Research Article
Research Article
Phylogenetic analysis of the Australian trans-Bass Strait millipede genus Pogonosternum (Carl, 1912) (Diplopoda, Polydesmida, Paradoxosomatidae) indicates multiple glacial refugia in southeastern Australia
expand article infoPeter Decker
‡ Senckenberg Museum für Naturkunde Görlitz, Görlitz, Germany
Open Access


This study documents the first detailed phylogenetic analysis of an Australian paradoxosomatid millipede genus. Two mitochondrial genes (partial COI and 16S) as well as partial nuclear 28S rDNA were amplified and sequenced for 41 individuals of the southeastern Australian genus Pogonosternum Jeekel, 1965. The analysis indicates that five species groups of Pogonosternum occur across New South Wales, Victoria and Tasmania: P. nigrovirgatum (Carl, 1912), P. adrianae Jeekel, 1982, P. laetificum Jeekel, 1982 and two undescribed species. P. coniferum (Jeekel, 1965) specimens cluster within P. nigrovirgatum. Most of these five species groups exhibit a pattern of high intraspecific genetic variability and highly localized haplotypes, suggesting that they were confined to multiple Pleistocene refugia on the southeastern Australian mainland. The phylogenetic data also show that northwestern Tasmania was colonized by P. nigrovirgatum, probably from central Victoria, and northeastern Tasmania by an as yet undescribed species from eastern Victoria.


Invertebrate, COI, 16S, 28S, genetic variability


Pogonosternum Jeekel, 1965 is the most widespread and species-rich genus of the millipede tribe Antichiropodini Brölemann, 1916 in Victoria, with the five described species Pogonosternum nigrovirgatum (Carl, 1902), P. coniferum Jeekel, 1965, P. adrianae Jeekel, 1982, P. laetificum Jeekel, 1982 and the subspecies P. nigrovirgatum infuscum Jeekel, 1982, all hitherto recorded from Victoria only. However, Jeekel (1982) and Mesibov and Churchill (2003) have recorded undescribed Pogonosternum species from Tasmania, and Car (2010) listed two undescribed Pogonosternum species from New South Wales.

Thus, Pogonosternum occurs on both sides of Bass Strait, which separates mainland Australia from Tasmania. The paradoxosomatid genus Somethus Chamberlin, 1920 also has a trans-Bass Strait distribution (Jeekel 2006), as do the paradoxosomatid species Dicranogonus pix Jeekel, 1982 and Notodesmus scotius Chamberlin, 1920 (Mesibov 2014).

Many soil invertebrates, including millipedes, have limited active dispersal capabilities. Phylogenetic studies of southeastern Australian soil invertebrates can give important insights into the impact of glacial periods during the Pleistocene (Byrne 2008, Endo et al. 2014, Garrick et al. 2004, Schultz et al. 2009, Sunnucks et al. 2006) and assist in identifying biogeographic barriers (Chapple et al. 2011). Unfortunately, phylogenetic studies of Australian millipedes are rare and restricted to a few taxa from a small number of localities (Adams and Humphreys 1993, Nistelberger et al. 2014, Wojcieszek and Simmons 2012). For the australiosomatine species Orocladosoma kosciuskovagum (Brölemann, 1913) from the Australian Alps a hypothesis of multiple glacial refugia has been proposed (Endo et al. 2014) to explain the results of such studies. Similarly, the australiosomatine genus Somethus in South Australia was found to have high morphological and genetic variability within species was discovered: it seems probable that isolation in multiple glacial refugia during the Pleistocene was the evolutionary driving force for this variability (Decker 2016).

The present study documents a molecular phylogenetic analysis of the antichiropodine genus Pogonosternum, using specimens from across the genus range, and with molecular evidence indicating past isolation in multiple Pleistocene refugia. Finally, the identity and origin of Tasmanian Pogonosternum populations are clarified.

Material and methods

Specimen collecting and preservation

Pogonosternum specimens were collected by hand in Victoria and New South Wales in August 2014 by the author, Karin Voigtländer and Robert Mesibov, and by Mesibov in Tasmania in May 2014 and May 2015 (Fig. 1). Most sites were searched for 1-5 hours with the aim of finding 1-3 adult males. At only a few localities were Pogonosternumfound to be abundant. Specimens were killed and stored in 95% ethanol, with a change of ethanol after 1–2 months. Full details of locality, date, collector, collection number and coordinates (WGS84 decimal degrees) are provided in Suppl. material 1.

Figure 1. 

Map of Southeast Australia showing the distribution of Pogonosternum sampling sites with site numbers (see Table 1 and Suppl. material 1 for further details). P. adrianae (light green), P. laetificum (green), P. nigrovirgatum s. l./coniferum (yellow), P. sp. A (red), P. sp. B (blue).

Table 1.

Site numbers, localities, GenBank accession numbers and repository accession numbers for all specimens analyzed. (See also Fig. 1) NMV = Museum Victoria, Melbourne, Victoria, Australia; QVMAG = Queen Victoria Museum and Art Gallery, Launceston, Tasmania, Australia; SAM = South Australian Museum, Adelaide, Australia; SMNG = Senckenberg Museum of Natural History Görlitz, Görlitz, Germany; NSW = New South Wales; SA = South Australia; TAS = Tasmania; VIC = Victoria. See Suppl. material 1 for further details.

Species Site No. Locality GenBank Acc. No. COI GenBank Acc. No. 16S GenBank Acc. No. 28S Voucher
Somethus scopiferus Jeekel, 2002 SA, Martin Washpool Conservation Park KT948674 KU833272 SMNG VNR016931
Somethus castaneus (Attems, 1944) SA, Adelaide, Upper Sturt KT964477 SAM OM2135
Archicladosoma magnum Jeekel, 1984 VIC, N Rawson KT948681 KU833273 SMNG VNR016994
Pogonosternum adrianae S58 VIC, S Dargo KU745235 KU745194 KU745185 NMV K-12203
Pogonosternum adrianae S59 VIC, W Balook KU745236 KU745195 NMV K-12204
Pogonosternum adrianae S62 VIC, NE Moe KU745237 KU745196 KU745186 NMV K-12207
Pogonosternum coniferum S67 VIC, Langwarrin KU745238 KU745197 NMV K-12212
Pogonosternum coniferum S71 VIC, NE Cape Schanck KU745239 KU745198 NMV K-12213
Pogonosternum laetificum S2 VIC, NE Tyaak KU745240 KU745199 NMV K-12095
Pogonosternum laetificum S5 VIC, SE Glenburn KU745241 KU745200 NMV K-12096
Pogonosternum laetificum S7 VIC, E Toolangi KU745242 KU745201 NMV K-12101
Pogonosternum laetificum S9 VIC, SE Healesville KU745243 KU745202 NMV K-12102
Pogonosternum laetificum S14 VIC, SE Narbethong KU745244 KU745203 KU745187 SMNG VNR016987
Pogonosternum laetificum S15 VIC, E Narbethong KU745245 KU745204 SMNG VNR016988
Pogonosternum laetificum S17 VIC, N Marysville KU745246 KU745205 NMV K-12109
Pogonosternum laetificum S18 VIC, S Eildon KU745247 KU745206 NMV K-12110
Pogonosternum laetificum S19 VIC, W Barjarg KU745248 KU745207 NMV K-12176
Pogonosternum laetificum S88 VIC, Mt Macedon KU745249 KU745208 NMV K-13113
Pogonosternum nigrovirgatum S60 VIC, SE Traralgon South KU745250 KU745209 KU745188 NMV K-12205
Pogonosternum nigrovirgatum S63 VIC, SW Trafalgar KU745251 KU745210 NMV K-12208
Pogonosternum nigrovirgatum S64 VIC, W Nyora KU745252 KU745211 SMNG VNR016989
Pogonosternum nigrovirgatum S65 VIC, SE The Gurdies KT948680 KU745212 KT964478 NMV K-12211
Pogonosternum cf. nigrovirgatum S77 VIC, NW Lorne KU745253 KU745213 SMNG VNR016990
Pogonosternum cf. nigrovirgatum S78 VIC, W Kennett River KU745254 KU745214 NMV K-13114
Pogonosternum cf. nigrovirgatum S81 VIC, N Apollo Bay KU745255 KU745215 KU745189 NMV K-13115
Pogonosternum cf. nigrovirgatum S83 VIC, SW Staughton Vale KU745256 KU745216 SMNG VNR016991
Pogonosternum nigrovirgatum S87 VIC, W Gisborne KU745257 KU745217 NMV K-13116
Pogonosternum cf. nigrovirgatum X2 TAS, S West Montagu KU745258 KU745218 QVMAG:2015:23:1
Pogonosternum sp. A S21 VIC, N Glenrowan KU745259 KU745219 NMV K-12177
Pogonosternum sp. A S22 VIC, NE Thoona I KU745260 KU745220 NMV K-12178
Pogonosternum sp. A S23 VIC, NE Thoona II KU745261 KU745221 NMV K-12179
Pogonosternum sp. A S24 VIC, SE Chiltern KU745262 KU745222 SMNG VNR016992
Pogonosternum sp. A S25 VIC, SSW Chiltern KU745263 KU745223 KU745190 NMV K-12181
Pogonosternum sp. A S31 NSW, E Talbingo I KU745264 KU745224 NMV K-12187
Pogonosternum sp. A S32 NSW, E Talbingo II KU745265 KU745225 NMV K-12188
Pogonosternum sp. A S42 VIC, NNW Bemm River KU745266 KU745226 NMV K-12192
Pogonosternum sp. A S47 VIC, E Orbost KU745267 KU745227 NMV K-12195
Pogonosternum sp. A S49 VIC, Buchan KU745268 KU745228 NMV K-12197
Pogonosternum sp. A S52 VIC, SW Nowa Nowa KU745269 KU745229 NMV K-12199
Pogonosternum sp. A X1 TAS, W Tomahawk KU745270 KU745230 KU745191 SMNG VNR016986
Pogonosternum sp. B S26 NSW, SE Holbrook KU745271 KU745231 NMV K-12182
Pogonosternum sp. B S27 NSW, W Tumbarumba KU745272 KU745232 NMV K-12183
Pogonosternum sp. B S28 NSW, NNE Tumbarumba KU745273 KU745233 KU745192 SMNG VNR016993
Pogonosternum sp. B S29 NSW, SE Batlow KU745274 KU745234 KU745193 NMV K-12185


Maps were created with ArcGIS 10. The final phylogenetic trees were edited using Adobe Illustrator CS4.

Molecular analysis

DNA was extracted from 2-4 legs from each of 41 Pogonosternum specimens and from the three paradoxosomatid species Archicladosoma magnum Jeekel, 1984, Somethus scopiferus Jeekel, 2002 and S. castaneus (Attems, 1944), which were chosen as outgroups (Table 1). Total genomic DNA was extracted using the Qiagen DNAeasy Blood&Tissue kit following the standard protocol except that tissue was incubated for 48h.

Glom primer cocktail pairs (Decker 2016, Macek et al. 2014) were used to sequence a 618 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Primer pairs 28S D1a (Fw) and 28S D3b (Rv) (Dell’Ampio et al. 2009) were used to amplify 1225 bp of the D2 fragment and adjacent areas of D1 and D3 on the nuclear 28S ribosomal RNA gene.

For PCR protocol and all primer sequences (COI, 28S) see Decker (2016).

Primer pairs 16Sar (Fw) (5’-CGCCTGTTTAACAAAAACAT-3’) and 16Sbr (Rv) (5’-CCGGTCTGAACTCAGATCACGT-3’) (Simon et al. 1994) were used to sequence a 566 bp fragment of the large-subunit ribosomal RNA (16S) gene. The following thermocycling profile was used to amplify fragments of 16S: pre-denaturation at 94°C for 4 min 30 sec, 35 cycles of 30 sec at 94°C, 30 sec at 49°C and 50 sec at 72°C, and the final extension step for 5 min at 72°C.

All PCR mixes had a total volume of 10 µl comprising 1 µl template, 0.2 µM of each primer, 4x0.2 mM dNTPs [Peqlab], 1 x PCR Buffer containing 1.5 mM MgCl2 [Peqlab], and 0.05u Polymerase [Peqlab].

All fragments were sequenced in both directions by the BiK-F Laboratory Centre, Frankfurt, Germany. All obtained sequences were checked via BLAST searches of GenBank; no contamination was discovered. The sequences were aligned by hand in ClustalX ver. 1.83 (Chenna et al. 2003) and uploaded to GenBank (Table 1).

Some homologisation problems in the 16S rRNA sequences arose mainly because of the highly variable expansion loops. As a result, selected alignment positions (272-297) were excluded from the 16S rRNA dataset for all further analyses using MEGA6.

The final alignments consisted of 618 bp of COI mtDNA, 540 bp of 16S rRNA and 1206 bp of 28S rRNA. The combined datasets after these exclusions consisted of 1158 bp for COI+16S. Individual partial alignments can be obtained from the author upon request. The alignment of the combined dataset can be found in the Suppl. material 2 as a FASTA file.

COI and 16S sequences were combined as a single dataset and incongruence assessed between the mtDNA intergenic spacer sequences with the incongruence length difference (ILD) test (Farris et al. 1994) implemented as the partition homogeneity test in PAUP* version 4.0b10 using a full heuristic search, 10 random taxon addition replicates, tree-bisection-reconnection (TBR) branch swapping, and with MaxTrees set to 100 (Swofford 2002). The best-fit model of nucleotide substitution for the individual COI and 16S dataset was determined by MrModelTest 2 (Nylander 2004). The best-fit model of nucleotide substitution selected using MrModelTest 2 was the General Time Reversible model with gamma distribution and proportion of invariant sites (Nei and Kumar 2000) for the individual COI and 16S dataset. The trees constructed from individual genes did not show significant conflicts in topology (nodes different among trees with support > 70% in ML) and no significant incongruence among the three genes was revealed by the ILD test (P > 0.83 in all of the pairwise comparisons), so the sequences were concatenated into a dataset containing 1158 characters for phylogenetic analysis.

The combined dataset of COI and 16S was analysed under maximum likelihood (ML) using MEGA6 (Tamura et al. 2011) and Bayesian inference (BI) using MrBayes version 3.2 (Ronquist et al. 2012). For ML analysis, three independent runs were performed with nodal support estimated from 1000 bootstrap (BP) pseudoreplicates using the best-fit model for the concatenated dataset. For Bayesian analysis, two independent runs were carried out with four differentially heated Metropolis-coupled Monte Carlo Markov chains for 10 000 000 generations started from a random tree and chains were sampled every 100 generations.

Multiple runs of ML and BI converged in trees with the same topology and similar likelihood score so that only the result of the first run is presented. The topology resulting from ML and BI analyses was largely congruent except for the arrangements of several terminal nodes with low support. Thus, results from the ML and BI analyses are shown together based on the ML tree with bootstrap (BP) and posterior probabilities (PP) of the major lineages shown on the corresponding branches with BP values > 70 (Fig. 2).

Figure 2. 

Maximum likelihood tree for the combined mitochondrial COI+16S dataset, 1000 bootstrap replicates, values below 70 not shown. The bootstrap values of ML and posterior probabilities of BI are given above and below the corresponding branches, respectively, for all major clades. Scale bar = substitutions per site. Coloured blocks indicate species groups. Color of branches refers to the major subregions shown in the map, Tasmanian branches thicker. General differences in male gonopod morphology are shown by sketches of the apical region of the right gonopod not drawn to scale. Coloured lines link those analysed specimens that have similar gonopod morphology. Posterior view = post.; lateral view = lat.; anterior view = ant.

An appropriate DNA substitution model was determined for 28S under the Bayesian Information Criterion (BIC) in Modeltest implemented in MEGA 6 (Tamura et al. 2011). The lowest Bayesian Information Criterion score (BIC) was obtained for 28S rRNA (BIC 3875.11) with the Tamura 3-parameter model (Tamura 1992).

A phylogenetic hypothesis was inferred for COI+16S and 28S by using the maximum likelihood method conducted in MEGA6 (Tamura et al. 2011). The phylogenetic tree with the highest log likelihood (COI+16S: -7237.4280; 28S: -1831.9238) is shown (Figs 2, 3). Initial trees for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach (Tamura et al. 2004). A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories (+G, parameter = COI+16S: 0.2338)). The bootstrap consensus tree inferred from 1000 replicates (Felsenstein 1985) is here used as the best estimate of the phylogeny of the analyzed taxa (Figs 2, 3).

Figure 3. 

Maximum likelihood tree for the nuclear 28S dataset, 1000 bootstrap replicates, values below 70 not shown.

Mean uncorrected pairwise distances between terminals (transformed into percentages) were determined using MEGA6 (Tamura et al. 2011) and can be found in Suppl. material 3.


Phylogenetic and distance analysis

The monophyly of the genus Pogonosternum is strongly supported (MLBP = 97; BIPP = 1.0) in the mitochondrial tree and shows five clades within Pogonosternum, resembling five species groups (Fig. 2).

One main clade includes three species from the mountainous area east and northeast of Melbourne: the undescribed species Pogonosternum sp. B (MLBP = 99; BIPP = 1.0), already mentioned by Car (2010) from New South Wales, P. laetificum (MLBP= 33; BIPP = 1.0) and P. adrianae (MLBP = 68; BIPP = 1.0), both not supported, the latter forming a sister clade (MLBP = 100; BIPP = 1.0) to P. sp. B. The latter two species show moderately large intraspecific distances ranging from 1.1 to 4.6% (P. sp. B) and 0.1 to 3.0% (P. adrianae), while P. laetificum shows high intraspecific distances (0.6–5.5%), even between geographically close (<10 km) populations.

Pogonosternum nigrovirgatum sensu lato with a trans-Bass Strait distribution formed a well-supported (MLBP = 89; BIPP = 1.0) sister clade to the new species P. sp. A (MLBP = 98; BIPP = 1.0) that also has a trans-Bass Strait distribution. Pogonosternum sp. A also occurs in New South Wales (Car 2010) and in northeast Tasmania (Mesibov & Churchill 2003). Pogonosternum nigrovirgatum s. l. occurs on mainland Australia (Otway Ranges to eastern Victoria) and in northwest Tasmania. Pogonosternum coniferum clusters with another form with intermediate gonopods (referred to as P. cf. nigrovirgatum in Fig. 2) between P. nigrovirgatum sensu stricto and P. coniferum. Both P. nigrovirgatum s. l. and P. sp. A show high intraspecific distances ranging from 1.8 to 6.8% within P. nigrovirgatum s. l. and 1.1 to 5.9% within P. sp. A.

Within the P. nigrovirgatum s. l. species-group, the greatest genetic distances were observed between populations in the Strzelecki Ranges (S60, S63; MLBP = 100; BIPP = 1.0) and more western populations, with values ranging from 5.0 to 6.8%. Specimens from the Otway Ranges (S77, S78, S81) all formed a well-supported cluster (MLBP = 86; BIPP = 1.0). The Tasmanian specimen (X2) was distinct from both the Strzelecki Ranges (5.4–6.0%) and central and western Victorian specimens (3.7–3.8%). In the case of Pogonosternum sp. A the largest distances (4.2–5.8%) were between the Eastern Gippsland populations (S42, S47; MLBP = 100; BIPP = 1.0) and all other specimens. The status of the northeast Tasmanian specimen is not well resolved; it is closest to a population from Kosciuszko National Park (S31, 3.0%), the two forming a poorly supported sister clade with a specimen from Gippsland (S52; MLBP = 55; BIPP = 0.6).

All species show considerable intraspecific genetic distances and high phylogeographic structure, especially P. laetificum, and, except in the case of P. adrianae, no haplotypes are shared between different populations. Additional one to three sequenced specimens from eight sampling sites (S14, S15, S22, S58, S59, S78, S83, S87) always showed the same haplotype in Pogonosternum (data not published).

Interspecific distances within the genus Pogonosternum are moderately large, varying from 5.5% (P. sp. A–P. nigrovirgatum s. l.) to 10.4% (P. nigrovirgatum s. l.–P. laetificum), except P. adrianae to P. laetificum with only 2.9%.

Owing to the general lack of variability within the nuclear 28S rRNA dataset, the phylogenetic relationships among species were largely unresolved. Distances for 28S rRNA within Pogonosternum are very low, with a maximum of three base pair differences noted for P. sp. B (Fig. 3). Only the two condensed sister clades of P. nigrovirgatum + P. sp. A and P. adrianae + P. laetificum, as well as P. sp. B are shown.


In a separate paper (Decker, in preparation), the morphology of the Pogonosternum species groups is described in detail and new species are described, based on the specimens used here and from ca 130 additional localities. Here I note briefly that several common morphological features were observed in the gonopods of P. nigrovirgatum s. l., P. laetificum, and P. sp. A: some specimens also showed intermediate states of those features (Fig. 2). It was found, however, when additional material was examined from each population that the morphology of each population was locally stable. It was only in rare cases in the Otway Ranges and NW Tasmania populations that two gonopod morphs occurred in one place.

Surprisingly, gonopod morphology did not appear to agree well with the phylogenetic tree (Fig. 2). Various gonopod forms were distributed with no apparent phylogeographical correlation. Only the species P. adrianae and P. sp. B showed stability in both gonopods and some other non-gonopodal characters over their distribution area, even when material from other museum collections was included (Decker, in preparation).


Phylogenetic analysis

The mitochondrial tree (Fig. 2) shows five main clades, suggesting five species. Pogonosternum coniferum clustered within P. nigrovirgatum, and its taxonomic status needs re-examination (Decker, in preparation).

The 28S tree shows little or only little resolution at the species level (Fig. 3), but was useful in identifying sister clades. This result contrasts with that from a study of the paradoxosomatid genus Somethus in South Australia, in which the 28S gene was used successfully for species identification (Decker 2016). Future studies on other Australian Paradoxosomatidae will reveal if 28S is useful as a diagnostic nuclear gene at the species level.

Morphological variability

With the exception of P. adrianae and P. sp. B, Pogonosternum species show significant variability in gonopod form, with local morphs occurring throughout each species’ distribution area.

Interestingly, P. adrianae is morphologically distinct (in size, spiracles, male tibiotarsal brushes and gonopods, female coxal process) from P. laetificum despite their close genetic distance.

Gonopod variability was also documented for some species of Somethus in South Australia (Decker 2016) and Stygiochiropus Humphreys & Shear, 1993 from Western Australia (Humphreys and Shear 1993). Another good example of variability is seen in the trans-Bass Strait (eastern Victoria, NE Tasmania) paradoxosomatid millipede, Dicranogonus pix: while this species shows only slight variability in gonopods there is marked variation in the development of their paranota. Individuals with no paranota are separated from those with keels by a gap between the Kent and Furneaux Groups of islands (Mesibov 2014).

This study has shown that in the area of southern and southeast Australia, there are at least two genera, Pogonosternum and Somethus (Decker 2016), which both show variability in morphology and genetics. Poor sampling and too few specimens could lead to incorrect conclusions and unnecessary multiple species descriptions.

Multiple glacial refugia in southeastern Australia

The results indicate that there is high intraspecific genetic divergence, with high genetic distances and haplotype diversity in the mitochondrial genes between populations of Pogonosternum, even those adjacent to each other. The P. laetificum clade, which has been sampled extensively in the Central Highlands, shows particularly high intraspecific genetic differences (mean genetic distance of 3.9%), apparently without corresponding geographic patterning, or morphological variation (Decker, in preparation).

The phylogenetic patterns with high intraspecific divergence, high genetic distances, and haplotype diversity with unique local haplotypes, resulting in long branches, shown by Pogonosternum, indicate multiple Pleistocene refugia according to Byrne (2008). These refugia provided suitably moist habitats in which species could persist during the dry, cold climate cycles of the Pleistocene period in southern Australia, while glaciation was limited to the alpine areas of the Great Dividing Range and Tasmania (Barrows et al. 2002). Moderate to high genetic diversity prior to these cycles can be assumed for poorly dispersing millipedes, through isolation by distance, and it is likely that populations were isolated within refugia, leading to further genetic diversification. In contrast, contractions to one or few major refugia during cold, arid periods would result in a low genetic diversity, few divergent lineages and low haplotype diversity, with few haplotypes in areas of postglacial recolonisation (Byrne 2008).

The phylogenetic patterns shown by Pogonosternum suggest that in Victoria and New South Wales there were large areas with multiple local refugia during the Pleistocene. No region in the study area on mainland Australia showed results which indicate rapid postglacial resettlement of Pogonosternum.

Evidence for multiple glacial refugia was also identified in the spirostreptidan millipede Atelomastix bamfordi Edward & Harvey, 2010 in Western Australia (Nistelberger et al. 2014) and for some species of Somethus in South Australia (Decker 2016). Similar phylogeographic patterns seem to occur in other soil invertebrates with limited dispersal capacities in southern Australia, for example flatworms (Sunnucks et al. 2006) and springtails (Garrick et al. 2004).

Endo et al. (2014) have suggested, however, that glacial periods have had less of an impact on the distribution and genetic diversity of invertebrate groups (Coleoptera, Orthoptera, Collembola, Diplopoda) in the Australian Alps than they have in alpine systems in the Northern Hemisphere.

However, further studies on genetic and morphological variability on a finer geographical scale could lead to a better understanding of the pattern and impact of isolation in multiple glacial refugia during the Pleistocene, also as an evolutionary driving force for morphological variability in some species.

Gippsland phylogeography

There is a notable high genetic distance gap within P. nigrovirgatum sensu lato between specimens from the Strzelecki Ranges (S60, S63), West Gippsland, and those sampled in the central and western regions in Victoria, but some specimens of adjacent populations from the latter (S64, S65) were morphologically indistinguishable from specimens from the Strzelecki Ranges. A similar genetic gap was observed in P. sp. A for the populations in Eastern Gippsland east of Orbost (S42, S47) and all other populations. These two cases indicate that these areas may have been isolated for long periods from neighboring regions, possibly before the Pleistocene, perhaps during a marine incursion in the Gippsland Basin and other parts of southeast Australia close to the Miocene–Pliocene boundary (Dickinson et al. 2002).

Trans-Bass Strait distribution

The genus Pogonosternum shows a trans-Bass Strait distribution and most likely originated in mainland southeast Australia, since the highest species diversity is found on the mainland and the two Tasmanian branches occupy only very subordinate positions on the tree (Fig. 2). Tasmanian populations of this genus are restricted to the northeast and northwest corners of the Tasmanian mainland and neighboring islands, and presumably dispersed from Victoria when it was largely connected with Tasmania during the Pleistocene (Lambeck and Chappell 2001). Mitochondrial data suggest that the sequenced population of P. nigrovirgatum s. l. in northwest Tasmania was most likely derived from one in central Victoria or the Otway Ranges. While the results for P. sp. A from northeast Tasmania do not show a close relationship to coastal Victorian populations, analysis of 16S (data not included here) including sequences from two other localities in the western part of East Gippsland showed the Tasmanian specimen clustering with the latter. This indicates that the settlement of Tasmania by this species started in the Gippsland region. A remarkably similar distribution to that of P. sp. A across Bass Strait is also known for the paradoxosomatid millipedes Dicranogonus pix and Notodesmus scotius (Mesibov 2014).

Further studies using more sampling localities in Tasmania and its islands could indicate points of origin in Victoria and the timing of millipede settlement of Tasmania.


Sincere thanks to Karin Voigtländer (SMNG), Ulrich Burkhardt (SMNG), Christiane Ritz (SMNG), Thomas Wesener (ZFMK) for technical advice as well as interesting discussions, Willy Xylander (SMNG) for institutional support and special thanks to Robert Mesibov (West Ulverstone) for help with collecting Pogonosternum. Cathy Car (Perth) provided suggestions on the draft manuscript and Henrik Enghoff, Sergei Golovatch, Robert Mesibov and one anonymous reviewer kindly improved this work. The field trip of the author and Karin Voigtländer was financially supported by the ‘Förderkreis Naturkundemuseum Görlitz’. The publication of this article was funded by the Open Access fund of the Leibniz Association.


  • Adams M, Humphreys WF (1993) Patterns of genetic diversity within selected subterranean fauna of the Cape Range peninsula, Western Australia: systematic and biogeographic implications. Records of the Western Australian Museum, Supplement 45: 145–164.
  • Attems CMT (1944) Neue Polydesmoidea. Zoologischer Anzeiger 144(11-12): 223–251.
  • Barrows TT, Stone JO, Fifield LK, Cresswell RG (2002) The timing of the last glacial maximum in Australia. Quaternary Science Reviews 21: 159–173. doi: 10.1016/S0277-3791(01)00109-3
  • Brölemann HW (1913) The Myriapoda in the Australian Museum. Part ii. Diplopoda. Records of the Australian Museum 10(6): 77–158. doi: 10.3853/j.0067-1975.10.1913.899
  • Brölemann HW (1916) Description of a new species of Myriapoda from New South Wales. Proceedings of the Linnean Society of New South Wales 40(4): 683–684. doi: 10.5962/bhl.part.18888
  • Byrne M (2008) Evidence for multiple refugia at different time scales during Pleistocene climatic oscillations in southern Australia inferred from phylogeography. Quaternary Science Reviews 27: 2576–2585. doi: 10.1016/j.quascirev.2008.08.032
  • Car CA (2010) Pine plantations and native millipedes (Diplopoda: Polydesmida: Paradoxosomatidae) in south-eastern New South Wales, Australia. Australian Journal of Entomology 49(4): 317–323. doi: 10.1111/j.1440-6055.2010.00771.x
  • Chamberlin RV (1920) The Myriapoda of the Australian Region. Bulletin of the Museum of Comparative Zoology 64(1): 1–269.
  • Chapple DG, Hoskin CJ, Chapple SNJ, Thompson MB (2011) Phylogeographic divergence in the widespread delicate skink (Lampropholis delicata) corresponds to dry habitat barriers in eastern Australia. BMC Evolutionary Biology 11: 191. doi: 10.1186/1471-2148-11-191
  • Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Research 31: 3497–3500. doi: 10.1093/nar/gkg500
  • Decker P (2016) Integrative taxonomic revision of the polymorphic flat-millipede genera Oncocladosoma and Somethus in South Australia (Diplopoda: Polydesmida: Paradoxosomatidae). Invertebrate Systematics: In press.
  • Dell’Ampio E, Szucsich NU, Carapelli A, Frati F, Steiner G, Steinacher A, Pass G (2009) Testing for misleading effects in the phylogenetic reconstruction of ancient lineages of hexapods: influence of character dependence and character choice in analyses of 28S rRNA sequences. Zoologica Scripta 38(2): 155–170. doi: 10.1111/j.1463-6409.2008.00368.x
  • Dickinson JA, Wallace MW, Holdgate GR, Gallagher SJ, Thomas L (2002) Origin and timing of the Miocene-Pliocene unconformity in southeast Australia. Journal of Sedimentary Research 72: 288–303. doi: 10.1306/082701720288
  • Edward KL, Harvey MS (2010) A review of the Australian millipede genus Atelomastix (Diplopoda: Spirostreptida: Iulomorphidae). Zootaxa 2371: 1–63. doi: 10.11646/%25x
  • Endo Y, Nash M, Hoffmann AA, Slatyer R, Miller AD (2014) Comparative phylogeography of alpine invertebrates indicates deep lineage diversification and historical refugia in the Australian Alps. Journal of Biogeography 42: 89–102. doi: 10.1111/jbi.12387
  • Felsenstein J (1985) Confidence Limits on Phylogenies: An Approach Using the Bootstrap. Evolution 39: 783. doi: 10.2307/2408678
  • Garrick RC, Sands CJ, Rowell DM, Tait NN, Greenslade P, Sunnucks P (2004) Phylogeography recapitulates topography: very fine-scale local endemism of a saproxylic ‘giant’ springtail at Tallaganda in the Great Dividing Range of south–east Australia. Molecular Ecology 13: 3329–3344. doi: 10.1111/j.1365-294X.2004.02340.x
  • Humphreys WF, Shear WA (1993) Troglobitic millipedes (Diplopoda: Paradoxosomatidae) from semi-arid Cape Range, Western Australia: systematics and biology. Invertebrate Taxonomy 7: 173–195. doi: 10.1071/IT9930173
  • Jeekel CAW (1965) A new genus and a new species of the family Paradoxosomatidae from Australia (Diplopoda, Polydesmida). Entomologische Berichten 25: 7–14.
  • Jeekel CAW (1982) Millipedes from Australia, 2: Antichiropodini from Victoria (Diplopoda, Polydesmida, Paradoxosomatidae). Bulletin Zoölogisch Museum, Universiteit van Amsterdam 8(24): 201–212.
  • Jeekel CAW (1984) Millipedes from Australia, 6: Australiosomatini from Victoria (Diplopoda, Polydesmida, Paradoxosomatidae). Records of the Australian Museum 36: 19–44. doi: 10.3853/j.0067-1975.36.1984.323
  • Jeekel CAW (2002) Millipedes from Australia, 14: A third contribution to South Australian Paradoxosomatidae (Diplopoda, Polydesmida). Myriapod Memoranda 5: 60–77.
  • Jeekel CAW (2006) Millipedes from Australia, 18: Tasmanian Paradoxosomatidae (Diplopoda, Polydesmida) (Genera Somethus Chamb., Notodesmus Chamb. and Aethalosoma nov.). Myriapod Memoranda 8: 75–89.
  • Lambeck K, Chappell J (2001) Sea level change through the last glacial cycle. Science 292: 679–686. doi: 10.1126/science.1059549
  • Macek O, Bartel D, Szucsich N, Pass G (2014) Cocktails and pills: A COI primer cocktail for pill millipedes. In: Tuf IH, Tajovský K (Eds) 16th International Congress of Myriapodology. Book of Abstracts. Institute of Soil Biology, BC ASCR & Faculty of Science, Palacký University, Olomouc, CZ, 50.
  • Mesibov R (2014) The Australian millipede Dicranogonus pix Jeekel, 1982 (Diplopoda, Polydesmida, Paradoxosomatidae): a species with and without paranota. ZooKeys 454: 29–39. doi: 10.3897/zookeys.454.8625
  • Mesibov R, Churchill TB (2003) Patterns in pitfall captures of millipedes (Diplopoda: Polydesmida: Paradoxosomatidae) at coastal heathland sites in Tasmania. Australian Zoologist 32(3): 431–438. doi: 10.7882/AZ.2002.021
  • Nistelberger H, Byrne M, Coates D, Roberts D (2014) Strong Phylogeographic Structure in a Millipede Indicates Pleistocene Vicariance between Populations on Banded Iron Formations in Semi-Arid Australia. PLoS ONE 9(3): e93038. doi: 10.1371/journal.pone.0093038
  • Nylander JAA (2004) MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space. Systematic Biology 61(3): 539–542. doi: 10.1093/sysbio/sys029
  • Schultz MB, Smith SA, Horwitz P, Richardson AMM, Crandall KA, Austin CM (2009) Evolution underground: A molecular phylogenetic investigation of Australian burrowing freshwater crayfish (Decapoda: Parastacidae) with particular focus on Engaeus Erichson. Molecular Phylogenetics and Evolution 50: 580–598. doi: 10.1016/j.ympev.2008.11.025
  • Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994) Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annals of the Entomological Society of America 87: 651–701. doi: 10.1016/j.ympev.2008.11.025
  • Sunnucks P, Blacket MJ, Taylor JM, Sands CJ, Ciavaglia SA, Garrick RC, Tait NN, Rowell DM, Pavlova A (2006) A tale of two flatties: different responses of two terrestrial flatworms to past environmental climatic fluctuations at Tallaganda in montane southeastern Australia. Molecular Ecology 15: 4513–4531. doi: 10.1111/j.1365-294X.2006.03107.x
  • Swofford DL (2002) PAUP*: Phylogenetic analysis using parsimony (*and other methods). v.4.0b10. Sinauer & Associates, Sunderland, Massachusetts. doi: 10.1111/j.0014-3820.2002.tb00191.x
  • Tamura K (1992) Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G + C-content biases. Molecular Biology and Evolution 9: 678–687.
  • Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences of the United States of America 101: 11030–11035. doi: 10.1073/pnas.0404206101
  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731–2739. doi: 10.1093/molbev/msr121
  • Wojcieszek JM, Simmons LW (2012) Evidence for stabilizing selection and slow divergent evolution of male genitalia in a millipede (Antichiropus variabilis). Evolution 66(4): 1138–1153. doi: 10.1111/j.1558-5646.2011.01509.x

Supplementary materials

Supplementary material 1 

Full data of sequenced specimens

Peter Decker

Data type: Tab-delimited text file

Explanation note: Full details of sequenced specimens, including locality, date, collector, collection number and coordinates.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (12.06 kb)
Supplementary material 2 

Alignment of combined dataset

Peter Decker

Data type: FASTA file

Explanation note: Alignment of the combined COI mtDNA and 16S rRNA dataset

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (49.92 kb)
Supplementary material 3 

P-distances of combined COI and 16S dataset

Peter Decker

Data type: CSV File

Explanation note: Mean uncorrected pairwise distances between terminals (transformed into percentages) of the combined COI mtDNA and 16S rRNA dataset.

This dataset is made available under the Open Database License ( The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (7.55 kb)
login to comment