LECHYTIIDAE CHTHONIIDAE Figure 32. Maximum Likelihood of representative COI haplotypes of pseudoscorpionida from this study and a sub-set of reference specimens from WAM, Helix and GenBank. Proposed lineages from this study is indicated by the shaded, coloured boxes. Report by Western Australian Museum (Molecular Systematics Unit) PSEUDOSCORPIONES In this study a total of three specimens were extracted for molecular analysis, all of which produced good quality COI sequences. A summary of specimen identifications is depicted in Table 11. A total of three distinct lineages/species belonging to 2 different families, were detected using Maximum likelihood analysis (Figure 32), none of which matched known lineages. The two chthoniid lineages, identified by Bennelongia as belonging to the genus Tyrannochthonius, ranged between 11.4–15.4% genetic difference between Helix lineage GC1 (Koodaideri) and an unknown species from GenBank respectively. The lineage of Lechytia (LECHYTIIDAE) was also unique and differed from described species from GenBank (Lechytia sini; USA) by 19.2%. The family Lechytiidae includes only a single genus, Lechytia, which currently contains 24 described species worldwide, and only one species described from Australia (Lechytia libita; QLD; Harvey, 2006). Unfortunately there are no available sequences of Lechytia from Australia or from a number of undescribed species lodged with the WA Museum. The distribution of these lineages within the project site is depicted in Figure 33. Table 11. Summary of pseudoscorpion 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 Chthoniidae Tyrannochthonius WAM-CHTH001 1 11.4% (GC1:Helix; Koodaideri) RT43 Chthoniidae Tyrannochthonius WAM-CHTH002 1 15.4% (Austrochthonius EU559513.1) RT56 Lechytiidae Lechytia WAM-LECYT001 1 19.2% (Lechytia sini KX263367.1) RT56 Figure 33. Map of pseudoscorpion lineages (CHTHONIIDAE & LECHYTIIDAE) detected within the project area. Figure 34. Maximum Likelihood of representative COI haplotypes of Schizomida from this study and a sub-set of reference specimens from WAM, Helix and GenBank. Proposed lineages from this study is indicated by the shaded, coloured box. Report by Western Australian Museum (Molecular Systematics Unit) SCHIZOMIDA A summary of the schizomid specimen identifications is depicted in Table 12. In this study, a total of three specimens were extracted for molecular analysis and produced good quality COI sequences. One new distinct lineage was detected from the project area using Maximum likelihood analysis (Figure 34). Draculoides `WAM-DRAC001` is 13% different to the next closest lineage (EU272697.1) and around 14.2% different from described species Draculoides julianneae (EU272696.1) and Draculoides bramstokeri (EU272684.1). The distribution of this lineage within the project site is depicted in Figure 29. Table 12. Summary of the Schizomida lineage detected within the Project Area. % Divergence from closest known lineage is shown with lineage name in parentheses. Family Genus Species/Lineage Specimen Divergence RTIO Hubbardiidae Draculoides WAM-DRAC001 3 13% (EU272697.1 P sp) RT43 MYRIAPODA CHILOPODA & DIPLOPODA Specimens of Chilopoda or Diplopoda collected either failed to sequence or were not sub-sampled for molecular analysis and were identified taxonomically by Bennelongia (Appendix I) PAUROPODA (TETRAMEROCERATA) A summary of Pauropoda molecular identifications is depicted in Table 13. In this study 10 specimens likely belonging to the genus Decapauropus (Pauropodidae) were extracted for molecular analysis, and eight produced a good quality sequences. Five lineages were detected with large genetic divergences between species, ranging from 12.4–26.6% genetic divergence between the next closest lineage (Figure 35; Appendix II). Very little molecular work has been undertaken for Pauropoda and there are no published papers on the molecular systematics of Pauropoda in Australia. GenBank sequences have been utilised from research undertaken by Qian et al. (unpublished), who examined Pauropoda from China and found deep barcode divergences between species. The distribution of this lineage within the project site is depicted in Figure 36. Figure 35. Maximum Likelihood of representative COI haplotypes of Pauropoda from this study and a sub-set of reference specimens from GenBank. Proposed lineages from this study is indicated by the shaded, coloured boxes. Report by Western Australian Museum (Molecular Systematics Unit) Table 13. Summary of Pauropoda lineages detected from the Project area. % Divergence from closest known lineage is shown with lineage name in parentheses. Family Genus Species/Lineage Specimen Divergence RTIO Pauropodidae Decapauropus WAM-PAUD001 1 17.4% (WAM-PAUD002) RT43 Pauropodidae Decapauropus WAM-PAUD002 4 17.4% (WAM-PAUD001) RT43 Pauropodidae Decapauropus WAM-PAUD003 1 26.6% (Decapauropus crassescens JN974348.1) RT43 Pauropodidae Decapauropus WAM-PAUD004 1 12.4% (WAM-PAUD005) RT43 Pauropodidae Decapauropus WAM-PAUD005 1 12.4% (WAM-PAUD004) RT43 Figure 36. Map of Pauropoda lineages (PAUROPODIDAE) detected within the project area. Report by Western Australian Museum (Molecular Systematics Unit) SYMPHYLA (CEPHALOSTIGMATA) A summary of the symphylan lineages detected from the project area is depicted in Table 14. In this study, seven specimens that were submitted for molecular analysis produced viable COI sequences after we optimised PCR protocols (unpublished; Figure 37). One additional specimen showed contamination and was eliminated from the analysis, one was not sub-sampled, and another was missing from the vial (WAMT146615). A distribution map of the lineages detected from the project is depicted in Figure 38, and images of WAMT144260 from Channar Mine is depicted in Figure 39. Table 14. Summary of Symphylan 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 Task Scutigerellidae Scutigerella `WAM-SCUTI002` 1 25.6% (WAM-SYMPH001, Robe River) RTIO035 Scutigerellidae Scutigerella `WAM-SCUTI003` 2 16.1% (WAM-SCUTI005) RTIO043 Scutigerellidae Scutigerella `WAM-SCUTI004` 1 18.5% (WAM-SCUTI005) RTIO043 Scutigerellidae Scutigerella `WAM-SCUTI005` 1 16.1% (WAM-SCUTI003) RTIO035 Scutigerellidae Symphyella `WAM-SYMPH002` 1 25.6% (WAM-SYMPH001, Robe River) RTIO046 It is clear from the analysis that these specimens represent five distinct lineages from two genera and differed from the closest lineage from between 16.1–25.6% (Figure 37; Appendix II). Two specimens of the lineage WAM-SCUTI003 (WAMT145461 & T145462) differed from each other by 5.3% and differed from the next closed lineage (WAM-SCUTI005) by 16.1%. The closest lineage to Symphyella WAM-SYMPH002 (WAM144298) was Symphyella WAM-SYMPH001 (WAMT145913) from the Robe River Valley Mesa C, and differed by 25.6% (Figure 37; Appendix II). Helix Molecular Solutions have also sequenced a number of symphylan specimens from the Robe River Valley and determined a number of distinct lineages (Unpublished). These lineages however cannot be compared to sequences produced from this molecular project as they used different primer pairs which do not overlap the Barcoding Gene region and were designed specifically for symphylans. Very little molecular and morphological research has been undertaken for symphylans, and therefore results from this phylogenetic analysis should be used with caution. Very little inference can be made to the higher level classifications or relationships within this group until further molecular and morphological work has been undertaken, within the Pilbara region of WA. Generic level classifications of the Symphyla are based on morphological identifications undertaken by Bennelongia, and are supported at some level with the molecular analysis. Symphyella Scutigerella Figure 37. Maximum Likelihood of all COI haplotypes of Symphylans (SCUTIGERELLIDAE) from the study area 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) Figure 38. Distribution map of the Symphylan lineages (SCUTIGERELLIDAE) detected within the project area. Figure 39. Automontage image a Symphylan lineage, `WAM-SCUTI002` (WAM T14460) collected from Channar Mine, approx. 14km SE Paraburdoo. Dorsal (top) and Lateral (Bottom) Report by Western Australian Museum (Molecular Systematics Unit) REFERENCES Bradbury, J.H. & Williams, W.D. 1996. Freshwater amphipods from Barrow Island, Western Australia. Records of the Australian Museum 48: 33-74. Bradbury, J.H. & Williams, W.D. 1997. The amphipod (Crustacea) stygofauna of Australia: description of new taxa (Melitidae, Neoniphargidae, Paramelitidae), and a synopsis of known species. Records of the Australian Museum 49 (3): 249-341. Brown, L., Finston, T., Humphreys, G., Eberhard, S & Pinder, A. 2015. Groundwater oligochaetes show complex genetic patterns of distribution in the Pilbara region of Western Australia. Invertebrate Systematics 29: 405-420. Castresana, J. 2000. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17, 540-552. Cooper, S.J.B., Bradbury, J.H., Saint, K.M., Remko Leys, S., Austin A.D. & Humphreys, W.F. 2007. Subterranean archipelago in the