Corresponding author: Peter Decker (
Academic editor: R. Mesibov
This study documents the first detailed phylogenetic analysis of an Australian paradoxosomatid millipede genus. Two mitochondrial genes (partial
Decker P (2016) Phylogenetic analysis of the Australian trans-Bass Strait millipede genus
Thus,
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 (
The present study documents a molecular phylogenetic analysis of the antichiropodine genus
Map of Southeast Australia showing the distribution of
Site numbers, localities, GenBank accession numbers and repository accession numbers for all specimens analyzed. (See also Fig. = Museum Victoria, Melbourne, Victoria, Australia = Queen Victoria Museum and Art Gallery, Launceston, Tasmania, Australia = South Australian Museum, Adelaide, Australia = Senckenberg Museum of Natural History Görlitz, Görlitz, Germany = New South Wales = South Australia = Tasmania = Victoria
Species | Site No. | Locality | GenBank Acc. No. |
GenBank Acc. No. 16S | GenBank Acc. No. 28S | Voucher |
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Maps were created with ArcGIS 10. The final phylogenetic trees were edited using Adobe Illustrator CS4.
DNA was extracted from 2-4 legs from each of 41
Glom primer cocktail pairs ( cytochrome
For PCR protocol and all primer sequences (
Primer pairs 16Sar (Fw) (5’-CGCCTGTTTAACAAAAACAT-3’) and 16Sbr (Rv) (5’-CCGGTCTGAACTCAGATCACGT-3’) (
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 (
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
incongruence length difference tree-bisection-reconnection
The combined dataset of maximum likelihood Bayesian inference bootstrap
Multiple runs of bootstrap posterior probabilities
Maximum likelihood tree for the combined mitochondrial
An appropriate DNA substitution model was determined for 28S under the Bayesian Information Criterion Bayesian Information Criterion score
A phylogenetic hypothesis was inferred for Maximum Composite Likelihood
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 (
The monophyly of the genus
One main clade includes three species from the mountainous area east and northeast of Melbourne: the undescribed species
Within the
All species show considerable intraspecific genetic distances and high phylogeographic structure, especially
Interspecific distances within the genus
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
In a separate paper (Decker, in preparation), the morphology of the
Surprisingly, gonopod morphology did not appear to agree well with the phylogenetic tree (Fig.
The mitochondrial tree (Fig.
The 28S tree shows little or only little resolution at the species level (Fig.
With the exception of
Interestingly,
Gonopod variability was also documented for some species of
This study has shown that in the area of southern and southeast Australia, there are at least two genera,
The results indicate that there is high intraspecific genetic divergence, with high genetic distances and haplotype diversity in the mitochondrial genes between populations of
The phylogenetic patterns with high intraspecific divergence, high genetic distances, and haplotype diversity with unique local haplotypes, resulting in long branches, shown by
The phylogenetic patterns shown by
Evidence for multiple glacial refugia was also identified in the spirostreptidan millipede
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.
There is a notable high genetic distance gap within
The genus
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 (
Full data of sequenced specimens
Tab-delimited text file
Full details of sequenced specimens, including locality, date, collector, collection number and coordinates.
Alignment of combined dataset
FASTA file
Alignment of the combined
P-distances of combined
CSV File
Mean uncorrected pairwise distances between terminals (transformed into percentages) of the combined