Pogonosternum cf. nigrovirgatum (Carl, 1912) “Dargo”: 7 males, 1 female, 1 juvenile (NMV K-12202, K-13866, K-13867, K-13474, K-13868, SMNG VNR018276, VNR018277) were collected by hand in forest on Dargo Road, SSW of Dargo, central Gippsland, Victoria, 37.595S, 147.193E by P. Decker, K. Voigtländer and R. Mesibov on 14 August 2014.

Somethus castaneus (Attems, 1944): specimens were collected by hand at two localities in Mount Osmond Reserve, Adelaide, South Australia by P. Decker and K. Voigtländer : 1 male (SMNG VNR016973) and 3 females (SAM OM2149, SMNG VNR016975 and VNR016976) on a southwestern slope (34.969S, 138.654E, 27 August 2014, site number S110), and 5 males (SAM OM2138, SAM registration in progress, SMNG VNR018275) and 1 female (SMNG VNR018274) on a northern slope (34.962S, 138.659E, 23 August 2014, site number S90).

Specimens were killed and stored in 95% ethanol, with a change of ethanol after 1–2 months. One male of Somethus castaneus from Mt. Osmond (SAM OM2138) was found dead in the field. DNA was not obtained from this specimen. The material is deposited in the Museums Victoria, Melbourne, Victoria, Australia (NMV), the South Australian Museum, Adelaide, Australia (SAM) and the Senckenberg Museum of Natural History Görlitz, Görlitz, Germany (SMNG).

Preserved specimens were imaged with a Leica Z6 Apo stereo microscope and Leica DFC420 camera. Focus-stacked images were assembled from 25–40 source images using the software package Leica Application Suite 4.5. All images were later edited using Adobe Photoshop CS4 and assembled into plates. The distribution maps were created with ArcMap 10.

DNA was extracted from 2–4 legs from each of four male Pogonosternum cf. nigrovirgatum “Dargo” and nine Somethus castaneus specimens from Mt Osmond (Table 1). Total genomic DNA was extracted using a Qiagen DNAeasy Blood & Tissue kit following the standard protocol with an incubation of tissue for 48h. Glom primer cocktail pairs (Decker 2016a, 2016b, 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. 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. For PCR protocol and all primer sequences (COI, 16S, 28S) see Decker (2016a, 2016b). Fragments were sequenced in both directions by the BiK-F Laboratory Centre, Frankfurt, Germany. All obtained sequences were checked with BLAST in GenBank and no contamination was apparent. The sequences were aligned by hand in ClustalX ver. 1.83 (Chenna et al. 2003) and uploaded to GenBank (Table 1). In addition, 41 previously published (Decker 2016a for more information) sequences for five Pogonosternum species P. nigrovirgatum, Pogonosternum adrianae Jeekel, 1982, Pogonosternum laetificum Jeekel, 1982, Pogonosternum jeekeli Decker, 2017, and Pogonosternum montanum Decker, 2017 (COI: Genbank accession numbers KU745235–KU745274, KT948680; 16S: KU745194–KU745234) were used for the phylogenetic analysis of the Pogonosternumcf. nigrovirgatum “Dargo” sequences. For the molecular analysis of Somethus castaneus “Mt Osmond” sequences the following published (Decker 2016b for more information) sequences were included: 16 COI sequences for the five Somethus species S. castaneum, Somethus inflatus (Jeekel, 2002), Somethus lancearius Jeekel, 2002, Somethus scopiferus Jeekel, 2002, and Somethus grossi Jeekel, 1985 (GenBank accession numbers KT948655–KT948656, KT948658, KT948662–KT948670, KT948672–KT948676) and 15 28S sequences of the four species S. castaneum, S. inflatus, S. lancearius, and S. grossi (GenBank accession numbers KT964457–58, KT964457). Archicladosoma magnum Jeekel, 1984 (KT948681) was used as outgroup. Primary homologisation problems in the 16S rRNA sequences of the Pogonosternum dataset arose because of the highly variable expansion loops. As a result, selected alignment positions (272–297) were excluded from the 16S rRNA dataset. COI and 16S sequences were combined in Pogonosternum as a single dataset and incongruence assessed between them 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).

Phylogenetic hypothesis was inferred for COI+16S, COI and 28S by using the maximum likelihood method conducted in MEGA6 (Tamura et al. 2011). The phylogenetic trees with the highest log likelihood (COI+16S: -5141; COI: -2565; 28S: -2328) are shown (Figs 3, 5). Initial trees for the heuristic search were obtained by applying the neighbour-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: 0.6793; COI: 0.1017; 28S: 0.0500)). The bootstrap consensus tree inferred from 1000 replicates (Felsenstein 1985) is here used as the best estimate of the phylogeny of each of the analysed taxa (Figs 3, 5). Mean uncorrected pairwise distances between terminals (transformed into percentages) were determined using MEGA6 (Tamura et al. 2011).

The final alignments consisted of 618 bp of COI mtDNA and 1206 bp of 28S rRNA in Somethus, and 1158 bp for COI+16S in Pogonosternum. Individual alignments are available upon request from the author. 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 > 0.81 in all of the pairwise comparisons), and the sequences were concatenated into a dataset comprised 1158 characters for phylogenetic analysis in Pogonosternum.

Pogonosternum nigrovirgatum and P. jeekeli are very similar in somatic morphology, and the “Dargo” form agrees with both species in size, colouration, spiracle morphology and form of the leg pair 2 coxa in females. The “Dargo” form (Fig. 1) agrees with typical P. nigrovirgatum (Fig. 2) in having an elongated gonopod femorite and differs from P. jeekeli in gonopods (Fig. 3) and male tarsal and tibial brushes only on leg pairs 1–7. See also Decker et al. (2017) for a detailed (re)description of P. nigrovirgatum and P. jeekeli and the considerable gonopod variability in these species as well as Decker 2016a for phylogeographic distribution of similar gonopod morphology. However, the four sequenced “Dargo” males are all phylogenetically closer to P. jeekeli in the combined COI and 16S dataset and form a well-supported group (Fig. 3; bootstrap value 98% and with uncorrected percent difference of 2.4%), and appear in the tree next to a population of P. jeekeli from northeastern Tasmania. The Dargo collection site is close to the observed species distribution boundary between P. jeekeli and P. nigrovirgatum (Fig. 4).

Figure 1. 

Pogonosternum cf. nigrovirgatum “Dargo”, male, right gonopod (NMV K-12202). A Posterior view B Lateral view C Anterior view D Mesal view. Abbreviations: fp1 = femoral process 1; fp2 = femoral process 2; lp = lateral process; prof = prolongation of femorite; S = solenomere; F = femorite. Scale bar: 0.5 mm.

Figure 2. 

Pogonosternum nigrovirgatum (Carl, 1902), ♂, right gonopod. A, B AMS KS96017 Ferntree Gully C, DNMV K-10248 from Sandringham and Brighton. Pogonosternum jeekeli Decker, sp. n., ♂, right gonopod E, FNMV K-10252 from Dyer Creek G, HNMV K-10250 from Bemm River. A, C Posterior view B, D Mesal view. Scale bar: 0.5 mm.

Figure 3. 

Bootstrap consensus tree for the combined mt COI+16S dataset in Pogonosternum; maximum likelihood, 1000 bootstrap replicates. Coloured dotted lines indicate specimens with similar gonopod morphology. Coloured bars indicate last male leg pairs with tibial and tarsal brushes (leg pair 7 = blue, leg pair 9 = green).

All six male specimens from Mt Osmond fully agree in gonopod morphology with S. castaneus and lack a medial prefemoral process (Decker 2016b). In the 28S phylogenetic analysis, all nine sequenced specimens (uncorrected p-distance of 0–0.08%) occur within a clade of S. castaneus and are closest to specimens from Onkaparinga, Horsnell Gully Conservation Park, and Brownhill Creek Recreation Park (Fig. 5). In the COI analysis (Fig. 5), three specimens from the northern slope of Mt Osmond (site S90) and three from the southwestern slope (site S110) occur with S. castaneus from Morialta Conservation Park, Brownhill Creek Recreation Park, Horsnell Gully Conservation Park, and Belair National Park (uncorrected p-distances 0.3–1.1%).

Figure 4. 

Distribution map of Pogonosternum nigrovirgatum (red circles), P. jeekeli (orange diamonds) and P. cf. nigrovirgatum “Dargo“ (yellow star) in southeastern Australia. Abbreviation: ACT = Australian Capital Territory.

Figure 5. 

COI and 28S bootstrap consensus trees for Somethus species; maximum likelihood, 1000 bootstrap replicates.

However, two specimens from site S90 and one from site S110 form a separate, well-supported clade (100% bootstrap support) within Somethus, with genetic p-distances of 4.8–6.9% to S. lancearius, 5.3–6.9% to S. castaneus and 7.2–8.8 % to S. inflatus. The two Mt Osmond localities are in the centre of the S. castaneus distribution (Fig. 6).

Figure 6. 

Map of South Australia and southern Mt Lofty Ranges showing localities for Somethus species: S. castaneus (filled triangle), S. inflatus (open triangle), S. lancearius (square), and S. castaneus from Mt. Osmond (yellow star).

Despite the clear phylogenetic placement of this form within P. jeekeli as indicated by COI+16S gene trees (Fig. 3), the gonopod morphology of the seven Dargo specimens is closest to that of P. nigrovirgatum. In addition, tibial and tarsal brushes in the Dargo males extend only to leg pair 7, whereas in P. jeekeli the brushes reach leg pair 9 (74 studied males, Decker et al. 2017). In P. nigrovirgatum male brushes typically extend to leg pair 7, and infrequently to leg pair 9 in the area around Port Philip Bay.

Pogonosternum nigrovirgatum and P. jeekeli form a sister clade (Decker 2016a) and the Dargo site is located close to the presumed boundary between the species (Fig. 4). The observed discordance between morphology and genetics could be the result of introgressive hybridisation of P. jeekeli and P. nigrovirgatum, resulting in a P. nigrovirgatum-type gonopod phenotype but a P. jeekeli mitochondrial COI and 16S genotypes. Furthermore, the finding of seven discordant males with a P. nigrovirgatum-type gonopod shows that the form is not the result of a developmental aberration in an individual within a P. jeekeli population. Another possibility is that P. cf. nigrovirgatum “Dargo” is simply a local variant of P. jeekeli, a species which is known to have variable gonopods in a genus that exhibits high gonopod variability (Decker 2016a, Decker et al. 2017). However, P. jeekeli is the only Pogonosternum species with tibial and tarsal brushes always ranging to leg pair 9. In contrast, brushes to leg pair 7 are found in P. adrianae Jeekel, 1982, P. montanum Decker, 2017, most males of P. nigrovirgatum and some males of P. laetificum Jeekel, 1982. This leg pair 7 limit is observed in four out of the five species in the genus Pogonosternum and likely to be plesiomorphic. Thus the hypothetical local variation in P. jeekeli resulting in the “Dargo” phenotype would involve both a change in gonopod morphology and a regression from apomorphy or common distribution of tibial and tarsal brushes in P. jeekeli. However, the author favours the hypothesis that two closely related Pogonosternum species have undergone introgressive hybridisation in an area of range overlap near Dargo. To distinguish between hypotheses of introgression versus gene tree lineage sorting, further gene sampling and analysis is needed of Pogonosternum individuals near the Dargo site and along near the presumed distribution boundary between P. nigrovirgatum and P. jeekeli. In addition, sampling of P. jeekeli and P. nigrovirgatum far from sympatry are needed.

Somethus castaneus was sampled for sequencing at 13 localities covering most of the known species range, and genetic variability was shown to be low (up to 3.8% in uncorrected p-distances in COI) with three phylogenetic lineages (Decker 2016b). The morphologically typical S. castaneus from Mt Osmond, in the centre of the species range, includes three individuals whose COI sequences are not close to those of either the other S. castaneus, S. inflatus or S. lancearius, although they are slightly closer to those of S. lancearius (in % bp difference). Somethus lancearius is distributed in the north-eastern and eastern part of the Adelaide Hills, with some scattered, possibly introduced, occurrences of S. castaneus within its distributional area. The closest record of S. lancearius is about 17 km from Mt Osmond. Genetic variability within S. lancearius from five sampled localities is 1.6–5.8% (in uncorrected p-distances in COI) and with unique haplotypes that correspond to geographical areas (Decker 2016b). Widespread sampling of this species is no longer possible, as natural vegetation has largely been cleared within its range and S. lancearius is now restricted to scattered conservation areas and tiny remnants.

Several of the paratypes of S. inflatus were collected in the Adelaide suburb of Glen Osmond, near Mt Osmond, in 1969 (Jeekel 2002). These paratypes appear to have been lost and could not be compared with recently collected S. inflatus (Decker 2016b), and no S. inflatus have since been found on Mt Osmond. S. inflatus is distributed to the southwest of Adelaide on the Fleurieu Peninsula and on Kangaroo Island (Fig. 6). Jeekel’s S. inflatus paratypes might represent a non-permanent introduction.

The three discordant S. castaneus found on Mt Osmond might be evidence for past introgression of mitochondrial DNA following hybridisation with another South Australian Somethus species. Alternatively, the anomalous individuals might represent a distinct and distantly related S. castaneus lineage which is either naturally occurring on Mt Osmond or introduced from another locality within the Adelaide Hills. There is no support for both hypotheses, but it seems that the likelihood of the presence (or former existence) on Mt Osmond of the in COI and 28S variable S. lancearius or S. inflatus is higher than that of a fourth distinct COI and 28S lineage in S. castaneus.

Future investigations with additional molecular markers and more individuals of from S. lancearius may not assist in clarifying the situation, as much of the former genetic variation of S. lancearius has probably been lost due to habitat loss and local extinctions. If the missing paratypes of S. inflatus are found in future, it might be possible to extract DNA and obtain sequences from them which could reveal whether S. inflatus in the Mt Osmond area has contributed mitochondrial COI to the local S. castaneus population.