A new species of Phryssonotus (Diplopoda: Synxenidae) from north-eastern New South Wales, Australia

Introduction

The family Synxenidae (Polyxenida) comprises two genera: Phryssonotus Scudder, established in 1885 (Scudder 1885), and Condexenus, described by Nguyen Duy-Jacquemin (2006). One species, Phryssonotus novaehollandiae, endemic to Australia, was first identified by Silvestri (1923) from a single juvenile specimen in the stadium V, featuring eight pairs of legs and 10 ommatidia, collected at Mount Lofty, South Australia. Diagnostic features for juvenile Phryssonotus, based on the number of ommatidia, were subsequently applied to adult specimens, leading to confusion in distinguishing between P. novaehollandiae and P. capensis, both of which possess 10 ommatidia. In 1984, this resemblance led Condé and Nguyen Duy-Jacquemin (1984) to mistakenly identify specimens from Australia and Papua New Guinea as P. capensis. Short and Huynh (2009) later confirmed that P. novaehollandiae was the only species present in Australia; this species possesses 11 ommatidia in the adult stage, which is clearly different from P. capensis. Despite this, Phryssonotus populations collected from different locations in Australia are biologically and morphologically distinct, displaying variations in sexual and asexual populations and in the patterns of tergal scale trichomes. This diversity led to the description of two distinct species, Phryssonotus australis and P. occidentalis, by Huynh and Veenstra (2017). Recently a new Phryssonotus species collected from north-eastern New South Wales exhibited distinct white colour patterns on its tergites, contrasting with the black colouration observed in its predecessors (Fig. 1). The combination of colour pattern characteristics and genetic evidence supports the description of Phryssonotus borealis, sp. nov. as a novel species within the family Synxenidae.

Fig. 1.

Image of live Phryssonotus borealis, sp. nov. showing a distinctive white medial band (T) on tergal scale trichomes and small lateral white stripes (S).

Materials and methods

Collection

Fifteen live Phryssonotus specimens, subadult stage with 14 pairs of legs, were collected from leaf litter at the base of a river red gum tree (Eucalyptus camaldulensis), approximately 100 m from the entrance of the Manilla Rivergum Caravan Park (30.743830S, 150.729166E; elevation 361 m) in Manilla, New South Wales, Australia, on 20 October 2022, by author Cuong Huynh (Fig. 2). The live specimens were subsequently reared to adult – 17 pairs of legs stage – to observe the dorsal arrangement patterns and colour variations of their tergal scale trichomes. Additionally, moulted exoskeletons were collected and used for detailed scale trichome measurements.

Fig. 2.

Phryssonotus specimen collection sites (▲) in the State of New South Wales, the location within Australia indicated on the inserted map (map source: d-maps.com).

Morphological study

Morphological characteristics of Phryssonotus specimens were examined following the methods of Huynh and Veenstra (2017). The sex of adult specimens (17 pairs of legs) was determined by the presence of reproductive organs on the second coxal plates: vulva in females and penes in males (Fig. 3a, b). Body length (mm) was measured from the vertex of the head to the tip of the telson. The tergal scale trichomes were measured for length-to-width ratios (μm), and their patterns and colour were recorded photographically (Figs 1 and 5).

Fig. 3.

The second legs of Phryssonotus species showing the sex organs. (a) Male sex organ – a short and pointed end penis; (b) female sex organ – a long and rounded end vulva.

The taxonomic classification of the species was based on the presence of a long frontal trichome A and the short frontal trichome B sockets next to the sensory organs – trichobothria located near the eye region of the head (Fig. 4), following the classification criteria of Short and Huynh (2009).

Fig. 4.

Scanning electron microscope image of a Phryssonotus borealis, sp. nov, head capsule showing nine ommatidia (*), two not being visible from this angle; sensory organ – trichobothria (a, b and c), a long frontal trichome A socket and positions of four short frontal trichome B sockets (B1–B4).

Results

Both the live Phryssonotus specimens and those from the museum consisted exclusively of females, suggesting that this population is parthenogenetic in this geographic region. Adult Phryssonotus specimens showed diagnostic morphological features, including 11 ommatidia, a sensory organ – a long frontal trichome A, and short frontal trichomes B (B1–B5) are adjacent to trichobothrium c(Fig. 4). The characteristics of these structures align with taxonomically important morphological features previously identified in Australian Phryssonotus species, consistent with findings of Short and Huynh (2009). The body length of live specimens ranged from 3.2 to 3.4 mm (n = 14), closely matching the size of Phryssonotus occidentalis (3.4 mm) from western Australia but slightly shorter than P. australis (range: 3.6–3.8 mm), parthenogenetic populations, from Victoria and Tasmania, and P. novaehollandiae (3.8 mm) from Victoria and South Australia (Huynh and Veenstra 2017). Additionally, adult Phryssonotus specimens showed scale trichome patterns differing from those of P. australis, P. novaehollandiae and P. occidentalis, with distinctive tergal scale trichomes forming a white medial trapezoid band, with small white stripes laterally on the body (Fig. 5).

Fig. 5.

Variation in scale trichome patterns on live specimens and illustrations of tergal scale trichome patterns seen in Phryssonotus species in Australia. (a) P. borealis, sp. nov., (b) P. australis, (c) P. novaehollandiae, (d) P. occidentalis.

A comparison of scale trichome length-to-width ratios in this new Phryssonotus species gave a ratio of 1:60 (n = 100 scale trichomes), which resembles that of P. occidentalis (1:60) but is smaller than those of P. australis (1:64) and P. novaehollandiae (1:75) (Huynh and Veenstra 2017).

The 18S maximum-likelihood phylogenetic tree, constructed using 1000 bootstrap replications, yielded a strongly supported clade comprising four Phryssonotus species, confirming their classification within the same genus (Fig. 6a). A similar analysis of the CO1 region also supported this grouping with high bootstrap values, linking Phryssonotus borealis, sp. nov. to sequences from three Phryssonotus species in the same clade (Fig. 6b). Pairwise genetic distances based on the 18S region showed minimal divergence for P. borealis, sp. nov. relative to P. novaehollandiae (0.19%), P. australis (0.23%), and P. occidentalis (0.23%). In contrast, CO1 sequences revealed significantly greater divergence: P. borealis, sp. nov. differed from P. novaehollandiae (11%), P. australis (11%), and P. occidentalis (10%) (Table 3).

Fig. 6.

The 18S (a) and CO1 (b) bootstrapping maximum-likelihood trees of Phryssonotus borealis, sp. nov. were generated with 1000 bootstrap replications. Support values >50% are shown on the nodes.

Taxonomy

Discussion

The Australian Phryssonotus species show high speciation and diversity. Despite their similarities in taxonomic characteristics, these species differ significantly in both morphology and genetic makeup. Notably, the variation in tergal scale trichome colouration serves as a key distinguishing feature. Most previously described Phryssonotus species are found in South Australia, Victoria and Western Australia, these regions collectively being considered part of southern Australia. The species have black tergal scale trichomes that stand out against the white background of the sides of the body (Fig. 5). In contrast, Phryssonotus borealis has scale trichomes forming a white medial trapezoid band and small lateral strips, forming the white pattern against the black background of its body. This species is found in north-eastern regions of New South Wales, extending to the border of Queensland. Because of its tergal trapezoid-shaped patterns it has a similar appearance to P. australis, a southern species primarily found in coastal habitats in Victoria and northern Tasmania. However, they differ in colouration and in the length–width ratios of the scale trichomes. Phryssonotus borealis is slightly shorter in body length and has smaller-scale trichome ratios than P. australis, and its measurements are similar to those of P. occidentalis from Western Australia. Notably, the length–width ratios of the scale trichomes among these Phryssonotus species are not a reliable characteristic to differentiate among these species, as body length of the species significantly influences the outcome of the scale trichome ratio. Instead, the colouration of the tergal scale trichomes serves as a distinct morphological character that differentiates among these Phryssonotus species.

Most Phryssonotus populations consist of both males and females. However, there is an exception in reproduction, as seen in the case of Phryssonotus australis, which reproduces through parthenogenesis. P. borealis is another example of an asexual population found in Australia. Both these species are parthenogenetic, consisting of a female-only population. Parthenogenesis is an effective survival strategy, allowing these species to adapt and maintain population stability in geographic isolation where habitats are favourable despite the absence of gene flow. This reproductive mode enables them to occupy diverse habitats across vast areas of Australia.

In this study, Phryssonotus species showed genetic differences. Phylogenetic trees indicated that P. borealis belongs to the genus Phryssonotus based on 18S analysis (Fig. 6a), and CO1 gene analysis (Fig. 6b) indicated significant genetic divergence from other Phryssonotus species. The genetic distance of P. borealis compared to its closest relatives (Table 3) exceeds the accepted species level in millipede taxonomy and validates its distinctiveness.

In conclusion, a combination of taxonomic and genetic studies confirmed that Phryssonotus borealis is a newly identified species. Its morphological and genetic differences from related species demonstrated ongoing speciation and adaptation within the genus Phryssonotus in Australia.