8242 Web Site: https://wildlifelicensing.dpaw.wa.gov.au Correspondance: Locked Bag 30 PAGE 1 Bentley Delivery Centre WA 6983 NO. 08-000722-2

Wildlife Conservation Act 1950 REGULATION 17 Regulation 17 – Licence to take fauna for scientific purposes (Regulation 17 - Standard) The undermentioned person may take fauna for research or other scientific purposes and where authorised, keep it in captivity, subject to the following and attached conditions, which may be added to, suspended or otherwise varied as considered fit. Director General

Conditions

1 The licensee must comply with the provisions of the Wildlife Conservation Act 1950, Wildlife Conservation Regulations 1970 and any Notices in force under this legislation. 2 The licensee shall take fauna only in the manner stated on the endorsed Regulation 17 licence application form and endorsed related correspondence. 3 Unless specifically authorised in the conditions of this Licence or otherwise in writing by the Director General, species of fauna declared as likely to become extinct, rare or otherwise in need of special protection shall not be taken. 4 Any by-catch of fauna, which is declared to be rare, likely to become extinct, or otherwise in need of special protection shall be released immediately at the point of capture. Where such fauna taken under this licence is injured or deceased, the licensee shall contact the Department’s Wildlife Licensing Section for advice on disposal. Records must be kept of any such fauna so captured and details are to be included in the report required under further condition below. 5 Any interaction involving Gazetted Threatened Fauna that may be harmful to the fauna and/or invasive may require approval from the Commonwealth Department of the Environment ph 02 6274 1111. Interaction with such species is controlled by the Commonwealth Environment Protection and Biodiversity Conservation Act 1999 and Environment Protection and Biodiversity Conservation Regulations 2000 as well as the Wildlife Conservation Act 1950 and Wildlife Conservation Regulations 1970. 6 No fauna shall be taken in areas where it would impinge on pre-existing scientific research programs. 7 Except in the case of approved lethal traps, the licensee shall ensure that measures are taken in the capture and handling of fauna to prevent injury or mortality resulting from that capture or handling. Where traps or other mechanical means or devices are used to capture fauna these shall be deployed so as to prevent exposure of trapped animals to ants and debilitating weather conditions and inspected at regular intervals throughout each day of their use. At the conclusion of research all markers used, and signs and structures erected by the licensee shall be removed and the environment returned to its original condition. 8 Not more than ten specimens of any one protected species of fauna shall be taken and removed from any location less than 20km apart. Where exceptional circumstances make it necessary to take a larger number of specimens from a particular location in order to obtain adequate statistical data, the collector must proceed with circumspection and justify their actions to the Director General in advance. 9 The licensee shall not release any fauna or their progeny in any area where it does not naturally occur, nor hand such fauna over to any other person or authority unless approved by the Director General, nor dispose of the remains of such fauna in any manner likely to confuse the natural or present day distribution of the species. 10 Bioprospecting involving the removal of sample aquatic and terrestrial organisms for chemical extraction and bioactivity screening shall not be conducted without specific written approval by the Director General. 11 No fauna is to be taken from any CALM land, as defined in the Conservation and Land Management Regulations 2002, without prior written approval of the Director General. No fauna is to be taken from any public land without the prior written approval of the Government Authority managing that land. 12 The licensee must not enter upon any private property or pastoral lease for the purposes of this licence, nor take any fauna from any private land or pastoral lease without the prior consent in writing of the owner or occupier. Similarly, in the case of Aboriginal lands, the licensee must not enter upon or take fauna from such lands without the written approval of the Department of Aboriginal Affairs and/or the relevant native title holders or applicants. 13 Copies of this licence and any written approval or consent required by conditions of this licence must be carried by the licensee and any person/s authorised under the licence at all times when conducting activities relevant to the licence DEPARTMENT OF PARKS AND WILDLIFE

Enquiries: 17 DICK PERRY AVE, KENSINGTON, WESTERN AUSTRALIA Telephone: 08 9219 9000 Facsimile: 08 9219 8242 Web Site: https://wildlifelicensing.dpaw.wa.gov.au Correspondance: Locked Bag 30 PAGE 2 Bentley Delivery Centre WA 6983 NO. 08-000722-2

and must be presented to an authorised officer of the Department upon request. 14 All holotypes and syntypes and a half share of paratypes of species or subspecies permitted to be permanently taken under this licence shall be donated to the Western Australian Museum. Duplicates (one pair in each case) of any species collected, which represents a significant extension of geographic range shall upon request be donated to the Western Australian Museum. 15 To prevent any unnecessary collecting in this State, all specimens and material taken and retained under the authority of this license shall, upon request, be loaned to the Western Australian Museum. Any unused portion or portions of any specimen collected under the authority of this license shall be offered to the Western Australian Museum for inclusion in its collection or made available to other scientific workers if so required. 16 Within one month of the expiration of this licence, the holder shall submit an electronic return into the department’s Wildlife Licensing System, detailing the locality, site, geocode, date and number of each species of fauna captured, sighted or vouchered during the currency of the licence. A copy of any paper, report or thesis resulting from the research shall upon completion be lodged with the Director General.

Purpose

Subterranean fauna survey sampling of troglofauna will be via PVC leaf-litter traps, net haul scrapings and stygofauna haul nets for Rio Tinto Iron Ore Paraburdoo in accordance with EPA guidance statement 54a.

Locations

The Greater Paraburdoo Study Area is located in the central Pilbara region, approximately 9 km south west of the town of Paraburdoo

Authorised Person Surname Given name(s) Durrant Bradley Volschenk Erich Main Dean Rodman Syngeon

Date of Issue 06/06/2018 Valid From 08/06/2018 Date of Expiry 07/06/2019

Licensee: Mr Shae Callan Address Office G 162 Rokeby Road Subiaco WA 6008 Australia

Issued by a Wildlife Licensing Officer of the Department of Parks and Wildlife under delegation from the Minister for Environment pursuant to section 133(1) of the Conservation and Land Management Act 1984.

Greater Paraburdoo Subterranean Fauna Survey

APPENDIX 2: Conceptual methodology for subterranean fauna habitat modelling

Appendix Page | 7

Conceptual Methodology for Subterranean Fauna Habitat Modelling

Greater Paraburdoo, Q1, 2019

1. Define the location

2. Collate available drill log data

 Data covers all deposits  Projected into 3D

3. Assign prospectivity to each geology

Different ‘Strand’(stratigraphy) and ‘Tag’ (mineralisation) combinations were assigned different prospectivities based on:  Physical characteristics = (high/med/low)  Previous records of subfauna in that geology (certain/uncertain) ‘High certain’ and ‘medium certain’ are the main subfauna habitats. 4. Determine water contours before and after proposed mining

Habitat extent can be compared before and after proposed dewatering

5. Determine pit shell before and after proposed mining

Habitat extent can be compared before and after proposed mining.

6. Determine high and low confidence areas

High confidence limited to 300m from drill holes 7. Model ‘high certain’ and ‘medium certain’ habitats

Restricted to the ‘high confidence’ area 3D representation of most likely available habitat. AB A: cross section B: side view C: plan view

C

8. Model habitat during different time periods

Pit shells and water contours are included for relevant time periods A

Separate analyses for AWT and BWT habitats

A: pre‐mining B: current C: proposed additional impacts B

C

8. Create 3D surface/ raster map of above water table habitat thickness

Use spatial interpolation of point data combined with confidence interval boundary.

‘High certain’ and ‘medium certain’ habitats are combined together to give a single habitat thickness.

Greater Paraburdoo Subterranean Fauna Survey

APPENDIX 3: DNA Report (WA Museum)

Appendix Page | 11

Report by Western Australian Museum (Molecular Systematics Unit)

RTIO035, 43, 46 & 56---: WAM Molecular Systematics Unit Molecular systematics of subfauna from Greater Paraburdoo– Final Report

(Rio Tinto – Greater Paraburdoo)

Report to Rio Tinto

21 Dec 2018

Karen Cullen & Mark Harvey

Department of Terrestrial Zoology, Western Australian Museum, Locked Bag 49, Welshpool DC, Western Australia 6986, Australia

Although identifications in this report were consistent with the best available information and current scientific thinking at the time of identification the use of this report is at the risk of the user. Any liability to users of this report for loss of any kind arising out of the use of this report or the information and identifications it contains is expressly disclaimed.

Report by Western Australian Museum (Molecular Systematics Unit)

SUMMARY

A subset of 213 subterranean fauna samples from the Greater Paraburdoo Region, originally collected by Biologic on four separate collecting trips during 2017–2018, were requested to be sequenced for Rio Tinto. These specimens belonged to 11 different classes of subterranean fauna, including 20 orders, 28 families, and over 60 distinct lineages. The majority of specimens were sorted and morphologically identified by Bennelongia.

The Molecular Systematics Unit (MSU) extracted DNA, sequenced for variation at the mitochondrial COI gene, or the mitochondrial small ribosomal RNA gene (12S) in the case of Zygentoma, and undertook molecular analyses with the aim to determine the lineage of each specimen and develop a base line of subterranean faunal lineages from the Greater Paraburdoo Region. Initial sampling of stygofauna was undertaken in the Greater Paraburdoo area from 1998–2003. However, very little molecular research has been conducted on subterranean fauna within this region, apart from a few key papers published looking at genetic patterning in amphipods (Finston et al. 2007; Finston & Johnson 2004) and oligochaete worms (Brown et al. 2015) in the Pilbara Region which relate to this study site. This report summarises all further sampling and molecular identification of species collected in this region.

We undertook a number of additional steps and processes in the attempt to increase DNA yield, and increase amplification of target DNA by trialling additional primers designed specifically for individual faunal groups.

In this study over 60 distinct genetic lineages/species were detected among the various groups, none of which have been formally described. Of these, four lineages/species matched those previously detected from the Pilbara (Finston et al. 2007; Cooper et al. 2007; Brown et al. 2015). A summary of lineages detected are shown in the Table below.

Report by Western Australian Museum (Molecular Systematics Unit)

Summary of lineages/species detected from this study

Matching Subphylum Class No. Orders known New lineages Total Lineages Amphipoda 2 9 11 Malacostraca Bathynellacea 0 1 1 Isopoda 0 4 4 Crustacea Cyclopoida 0 4 4 Maxillopoda Harpacticoida 0 4 4 Ostracoda Popocopida 0 1 1 Enchytraeida 2 3 5 Clitellata Oligochaeta Haplotaxida 0 4 4 Araneae 0 1 1 Palpigradi 0 2 2 Chelicerata Arachnida Pseudoscorpiones 0 3 3 Schizomida 0 1 1 Diplura 0 1 1 Entognatha Protura 0 1 1 Hexapoda Hemiptera 0 3 3 Insecta Zygentoma 0 8 8 Chilopoda Scolopendrida Failed to sequence Diplopoda Failed to sequence Myriapoda Pauropoda Tetramerocerata 0 5 5 Symphyla Cephalostigmata 0 5 5 Total 4 60 64

Report by Western Australian Museum (Molecular Systematics Unit)

Background and Objective Subterranean fauna belonging to five major taxonomic groups (Crustacea, Oligochaeta, Chelicerata, Hexapoda and Myriapoda) were collected from a number of different sites within the Greater Paraburdoo Region. Samples were collected by Biologic between 18 August 2017 and 4 July 2018 during four separate collecting trips. Of the material deposited at the Western Australian Museum over this period, a sub-set of 213 specimens were extracted for molecular analysis (Table1, Appendix 1).

The purpose of this project was to undertake sub-sampling, sequence for variation at the mitochondrial COI gene or 12S (Zygentoma), and compare molecular data of target samples with reference sequences to determine:

a) Lineages/species present,

b) Distributions of lineages/species within the Greater Paraburdoo Region, and

c) Affiliations with lineages previously identified by Helix Molecular Solutions, the Western Australian Museum (WAM), and/or reference sequences deposited on GenBank.

Methods Two hundred and thirteen specimens were subsampled at the WA Museum using Leica dissecting microscopes (MZ6, MZ16) and samples placed directly into ATL buffer for extraction using a commercially available DNA extraction kit (QIAGEN DN-easy). DNA samples were amplified by Polymerase Chain Reaction (PCR) using Folmer PCR primers (LCO1490, HCO2198; Folmer et al. 1994) to assess variation at the standard DNA barcoding region (Cytochrome Oxidase I; COI; Herbert et al. 2003). Zygentoma samples were amplified using 12S primers (M0112, M0113; MSU). Resulting PCR product was sequenced by the Australian Genomic Research Facility (AGRF) Perth node. Specimen, extraction, PCR and sequence data were managed using GENEIOUS software version 10.1.2 (Kearse et al. 2012).

The following additional steps were also undertaken in an attempt to increase DNA yield and amplification of target DNA:

 Concentrating final DNA extraction by eluting into 50µL of AE Buffer instead of 200µL

 Trial of different PCR protocols and annealing temperatures

 Trial of different PCR primer pairs:

o Chilopoda: LCO1490, HCOoutout (Prendini et al. 2005)

Report by Western Australian Museum (Molecular Systematics Unit)

o Myriapoda: LCO1490, CO!_antichir_R (J. Huey, WA Museum Molecular Systematics Unit)

Sequences were analysed against additional sequences obtained from GenBank, the Western Australian Museum (MSU, Cullen & Harvey, 2017; Cullen 2018), and Helix Molecular Solutions (Finston, et al. 2015, 2016a, 2016b, 2017).

Maximum likelihood analyses with 100 rapid bootstrap replicates was performed in RAxML (Randomised Accelerated Maximum Likelihood) version 8.2.7 software (Stamatakis, 2014), using the GTR Gamma model of sequence evolution and default settings. 12S sequence data was edited, aligned and run through Gblocks Version 0.91b software before Maximum likelihood analysis in RAxML. Gblocks is a computer program that eliminates poorly aligned positions and divergent regions of DNA or protein alignment so that it becomes more suitable for phylogenetic analysis (Castresana, 2000; Talavera & Castresana, 2007).

Lineages determined in this study were defined using molecular data with reference to morphological identifications undertaken by Bennelongia. These designations are dependent on the taxa and taxonomic certainty involved, and do not follow a standard sequence divergence threshold. These designations may alter if more data is collected, additional genes are sequenced, and if systematic revisions are undertaken for particular taxa.

Results A summary of specimen identifications together with lineage information are presented in tables in each taxa section (Tables 1-14). A consensus Maximum Likelihood Phylogenetic tree, including representative samples of available reference lineages, is shown in each section, as well as distribution maps of the main lineages detected within the project area and images of the specimens where available (Figures 1 -32). The list of specimens included in the molecular analysis component of the project is presented in Appendix I, which details the initial morphological identification alongside the Molecular ID results and comments. Specimens not listed here, were either not sub-sampled for molecular work, were missing, or not submitted. Complete tables of genetic distances (% difference) for each faunal group are supplied as supplementary material (Appendix II). See Appendix II for full sequence names of all sequences included in the analysis.

Report by Western Australian Museum (Molecular Systematics Unit)

CRUSTACEA

MALACOSTRACA:

AMPHIPODA & BATHYNELLACEA

A summary of the Amphipoda and Bathynellacea molecular identifications is depicted in Table 1. In this study, a total of 55 specimens were extracted for molecular analysis, with 44 of those producing viable COI sequences (Figure 1). A total of 11 distinct lineages/species of Amphipoda were detected using Maximum likelihood analysis (Figure 1), and 1 distinct lineage of Bathynellacea (Figures 1 & 2). Figure 1 shows the phylogenetic analysis of representative amphipod lineages from the Greater Paraburdoo Region in addition to 68 representative WAM, Helix and GenBank sequences. Daphnia pulicaria (DAPHNIIDAE: JN233925) and Geisha distinctissima (FLATIDAE: JN087438) were used as outgroups. Figures 3 and 4 shows maps with the known distributions of the lineages within the project area. Automontage images of the major lineages is depicted in Figures 5–7.

Table 1. Summary of Amphipod and Bathynellacea lineages detected this study. % Divergence from closest known lineage is shown with lineage name in parentheses.

Family Genus Species/Lineage Specimens Divergence from closest lineage RTIO #

25% (GenBank Pilbaranella De RTIO056 Bathynellidae Pilbaranella `WAM-BATH001` 2 Grey – MF074337) Bogidiellidae `WAM-AMPB001` 2 17% (WAM-AMPB003) RTIO35, 56 Bogidiellidae `WAM-AMPB002` 1 12% (Helix AMN002 - Robe R) RTIO056 Bogidiellidae `WAM-AMPB003` 1 17% (WAM-AMPB001) RTIO056 Paramelitidae Pilbarus `Sp. G` 7 8% (Pilbarus sp. H) RTIO035, 46, 56 Paramelitidae Pilbarus `Sp. H` 1 8% (Pilbarus sp. G) RTIO035 Paramelitidae `Yilgarus` `WAM-AMPP001` 16 14.6% (WAM-AMPP002) RTIO035, 43, 46, 56 Paramelitidae `Yilgarus` `WAM-AMPP002` 1 14.6% (WAM-AMPP001) RTIO035 11% (GenBank Yilgarus sp. RTIO046 Paramelitidae `Yilgarus` `WAM-AMPP003` 1 BES8834_Gifford) Eriopisidae Nedsia `WAM-AMPE001` 1 6.2% (WAM-AMPE002) RTIO056 Eriopisidae Nedsia `WAM-AMPE002` 1 6.2% (WAM-AMPE001) RTIO056 Eriopisidae Nedsia `WAM-AMPE003` 1 6.5% (WAM-AMPE002) RTIO046

BOGIDIELLIDAE

PARAMELITIDAE

ERIOPISIDAE

Figure 1. Maximum Likelihood of representative COI haplotypes of Amphipoda (PARAMELITIDAE, ERIOPISIDAE and BOGIDIELLIDAE) from this study and a sub-set of reference specimens from WAM, Helix Molecular solutions and GenBank. Proposed lineages from this study are indicated by the coloured, shaded boxes.

Figure 2. Maximum Likelihood of representative COI haplotypes of Bathynellacea (BATHYNELLIDAE) from this study and a sub -set of reference specimens from Genbank. Proposed lineage from this study is indicated by the coloured, shaded box. Report by Western Australian Museum (Molecular Systematics Unit)

Figure 3. Map of Pilbarus and ` Yilgarus` Amphipoda lineages detected in this study and their collecting locations within the project area.

Figure 4. Map of Pilbaranella (Bathynellacea), Nedsia and Bogidiellidae lineages (Amphipoda) detected in this study and their collecting locations within the project area.

Report by Western Australian Museum (Molecular Systematics Unit)

`WAM-AMPB001` Nedsia sp.

Figure 5. Automontage images of `WAM-AMPB001` (Bogidiellidae; WAM C72400) one of three lineages detected, and an unidentified Nedsia sp.(WAM C72401) collected from Paraburdoo Mine, approximately 12km SW of Paraburdoo.

`WAM-AMPP001` `WAM-AMPP002`

Figure 6. Automontage images of `Yilgarus` (PARAMELITIDAE) specimens detected in this study `WAM-AMPP001` (WAM C72412) from Seven Mile Creek, approx. 3km NE of Paraburdoo (Left); `WAM-AMPP002` (WAM C72407) from Turee Creek, approx. 25km SE of Paraburdoo (Right).

`Pilbarus. sp. G` `Pilbarus sp. H`

Figure 7. Automontage images of Pilbarus (PARAMELITIDAE) lineages detected in this study. `Pilbarus sp. G` (WAM C72413) from Channar Mine, approx. 14km SE of Paraburdoo (Left); `Pilbarus sp. H` (WAM C72414) from Turee Creek, approx. 25km SE of Paraburdoo (Right).

Report by Western Australian Museum (Molecular Systematics Unit)

Three new lineages of Bogidiellid amphipod were detected from the project area and did not closely match any available reference sequences. `WAM-AMPB001` is represented by two specimens and differed from closest representative by 17%. The other lineages were only represented by individual specimens. Currently there is only one described subterranean species of Bogidiellidae in Western Australia (Bogidomma australis Bradbury & Williams 1996), which occurs within carbonate karst anchialine systems on Barrow Island (Leijs et al. 2011; Eberhard et al. 2005). Unfortunately, no COI sequences are available for this species

Specimens of Nedsia (ERIOPISIDAE) proved to be difficult to sequence, with only four out of 14 specimens producing viable sequences, despite attempts at trouble-shooting (Fagan-Jeffries, 2012). However, from these four viable sequences, there appears to be three distinct lineages, differing from each other by around 6.2%. Figure 5 shows an automontage image of one specimen morphologically identified as Nedsia (ERIOPISIDAE: Bradbury & Williams 1997) that failed to sequence.

Of the five paramelitid amphipod lineages detected (Figures 6–7), two lineages closely matched known lineages Pilbarus sp. G (Seven Mile Creek; DQ256038.1, DQ256043.1 & DQ256046.1) and Pilbarus sp. H (Turee Creek; DQ256025.1, DQ256026.1, & DQ256022.1) on GenBank (Finston et al. 2007). These lineages were originally determined in a paper by Finston et al. (2007), which examined cryptic speciation in two widespread subterranean amphipod genera in the Pilbara Region of Western Australia. Within-species divergence ranged from between 0.5–4.6%, and <0.6% genetic divergence for Pilbarus sp. G and Pilbarus sp. H respectively. The two lineages differed from one another by 8.1–9.7%. Three new lineages of an undescribed genus `Yilgarus` (Paramelitidae Genus 2) were also detected in this study: `WAM-AMPP001` - `WAM-AMPP003` (Figures 1 & 6). Cooper et al. (2007) undertook a phylogenetic study of paramelitid and hyalid amphipods from the Yilgarn Region of central Western Australia and determined a number of lineages. It is clear that these specimens belong to the same genus as determined in Cooper et al. (2007), and are most closely related to Yilgarus sp. BES8850 from Wanna (17.4-19.0% genetic difference) and Yilgarus sp. BES8834 from Gifford (17.1 - 18.2%, Cooper et al. 2007).

Two specimens of Pilbaranella `WAM-BATH001` (Bathynellacea) were found in the project area. This lineage was very different to other lineages of Pilbaranella from the Pilbara that have been previously sequenced (Perina, et al. 2018). The closest lineage was a Pilbaranella located from the De Grey River Catchment.

ARMADILLIDAE

SCYPHACIDAE

PHILOSCIIDAE

TAINISOPIDAE

PLATYARTHRIDAE

Figure 8. Maximum Likelihood of all COI haplotypes of ISOPODA from the present study and a sub-set of reference specimens from WAM, Helix Molecular Solutions, and Genbank. Note: Higher level taxonomic identification is tentative as the analysis is based only on COI data, and thus should be treated with caution. Report by Western Australian Museum (Molecular Systematics Unit)

ISOPODA

A summary of the Isopoda molecular identifications is depicted in Table 2. In this study, a total of five specimens were extracted for molecular analysis, and all produced viable COI sequences. A total of four distinct lineages/species from three families were detected using Maximum likelihood analysis (Figure 8).

Table 2. Summary of Isopod lineages. % Divergence from closest known lineage is shown with lineage name in parentheses.

Specimen Family Genus Species/Lineage Divergence from closest lineage RTIO # s Armadillidae WAM-ARMD003 1 22.3% (Buddelundia sp. KX656281) RT43 Armadillidae Troglarmadillo WAM-ARMD004 3 19.9% (WAM-ARMD001_Dinner Camp) RT46, 56 Paraplatyarthru 11.8% Paraplatyarthrus sp5MJ Platyarthridae WAM-PARA001 1 RT56 s KR424578)

Tainisopidae Pygolabis WAM-PYGO001 8 16.4% (Pygolabis EU107646) RT56

Figure 8 shows the phylogenetic analysis of representative isopoda lineages from the Greater Paraburdoo Project area in addition to 80 representative sequences from WAM, Helix Molecular Solutions, and GenBank. Chydaekata acuminata (DQ838027) was used as an outgroup for the analysis. None of the five isopod lineages detected closely matched any known lineages previously detected by WAM or Helix.

Figure 9. Map of ISOPODA lineages detected within the project area.

Report by Western Australian Museum (Molecular Systematics Unit)

MAXILLOPODA:

CYCLOPOIDA & HARPACTIOIDA

A summary of the Copepod (Cyclopoida & Harpacticoida) molecular identifications is depicted in Table 3. In this study, a total of 36 specimens were extracted for molecular analysis, 21 of those produced viable COI sequences (Appendix I). A total of eight distinct lineages/species were detected using Maximum likelihood analysis (Figure 10). Figure 10 shows the phylogenetic analysis of representative amphipod lineages from the Greater Paraburdoo Region in addition to 41 representative GenBank sequences. Geisha distinctissima (JN087438) was used as an outgroup for the analysis. Figure 11 shows a map with the known distributions of the eight lineages within the project area. Automontage images of Schizopera `WAM-SCHZ001` and Diacyclops `WAM-CYLD002` and is depicted in Figures 12-13.

Table 3. Summary of Copepoda detected this study and their general distributions. % Divergence from closest known lineage is shown with lineage name in parentheses.

RTIO # Family Genus Species/Lineage Specimens Divergence from closest lineage

Cyclopidae Diacyclops cockingi 28 Could not sequence (Bennelongia Morph ID) RT46, 56 Cyclopidae Diacyclops `WAM-CYLD001` 1 8.2% different to Diacyclops `WAM-CLYD002` RT46 Cyclopidae Diacyclops `WAM-CYLD002` 231 8.2% different to Diacyclops `WAM-CLYD001` RT43, 46, 56 22.5% (GenBank Acanthocyclops profunus RT46 Cyclopidae Pescecyclops `WAM-CYLP001` 2 KT075061.1) 17.9% (GenBank Thermocyclops decipiens RT46 Cyclopidae Thermocyclops `WAM-CYLT001` 1 MF443204.1) Miraciidae Schizopera `WAM-SCHZ001` 4 13.9% different to Schizopera `WAM-SCHZ002` RT56 Miraciidae Schizopera `WAM-SCHZ002` 26 13.9% different to Schizopera `WAM-SCHZ001` RT56 18.7% different from Parastenocaris jane on RT56 Parastenocarididae Parastenocaris `WAM-PARA001` 11 GenBank (JN039164). 18.8% different to `WAM-PARA002` 19.4% different from Parastenocaris jane on RT56 Parastenocarididae Parastenocaris `WAM-PARA002` 4 GenBank (JN039164). 18.8% different to `WAM-PARA001`

MIRACIIDAE

HARPACTICOIDA S. roberiverensis

CANTHOCAMPTIDAE

P. jane

PARASTENOCARIDIDAE

CYCLOPIDAE

D. humphreysi

CYCLOPOIDA

Figure 10. Maximum Likelihood of all COI haplotypes of Cyclopoida (CYCLOPIDA) and Harpacticoida (MIRACIIDAE & PARASTENOCARIDAE) from the present study and a sub-set of reference specimens from WAM and GenBank. Note: Higher level taxonomic identification is tentative as the analysis is based only on COI data, and thus should be treated with caution. Bennelongia species identifications that represent more than one lineage are shown in blue. Report by Western Australian Museum (Molecular Systematics Unit)

Figure 11. Map of CYCLOPOIDA and HARPACTICOIDA lineages detected within the project area.

Within the family Parastenocarididae, two distinct lineages were detected, which were originally identified as Parastenocaris jane using morphology (Figure 10). However, these two specimens differed from the GenBank sequence (JN039164.1 Parastenocaris jane) between 18.7–19.4% and each other by 18.8%. Therefore, it appears that Parastenocaris jane is not as widespread as originally thought and there are likely more cryptic species present.

Further evidence of cryptic species can be seen within the family Cyclopidae and Miraciidae. Specimens originally identified as Diacyclops humphreysi could be split into two distinct lineages, which differ from each other by 8.2%. Similarly, specimens identified as Schizopera roberiverensis were split into two lineages, which differ from each other Schizopera `WAM-SCHZ001` by 13.9%. There was an additional species recorded based on morphology, but unfortunately could not be sequenced despite repeated attempts. This species was originally identified by Bennelongia as Diacyclops cockingi and included 28 individuals. However, it is likely that these specimens may represent more than one species/lineage and further efforts to obtain sequences should be sought. Figure 12. Automontage image of Schizopera `WAM-SCHZ001` (MIRACIIDAE; split from WAM C71571)

Report by Western Australian Museum (Molecular Systematics Unit)

Diacylops `WAM-CYLD002`

Figure 13. Automontage images of Diacyclops `WAM-CYLD002`(CYCLOPIDAE) detected in this study (split from WAM C71568, 71569)

OSTRACODA:

A summary of the ostracod molecular identifications is depicted in Table 4. In this study, a total of four specimens were extracted for molecular analysis and two of those produced viable COI sequences belonging to one distinct lineage/species of the genus Gomphodella (Figure 14). Figure 15 shows the location where the two samples were collected (BES3700_99RS2). Automontage images of Gomphodella `WAM-OSTR001` and is depicted in Figure 16.

Table 4. Summary of Ostracod lineages detected. % Divergence from closest known lineage is shown with lineage name in parentheses.

Order Family Genus Species/Lineage Specimens Divergence RTIO #

28.6% (MG449354.1_Ostracoda sp. Ostracoda Limnochytheridae Gomphodella WAM-OSTR001 2 RT43 CH-659-G11)

Figure 14 shows the phylogenetic analysis of representative ostracod lineages from the Greater Paraburdoo project area in addition to 13 representative WAM and GenBank sequences. Polydesmus angustus (HQ966174.1) was used as an outgroup for the analysis. There are very few sequences of subterranean ostracods available to compare against, so the analysis presented here is limited in its interpretation. There are currently nine species of Gomphodella described from bore-holes in the Pilbara region (Karanovic & Humphreys, 2004), but currently there are no sequences available to analyse against.

Figure 14. Maximum Likelihood of all COI haplotypes of Ostracoda (PODOCOPIDA) from the present study and a sub-set of reference specimens from WAM and GenBank. Note: Higher level taxonomic identification is tentative as the analysis is based only on COI data, and thus should be treated with caution. Report by Western Australian Museum (Molecular Systematics Unit)

Figure 15. Map of the single OSTRACOD lineage detected within the project area.

Gomphodella `WAM-OSTR001`

Figure 16. Automontage images of Gomphodella `WAM-OSTR001` (LIMNOCYTHERIDAE) detected in this study (split from WAM C71573) CLITELLATA

Report by Western Australian Museum (Molecular Systematics Unit)

OLIGOCHAETA:

ENCHYTRAEIDA & HAPLOTAXIDA

A summary of the oligochaete molecular identifications is depicted in Table 5. In this study 24 specimens were extracted for molecular analysis, and 16 produced viable COI sequences. Figure 17 shows the phylogenetic analysis of representative oligochaete lineages from the Greater Paraburdoo Region in addition to 78 representative WAM and GenBank sequences. Orobdella ketagalan (OROBDELLIDAE: AB704793.1) was used as an outgroup for the analysis. A total of nine distinct lineages were detected using Maximum likelihood analysis (Figure 17). The distribution of these lineages within the project site is depicted in Figure 18. Automontage image of Enchyraeidae sp. E6 is depicted in Figure 19.

Table 5. Summary of Oligochaeta: Clitellata lineages detected in this study and their distributions. % Divergence from closest known lineage is shown with lineage name in parentheses.

Family Genus Species/Lineage Specimens Divergence from closest lineage RTIO #

Enchytraeidae Enchytraeus `Enchyraeidae sp. E6(2-4)` 5 7.8% (Enchytraeidae sp. E6-5 KM206485) RT35, 43, 56 Enchytraeidae Enchytraeus `Enchyraeidae sp. E6-11` 1 8.5% (Enchytraeidae sp. E6-1 KM206478) RT35 Enchytraeidae Enchytraeus `WAM-ENCH001` 1 10.9% (Enchytraeidae sp.E8 KM206490) RT35 Enchytraeidae Enchytraeus `WAM-ENCH002` 2 10% (Enchytraeidae sp. E7 KM206487) RT56 7.9% (Enchytraeidae sp. E6-9 Enchytraeidae Enchytraeus `WAM-ENCH003` 3 RT56 KM206529) Naididae Pristina `WAM-NAIDP001` 1 15.5% (Nadidae sp. N3 KM206504) RT35 Naididae `WAM-NAID002` 1 19.3% (Nadidae sp. N3 KM206504) RT46 Phreodrilidae `WAM-PHRE001` 1 17% (Phreodrilidae sp. P5 KM206511) RT46 Phreodrilidae `WAM-PHRE002` 2 21% (Phreodrilidae sp. P9 KM206522) RT35

Two new lineages belonging to the family Naididae were detected in this analysis. These species were genetically distinct and related to Naidiae sp. N3 on GenBank (KM206493, KM206494, & 206504) from the Upper Fortescue Catchment (Figure 17; Brown et al. 2015).

Within the family Phreodrilidae, two distinct lineages were detected (`WAM-PHRE001` and `WAM-PHRE002`) which did not match any available reference sequences. These two lineages differed from each other greatly, ranging between 17–21% genetic divergence from closest lineages (Figure 17; Appendix II).

NAIDIDAE

ENCHYTRAEIDAE

PHREODRILIDAE

Figure 17. Maximum Likelihood of representative COI haplotypes of Oligochaetes (NAIDIAE, PHREODRILIDAE & ENCHYTRAEIDAE) from this study and a sub-set of reference specimens from WAM and GenBank. Proposed lineages from this study are indicated by the shaded, coloured boxes. Note: Higher level taxonomic identification is tentative as the analysis is based only on COI data, and thus should be treated with caution. Report by Western Australian Museum (Molecular Systematics Unit)

Figure 18. Map of the Oligochaete lineages (NAIDIDAE, PHREODRILIDAE & ENCHYTRAEIDAE) detected within the project area.

Of the nine oligocheate lineages detected in this study, two lineages closely matched previously identified lineages belonging within the highly divergence species complex `Enchyraeidae sp. E6` (Brown et al. 2015), which may consist of up to 11 lineages/species. This species complex requires further research, but it is likely that Enchyraeidae Sp. E6-2 to E6-4 may be considered one species and five specimens have been assigned this lineage (`Enchyraeidae Sp. E6 (2-4)`; Figure 19). The distribution extends from a bore in the Paraburdoo Mine to the Turner River in the Port Headland Coastal catchment (E. sp. E6-4) to Upper South Fortescue River and Kangeenareena Creek in the South Fortescue Catchment (Brown et al. 2015).

WAM V9279 matched lineage Enchyraeidae sp. E6-11 from Eel Creek in the De Grey River Catchment (Brown et al. 2015), with only 5.7% genetic divergence found between these two specimens. Figure 19. Automontage images of Enchytraeus sp. E6 (2-4) These lineages were originally determined in a detected in this study (split from WAMV8906)

CAMPODEIDAE

PARAJAPYGIDAE

JAPYGIDAE

Figure 20. Maximum Likelihood of representative COI haplotypes of Diplura from this study and a sub-set of reference specimens from WAM, Helix and GenBank. Proposed lineage from this study is indicated by the shaded, coloured box Report by Western Australian Museum (Molecular Systematics Unit)

paper by Brown et al., (2015), which examined complex genetic patterns of groundwater oligochaetes in the Pilbara Region of Western Australia.

An additional three new species of Enchytraeidae oligochaete (`WAM-ENCH001`, `WAM- ENCH002` & `WAM-ENCH003`) were detected in this study and did not closely match any available reference sequences, differing from closest lineages between 7.9-10%.

HEXAPODA

ENTOGNATHA: DIPLURA & PROTURA

A summary of Diplura and Protura molecular identifications is depicted in Table 6. In this study one dipluran and one proturan were extracted for molecular analysis and both produced good quality sequences. The Dipluran differed from the closest Helix Dipluran lineage (Helix DJA009), located in Mesa H in the Robe River Valley, by 13.4% (Figure 20; Appendix II). Unfortunately no Proturan sequences were available for comparison. The distribution of these lineages within the project site is depicted in Figure 21.

Table 6. Summary of Diplura and Protura lineages detected in the project area. % Divergence from closest known lineage is shown with lineage name in parentheses.

Specimens RTIO # Order Family/Genus Species/Lineage Divergence Diplura Japygidae WAM-DPLJ005 1 13.4% (HelixDJA009; Mesa H, Robe River) RT56 Protura WAM-PROT001 1 Not analysed RT56

Figure 21. Map of the Protura and Diplura lineages (ENTOGNATHA) detected within the project area.

MEENOPLIDAE

CIXIIDAE

Figure 22. Maximum Likelihood of representative COI haplotypes of Hemiptera from this study and a sub-set of reference specimens from WAM and GenBank. Proposed lineages from this study are indicated by the shaded, coloured boxes. Report by Western Australian Museum (Molecular Systematics Unit)

INSECTA: HEMIPTERA

A summary of the Hemiptera molecular identifications is depicted in Table 7. In this study 22 specimens were extracted for molecular analysis, and 20 produced viable COI sequences. Figure 22 shows the phylogenetic analysis of representative lineages from the Greater Paraburdoo Region in addition to 12 representative GenBank sequences. Geisha distinctissima (JN087438) was used as an outgroup for the analysis. A total of three distinct lineages from two families (CIXIIDAE and MEENOPLIDAE) were detected using Maximum likelihood analysis (Figure 22). The distribution of these lineages within the project site is depicted in Figure 23. An Automontage image of Phaconeura WAM-PHAC001` is

depicted in Figure 24. Figure 24. Image of Phaconeura `WAM-PHAC001` (MEENOPLIDAE; Split from WAME98566)

Table 7. Summary of Hemiptera lineages detected in this study. % Divergence from closest known lineage is shown with lineage name in parentheses.

Family Genus Species/Lineage Specimens Divergence from closest lineage RTIO #

Cixiidae Oliarus? WAM-CIXO001 5 16.1% (HF674830.1_Oliarus polyphemus) RT46 Meenoplidae Phaconeura WAM-PHAC001 16 14.1% (KF227250.1_Phaconeura sp. BOLD:AAI2489) RT43, 46, 56 Meenoplidae Phaconeura WAM-PHAC002 4 14.4% (KF227233.1_Phaconeura sp. BOLD:AAI2490) RT43, 56

Figure 23. Map of the Hemiptera lineages (CIXIIDAE & MEENOPLIDAE) detected within the project area.

Report by Western Australian Museum (Molecular Systematics Unit)

INSECTA: ZYGENTOMA

A summary of the Zygentoma molecular identifications is depicted in Table 8. In this study 35 specimens were extracted for molecular analysis, and 30 produced viable 12S sequences (using Primer pair: M0112/M0113). Figure 25 shows the phylogenetic analysis of representative Zygentoma lineages from the Greater Paraburdoo Region in addition to 36 representative WAM, Helix and GenBank sequences. Graphosoma lineatum (KM013315.1) was used as an outgroup for the analysis. A total of eight distinct lineages were detected from three different genera (Dodecastyla, Lepidospora and Trinemura) using Maximum likelihood analysis (Figure 25). The distribution of these lineages within the project site is depicted in Figure 26. An Automontage image of Tinemura WAM-ZYGS001` is depicted in Figure 27.

Table 8. Summary of Zygentoma lineages detected in this study and their distributions. % Divergence from closest known lineage is shown with lineage name in parentheses.

Family Subfamily Genus Species/Lineage Specimens Divergence from closest lineage RTIO # 7.8-9.3% Nicoletiidae Atelurinae Dodecastyla WAM -ZYGA001 7 RT43, 46 (JD12_RC13KOOD0213_Koodaideri) Nicoletiidae Coletiniinae Lepidospora WAM-ZYGC001 2 6.9% (MO18_GR15MEB0216_Robe River) RT43, 46 Nicoletiidae Subnicoletiinae Trinemura WAM-ZYGS001 7 5.3-7.2% (WAM-ZYGS004) RT43, 56 RT43, 46, Nicoletiidae Subnicoletiinae Trinemura WAM-ZYGS002 5 8.1% (WAM-ZYGS003) 56 Nicoletiidae Subnicoletiinae Trinemura WAM-ZYGS002-A 1 6.6-7.2% (WAM-ZYGS002) RT43 RT43, 46, Nicoletiidae Subnicoletiinae Trinemura WAM-ZYGS003 3 8.1% (WAM-ZYGS002) 56 Nicoletiidae Subnicoletiinae Trinemura WAM-ZYGS004 2 5.3-7.2% (WAM-ZYGS001) RT43 Nicoletiidae Subnicoletiinae Trinemura WAM-ZYGS005 2 5.9-6.2% (Helix TN014_Todd Bore) RT56

ATELURINAE

COLETINIINAE

SUBNICOLETIINAE

Figure 25. Maximum Likelihood of representative 12S haplotypes of Zygentoma from this study and a sub-set of reference specimens from WAM, Helix and GenBank. Proposed lineages from this study are indicated by the shaded, coloured boxes. Report by Western Australian Museum (Molecular Systematics Unit)

Figure 26. Map of the Zygentoma lineages (NICOLETIIDAE) detected within the project area.

Figure 27. Image of Trinemura `WAM-ZYGS001` (SUBNICOLETIINAE; split from WAME98575) detected within the project area.

Figure 28. Maximum Likelihood of all COI haplotypes of Araneae from the study area and a sub-set of reference specimens from WAM and GenBank. Proposed lineage from this study is indicated by the shaded, coloured box. Report by Western Australian Museum (Molecular Systematics Unit)

CHELICERATA

ARACHNIDA:

ARANEAE

Only one spider was collected during the sampling period and was extracted for molecular analysis (WAMT144292; Table 9). It is unknown whether this species is restricted to the subterranean environment or the Project area due to lack of comparative material. However, this specimen differed from the closest Helix lineage from Mesa B & C in the Robe Valley, by 10.1% (Figure 28). The distribution of Araneae and Schizomida lineages within the project site is depicted in Figure 29.

Table 9. Araneae lineage detected from the Project area. % Divergence from closest known lineage is shown with lineage name and location in parentheses.

Family Genus Species/Lineage Specimens Divergence from closest lineage RTIO #

Araneidae? unknown `WAM-ARAN001` 1 10.1% (Helix AA001; Robe Valley: Mesa B & C) RT43

Figure 29. Map of Araneae (WAM-ARAN001) and Schizomida (WAM-DRAC001) lineages detected within the project area.

Figure 30. Maximum Likelihood of representative COI haplotypes of Palpigradi from this study and a sub-set of reference specimens from GenBank. Proposed lineages from this study is indicated by the shaded, coloured box. Report by Western Australian Museum (Molecular Systematics Unit)

PALPIGRADI

A summary of the palpigradi molecular identifications is depicted in Table 10. In this study four specimens likely belonging to the genus Eukoenenia (EUKOENENIIDAE) were extracted for molecular analysis, and three specimens produced a good quality sequences. Figure 30 shows the phylogenetic analysis of representative lineages from the Greater Paraburdoo Region in addition to 12 representative GenBank sequences. Prokoenenia wheeleri (KF823874.1) was used as an outgroup for the analysis. Two distinct lineages/species of Eukoenenia were detected using Maximum likelihood analysis (Figure 30), and differed from each other by 24.2%. The distribution of these lineages within the project site is depicted in Figure 31. Palpigradi are often found in subterranean environments but are still poorly known, with very few molecular studies undertaken to date (Giribet, et al. 2014). Giribet et al., (2014) published the first phylogenetic hypothesis of palpigradi based on molecular data, but found similar difficulty in obtaining well-preserved specimens, or obtaining sufficient yields of DNA.

Table 10. Summary of Palpigradi lineages detected in the Project area. % Divergence from closest known lineage is shown with lineage name in parentheses.

Family Genus Species/Lineage Specimen Divergence RTIO Eukoeneniidae Eukoenenia WAM-PALE001 2 24.2% (WAM-PALE002) RT43 Eukoeneniidae Eukoenenia WAM-PALE002 1 24.2% (WAM-PALE001) RT43

Figure 31. Map of Palpigradi (EUKOENENIIDAE) lineages detected within the project area.