Description of five new Aname L. Koch, 1873 (Araneae, Anamidae) species collected on Bush Blitz expeditions

. Aname L. Koch, 1873 is an incredibly diverse genus of mygalomorph spiders endemic to Australia, occurring from coast to coast in tropical, semi-arid and arid bioregions. They are relatively gracile mygalomorph spiders that build open burrows, sometimes with a secondary entrance that functions as an escape chute. The genus currently contains 48 species, but the true diversity is likely to be closer to 200 species. Here we describe five new species ( A. ningaloo sp. nov., A. salina sp. nov., A. tatarnici sp. nov., A. tenuipes sp. nov. and A. wongalara sp. nov.), primarily based on specimens collected on Bush Blitz expeditions in Western Australia (Cape Range, 2019), South Australia (Great Victoria Desert, 2017) and the Northern Territory (Wongalara, 2012). We complement these descriptions with a molecular phylogenetic analysis to place all new species into an existing phylogenetic framework.


Introduction
Aname L. Koch, 1873 is an incredibly diverse mygalomorph spider genus endemic to Australia, occurring from coast to coast in tropical, semi-arid and arid bioregions. Spiders in the genus are quite gracile and

Morphology
Specimens examined in this study are lodged in the Western Australian Museum, Perth (WAM), the South Australian Museum, Adelaide (SAM), and the Museum and Art Gallery of the Northern Territory (MAGNT). Specimens were preserved in 75% ethanol, although one leg (usually left leg III)  Koch, 1873 was often removed and stored in 100% ethanol at -70°C for genomic DNA extraction. Digital automontage images were taken using a Leica M165C stereo microscope with mounted DFC425 digital camera and processed using Leica Application Suite ver. 3.7 software (Leica, Wetzlar, Germany). The left leg I and pedipalp were dissected from specimens for photography, except when the left appendage was damaged or lost, in which case the right appendage was used and the image reflected in the taxonomic plates for easy comparison. The right bulb (copulatory organ) of males was also dissected from the specimen for the two images of this structure. Plate structure is modified slightly from Harvey et al. (2022). Pedipalp and leg measurements and ratios were calculated using the terminology and reference points defined by Castalanelli et al. (2020). Species are treated in alphabetical order. When listing material, coordinates are rounded to the nearest minute, to prevent providing exact coordinates that could be used for poaching. The following abbreviations are used: D = dorsal; MID = metatarsus I depth (width); MIL = metatarusus I length; MIPEL = metatarsus I proximal excavation length; PDL = pedipalpal depression length; PL = prolateral; PTL = pedipalpal tibia length; RL = retrolateral; TID = tibia I depth (width); TIL = tibia I length; TIS = tibia I length to spur; TISH = tibia I spur height; V = ventral.

Molecular phylogenetics
The sequencing dataset used in this study consists of the complete multi-locus dataset published previously for Aname (see Rix et al. 2021), augmented with new sequences for some of the species described herein. Markers include three mitochondrial and four nuclear loci: cytochrome c oxidase subunit I (COI), 12S ribosomal RNA (12S), 16S ribosomal RNA (16S), histone H3 (H3), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S), and elongation factor 1-gamma (EF-1γ). New sequences (labeled in Supplementary File 1) were generated using primers and PCR conditions detailed in previous published studies of Anamidae by the authors (Harvey et al. 2018), in the WAM Molecular Systematics Unit. Editing of sequence chromatograms, alignment and concatenation were performed using the Geneious software package (Biomatters Ltd). Alignment was performed using the MAFFT ver. 1.3.6 plug-in in Geneious (Katoh et al. 2002), and all sequence data are deposited in GenBank (see Supplementary File 1). The full concatenated dataset is 5067 bp and includes sequence data for 190 specimens including 24 outgroup taxa and 166 specimens of Aname. A phylogenetic analysis of this expanded dataset was conducted using maximum likelihood (ML), in the WIQ-TREE online interface (Nguyen et al. 2015;Trifinopoulos et al. 2016). ModelFinder (Kalyaanamoorthy et al. 2017) was used to choose models of DNA evolution, and the alignment was partitioned by locus, and by codon for coding genes (COI, H3, EF-1γ). To assess node support, 1000 ultrafast bootstrap replicates (Minh et al. 2013)

Phylogenetic analysis
The best log-likelihood score recovered in the ML analysis was -72612.334, and the recovered topology generally agreed with that recovered previously, in Rix et al. (2021), with some minor differences in relationships between lineages at the base of the tree (Fig. 1). However, all major clades were recovered as they were in the original analysis. The five species described in this publication (see below) were recovered as separate species-level lineages, and belong to disparate evolutionary groups covering the breadth of the

Etymology
The specific epithet is a noun in apposition in reference to the type locality of this species within the Ningaloo Coast World Heritage Area, Western Australia.

Remarks
This species was previously known under the code name Aname 'MYG728'. It is a member of the mellosa group (Clade 8) sensu Rix et al. (2021) (Fig. 1).

Distribution and natural history
Aname ningaloo sp. nov. is only known from the Cape Range, with the habitat in this area dominated by spinifex grasslands on limestone hills, and coastal sandplains (Fig. 2).

Etymology
The species epithet refers to the coastal distribution of this species ('salina', Latin, for 'salt', 'salty') (Brown 1956

Remarks
This species was previously recognised under two different code names -Aname 'MYG627' for a female specimen from Exmouth, and 'MYG712' for male specimens from Ashburton salt (not included in previous phylogenies). It is a member of the mainae group (Clade 7) sensu Rix et al. (2021) (Fig. 1).

Distribution and natural history
Two reciprocally monophyletic populations of Aname salina sp. nov. are currently known (Fig. 2). One of these is around the town of Onslow (most specimens are from the west of the town, near the Ashburton River), the other is in Cape Range National Park. Although these were recovered as monophyletic groups (Fig. 1), average pairwise divergence in COI between them is just 5.15% (low for COI in mygalomorph spiders, see Castalanelli et al. 2014) and they show no evidence of morphological divergence based on the samples available to us. The habitat in the regions where this species occurs is a mix of bare sand and salt flats, tussock, and hummock (spinifex) grasslands, and coastal habitats. Based on when adult males of this species have been collected, they appear to mature in Autumn (March/April).  Colour (in alcohol) (Fig. 6). Carapace chocolate-brown; chelicerae slightly darker red-brown; legs and ventral prosoma lighter, orange-brown; dorsal abdomen grey-brown, ventral and lateral abdomen pale.

Remarks
This species was included in recent molecular phylogenies of the Anamidae under the code name Aname 'MYG644'. It is an unplaced member of the Temperate-Eastern Radiation sensu Rix et al. (2021), recovered here as the sister-species to the pallida group (Clade 3) (Fig. 1).

Distribution and natural history
The holotype male of this species was collected on the eastern side of the Great Victoria Desert, in central South Australia, the habitat in this region is a mix of mallee, Acacia and Casuarina woodlands. The distribution of the species around this location is currently unknown.

Diagnosis
Males of Aname tenuipes sp. nov. can be readily distinguished from thos of all other described species by their metatarsus I, which possesses no conspicuous proximal excavation, and is instead elongate and cylindrical along its entire length (Fig. 7Q). They can further be distinguished by their unique embolus, which is very short (about 0.8 × bulb length), and curved, with a basal flange or embolic apophysis ( Fig. 7K-M).

Etymology
The specific epithet is an adjective meaning 'thin-footed', formed from the Latin 'tenuis', meaning 'thin', and 'pes' meaning 'foot' (Brown 1956). This is in reference to the elongate, cylindrical metatarsus I of this species, which lacks a proximal excavation -a distinguishing feature of this species.

Remarks
This species was included in recent molecular phylogenies of the Anamidae under the code name Aname 'MYG223'. It is a member of the pulchella group (Clade 5) sensu Rix et al. (2021) (Fig. 1).

Distribution and natural history
Currently, specimens of Aname tenuipes sp. nov. are known from two quite disjunct areas, the first is around the type locality in Dundas Nature Reserve and areas relatively close by such as Lake Lefroy, in central-southern Western Australia. The second is in or near the Great Victoria Desert, inland, around the border of Western Australia and South Australia. The species is presumably also found in the region between these areas. The habitat in these areas includes eucalypt, Acacia and Casuarina woodlands and mallee. Based on when adult males of this species have been collected, they appear to mature in Spring (September-November).  Raven, 1985 andA. distincta (Rainbow, 1914)

Etymology
The specific epithet is a noun in apposition in reference to the Wongalara Wildlife Sanctuary, a conservation estate owned and managed by the Australian Wildlife Conservancy (AWC), where the holotype of this species was collected.

Remarks
This species was included in recent molecular phylogenies of the Anamidae under the code name Aname 'MYG261'. It is an unplaced member of the Tropical Radiation sensu Rix et al. (2021), recovered here as the sister-species to 'MYG443' (Fig. 1).

Distribution and natural history
Aname wongalara sp. nov. is only known from a single specimen, collected in Wongalara Wildlife Sanctuary. The habitat in the area is tropical eucalypt and Acacia woodland and grasslands.

Discussion
With this and other recent descriptions (Harvey et al. 2022), described species in the genus Aname now totals 53, and species have been described from all eight species groups identified in Rix et al. 2021. As new species are being described and added to the phylogenetic foundation, morphological characteristics of each of these groups may be identified that allow their diagnoses. For example, six species have now been described from the mainae group: A. lorica, A. aragog, A. exulans, A. mainae, A. mcleeryorum, and A. salinas sp. nov. and these have led to the recognition of the distinctive metatarsus shape, abdominal pattern and elongate sternal sigilla of that group. Other new species have extended the known variability encompassed by the genus, namely A. tenuipes sp. nov. with its cylindrical metatarsus, lacking the proximal excavation which is normally distinct and characteristic of the genus.
Two species described in this study, Aname ningaloo sp. nov. and A. salina sp. nov., appear to occur sympatrically in Cape Range National Park, Western Australia. As has become a recognised pattern in the Australian mygalomorph fauna (see Wilson et al. 2018;Rix et al. 2020Rix et al. , 2021, congeneric species in WILSON J.D. et al. Five new species of Aname L. Koch, 1873 sympatry are from distantly related radiations, which in this case diverged from one another about eight million years ago (Harvey et al. 2018;Rix et al. 2021).