A new species of Mouse Spider ( Actinopodidae , Missulena ) from the Goldfields region of Western Australia

A new species of Mouse Spider (family Actinopodidae Simon, 1892), Missulena harewoodi, is described from near the City of Kalgoorlie-Boulder in the Goldfields region of Western Australia. It differs from all other Missulena species by the unusual light grey colouration of the abdomen in combination with small body size and shiny carapace. A phylogenetic analysis of a fragment (658 bp) of the COI barcoding gene places M. harewoodi sp. n. in a clade with four Missulena species from the Pilbara region of Western Australia, more than 900 km away. Missulena harewoodi sp. n. is one of the many species in this genus that are currently only known from a single, or a very limited number of specimen, highlighting the paucity of fauna collections in many arid regions of Australia and the difficulties in sampling these cryptic spiders.


Introduction
The spider family Actinopodidae Simon, 1892 has a Gondwanan distribution with species found in Australia and South America (World Spider Catalog 2017).In Australia, these spiders are commonly called Mouse Spiders and widely known because they can be quite large (up to 3-4 cm in body size) and superficially resemble the venomous Funnel-web Spiders (family Hexathelidae Simon, 1892).Some species may have a potent venom although spiders appear to generally apply a dry bite in defense (e.g.Isbister 2004;Isbister and Gray 2004;Rash et al. 2000).The colour patterns in male Missulena belong to the most spectacular amongst all mygalomorph spiders.Most striking are the sometimes bright red fangs and/or carapace in males, iridescent blue abdomens, or an abdomen with light colour patterns (Framenau et al. 2014).Vivid colouration with typical warning colours such as red possibly relates to the diurnal activity of mate-searching Missulena males, whereas the majority of males in other mygalomorph families are strictly nocturnal (Harms and Framenau 2013).
The Australian fauna in this family currently comprises 16 species in the single genus Missulena Walckenaer, 1805 (Miglio et al. 2014;World Spider Catalog 2017).However, species-level diversity in this genus is clearly much higher, with dozens of undescribed species identified in recent barcoding analyses (e.g.Castalanelli et al. 2014).The fauna of Western Australia, where 14 of the 16 described species occur (Miglio et al. 2014), is particularly diverse.Recent barcoding projects focusing on the Pilbara region in the northwest of the state recovered 13 distinct genetic clades, only five representing described species (Castalanelli et al. 2014).The two most recent taxonomic studies (Harms andFramenau 2013, Miglio et al. 2014) on Missulena developed a protocol for the description of spe-cies after several decades of taxonomic neglect and added six new species to the Australian fauna.They also provided an updated key to the males of all Australian species (Harms and Framenau 2013;Miglio et al. 2014).
Recent collections as part of environmental assessment studies in the semi-arid Goldfields region of Western Australia recovered a small male of Missulena with the feature of light grey abdomen both as live (VWF pers.obs.) and preserved (Fig. 1A) specimen, another facet of the striking colour variations present within this genus.Light abdomen discolourations have previously only been described from two larger species with very different distribution in Australia (M. pruinosa Levitt-Gregg, 1966 from tropical northern Australia and M. bradleyi Rainbow, 1914 from the subtropical/temperate eastern parts of the country), but the current species is smaller and the pattern is more pronounced.
In this paper, we describe this unusual species of Missulena based on male morphology.A phylogenetic analysis of a fragment of the CO1 barcoding gene is used to explore the systematic position of this species and consolidate its taxonomic concept developed based on morphology.In documenting this species, we hope to raise further awareness for the Australian Mouse Spiders by documenting another morphological rarity in its diverse fauna.
Molecular analyses.Sequencing of the mitochondrial CO1 gene of the holotype followed the protocol described in a previous study (Harms and Framenau 2013).Phylogenetic analyses were undertaken on an alignment comprising all 84 Missulena sequences from previous studies and available on GenBank (Castalanelli et al. 2014;Harms and Framenau 2013), four specimens that were sampled in Western Australia since then and the holotype of M. harewoodi sp.n. (Table 1).COI sequences of a single specimen of Euagrus chisoseus Gertsch, 1939 (Dipluridae Simon, 1889) and five unidentified specimens of Conothele Thorell, 1878 (family Ctenizidae Thorell, 1887) were extracted from GenBank and included in the analyses as outgroups.A barcoding threshold of 9.5% pairwise sequence divergence was used for species delineation at a molecular level (Castalanelli et al. 2014).Phylogenetic analyses were executed using MrBayes Version 3.2.1 for Macintosh (Ronquist et al. 2012), with the GTR+I+G model of nucleotide substitution suggested by MrModeltest Version 3.7 (Posada and Crandall 1998) for an unpartitioned dataset.Four Markov chain Monte Carlo (MCMC) chains were run for 40,000,000 generations, sampling every 1,000 generations and discarding the first of 25% of sampled trees as 'burnin'.FigTree version 1.4.3 (Rambaut 2016) was used to visualise and edit the tree.pitfall trap, Goldfields Blackbutt low woodland over open scrub on loam (WAM T142820).

Systematics
Etymology.The specific epithet is a patronym in honour of Greg Harewood, the collector of the type specimen.
Diagnosis.The colouration of the holotype of M. harewoodi sp.n. is most similar to M. pruinosa due to the light dorsal discolouration of the abdomen, but the species differs in the lower number of spines of the rastellum (three vs ten), smaller size (male body length 8.0 mm vs 12.5 mm) (measurements from Faulder 1995a) and narrower pedipalp tibia.Male Missulena bradleyi also have a light pattern on the dorsal side of the abdomen, but it is restricted to an anterior light blue patch and the species is also larger (male body length 8.0 mm vs 10.9 mm) (measurements from Faulder 1995a).Otherwise, somatic morphology most closely resembles three species with a brown carapace, M. melissae Miglio, Harms, Framenau & Harvey, 2014, M. faulderi Harms & Framenau, 2013and M. rutraspina Faulder, 1995, but M. harewoodi sp. n. differs considerably in the colour pattern of the abdomen of both live and preserved specimens (brown in M. melissae, grey-brown in M. faulderi and blue-grey in M. rutraspina) and the much smoother carapace (Faulder 1995b;Harms and Framenau 2013;Miglio et al. 2014).
Sternum: 2.17 long, 2.00 wide; pear-shaped and rebordered (Fig. 1E), with dark setae of varying length, arranged irregularly but denser laterally and towards labium; 4 pairs of sigilla located more than three times their length from the border of the sternum, anterior and second pair (anterior-posterior) smallest and poorly defined, third pair bigger than 2 anterior pairs and poorly defined; posterior pair biggest, roughly oval and well defined, 3 posterior sigilla slightly depressed.
Phylogenetic analyses.Our phylogenetic analyses places M. harewoodi sp.n. as sister taxon to an undescribed Missulena species from the Pilbara region in Western Australia, Missulena 'DNA02' (WAM T124777) (Fig. 3; Table 1), although this relationship is not well supported.Sequence divergence between both specimens is 14.5%, which is considerably larger than the current operational sequence divergence of 9.5%, employed to differentiate species in the Actinopodidae (Castalanelli et al. 2014).Morphological comparison between both species is not possible as the Pilbara species is represented by a single juvenile specimen.Missulena harewoodi sp.n. from the Goldfields nests within a clade of three putative new species, Missulena 'DNA02', 'DNA03' and 'MYG290' and Missulena faulderi, that are all from the Pilbara bioregion of Western Australia, situated more than 900 km north-west of the City of Kalgoorlie-Boulder (Fig. 3).The Pilbara is geologically, floristically and climatically distinct from the Goldfields and it is unclear whether the results of our analyses are caused by taxon bias in the set of DNA sequences or reflect a true biogeographic pattern.Only more comprehensive analyses with additional representatives from southern and central Western Australia and including additional genes can clarify the phylogeographic patterns in Missulena.
Distribution.Missulena harewoodi sp.n. is currently only known known from the type locality, ca.20 km East of the City of Kalgoorlie-Boulder in the Goldfields region of Western Australia (Fig. 4).
Habitat.The type specimen was collected alive in a 10-litre, dry bucket pitfall trap targeting vertebrates.The collecting site is described as Goldfields Blackbutt (Eucalyptus lesouefii) low woodland over open scrub on loamy soil (G.Harewood, personal communication to VWF).Similar to many other Missulena species, M. harewoodi sp.n. appears to mature in autumn (collected in April), contradicting the assumption that many mygalomorph spiders in arid and semi-arid Australia reproduce in the months with highest rainfall (e.g.January/February in the Goldfields near Kalgoorlie) (BoM 2017; EPA 2016).
Remarks.Missulena harewoodi sp.n. is the seventeenth named species of this genus in Australia and within a radi-  1).Small black dots represent all Missulena records from Western Australia (as downloaded from the Atlas of Living Australia -http://www.ala.org.au;accessed 10 April 2017).
us of about at least 100 km of its type locality, the only described species in the genus with the exception of the widespread M. occatoria (Walckenaer, 1805) (based on data of the Atlas of Living Australia; http://ala.org.au;accessed 10 April 2017).The species is yet another example of the extremely diverse invertebrate fauna of the semi-arid Goldfields region of Western Australia that is currently poorly studied in relationship to its invertebrate fauna when compared to other bioregions in Western Australia, e.g. the Pilbara (Durrant et al. 2010;McKenzie et al. 2009;Volschenk et al. 2010).Whilst comprehensive biological studies have been conducted in the Goldfields more than two decades ago, these rarely considered invertebrates (Biological Surveys Committee 1984; Keighery et al. 1995).Recent studies on terrestrial snails in the genus Bothriembryon Pilsbry, 1894 (Breure and Whisson 2012), millipedes in the genus Antichiropus Attems, 1911 (Car andHarvey 2013, 2014) and trapdoor spiders of the family Idiopidae Simon, 1892 (Rix et al. 2017) have highlighted both extreme diversity and endemism in invertebrates within the vast woodlands of the Goldfields and numerous unpublished reports as part of environmental impact assessments have supported these general findings for other taxonomic groups.The fact that M. harewoodi sp.n. has never been sampled before near the regional centre of Kalgoorlie-Boulder and remains only known from a single specimen that was collected as by-catch during a vertebrate trapping survey, highlights the need for a comprehensive invertebrate fauna survey of this region that includes both its woodlands and isolated ranges of banded ironstone.

Figure 3 .
Figure 3. Topology of Missulena species based on COI (fragment of 658 bp).Nodal support is indicated in squares (posterior probabilities).Branches of more of three specimens of the same species are collapsed.Morphotype designations of undescribed species reflects those databased at the Western Australian Museum.See Castalanelli et al. (2013) for details on the 'MYG'-coding system and museum registration numbers.

Figure 4 .
Figure 4. Distribution records of specimens sequenced at the COI gene fragment for this study (see Table1).Small black dots represent all Missulena records from Western Australia (as downloaded from the Atlas of Living Australia -http://www.ala.org.au;accessed 10 April 2017).

Table 1 .
Missulena specimen sequenced for this study.