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18 December 2015 Do cytochrome c oxidase 1 Gene Sequences Differentiate Species of Spirostreptid Millipedes (Diplopoda: Spirostreptida: Spirostreptidae)?
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Abstract

The aim of this study was to use cytochrome c oxidase 1 (CO1) sequences to recover a phylogeny for seven morphologically described spirostreptid millipede taxa from southern Africa, and to evaluate the correspondence between morphological and molecular phylogenies. Genetic p-distance generally increased with taxonomic divergence: inter-specific mean 15.33 % (14.09 % –17.02 %), inter-generic mean 18.43 % (6.83 %–26.81 %) and inter-order mean 24.16 % (range 18.56 %–30.77 %). Congruent Bayesian, maximum parsimony and neighbour-joining analyses of 520 nucleotides of the CO1 gene resolved the orders Spirostreptida, Julida and Callipodida. Members of genera within the Spirostreptidae (Archispirostreptus, Bicoxidens, Cacuminostreptus, Doratogonus, Orthoporoides, Plagiotaphrus and Spirostreptus) formed a single clade within which a sample of Thyropygus (family Harpagophoridae) was paraphyletically nested. Phylogenetic analyses failed to recover support for the genera Doratogonus, Bicoxidens, Archispirostreptus and Spirostreptus, as representatives of these genera were not monophyletic. Samples morphologically identified as the same species (Bicoxidens flavicollis) were part of two different clades, one of which was well supported and otherwise contained members of Doratogonus. This high level of divergence (mean 12.64 %) between morphologically identified spirostreptid millipede sister species could indicate that changes in genital morphology occur rather slowly relative to CO1 sequence substitution, and may underestimate species diversity.

INTRODUCTION

Millipedes are a major group of soil detritivores. About 15 % of the estimated 80 000 species have been described (Sierwald & Bond 2007). In Africa, the orders Spirostreptida, Polydesmida, Polyzoniida, Siphonophorida, Sphaerotheriida, Spirobolida and Stemmiulida have been recorded (Sierwald & Bond 2007). According to Hamer (2000) the Spirostreptida is the most common and conspicuous. The Spirostreptidae is one of four families in the Spirostreptida. Of approximately 45 African genera that belong to the Spirostreptidae (Krabbe 1982), 23 occur in southern Africa, south of the Kunene and Zambezi rivers (Hamer 2000). Among them are the genera Archispirostreptus Silvestri, 1895, Doratogonus Attems, 1914, Cacuminostreptus Mwabvu, 2010, Spirostreptus Brandt, 1833, Plagiotaphrus Attems, 1914, and Bicoxidens Attems, 1928, which contain large-bodied species.

Although male genitalia are central in spirostreptid millipede taxonomy (see Hoffman 2008; Hamer 2009; Mwabvu et al. 2010) because they are divergent and species specific (Bond et al. 2003), morphology-based classifications may be too inclusive (Adams et al. 2009; Ács et al. 2010) and may hide considerable variation. Molecular data have demonstrated in many arthropod groups that molecular divergence may not reflect divergence in morphology, thus resulting in an underestimation of diversity (Brewer et al. 2012). Derkarabetian et al. (2011) also reported a lack of divergence in reproductive morphology in Opiliones, although genetic divergence is significant. Other studies, for example, in Anadenobolus millipedes (Bond et al. 2003) and spiders (Huber et al. 2005), also suggest that genetic and genitalic divergence are decoupled. Hence, groups that were described on the basis of morphological characters are being re-evaluated using appropriately variable DNA sequence data.

The use of DNA sequences to distinguish taxa, identify new species and estimate phylogeny is becoming prominent in invertebrate systematics (Hebert et al. 2004), including arthropods (Brewer et al. 2013). For example, mitochondrial cytochrome c oxidase 1 (CO1) gene sequences have been used to differentiate species in pelagic sea snails (Hunt et al. 2010), crustaceans (Witt et al. 2006), lepidopterans of North America (Hebert et al. 2010), tropical butterflies (Elias et al. 2007; Escalante et al. 2010) and arachnids (Boyer et al. 2005). Furthermore, mitochondrial 16S rRNA gene sequences have also been used to discriminate members of the orders Polydesmida (Marek & Bond 2007), Spirobolida (Bond & Sierwald 2002), Glomerida (Wesener 2012) and Julida (Enghoff et al. 2011, 2013). Moreover, most of the centipede and millipede faunas of Bavaria, southern Germany (120+ species representing all major orders and families) have been subjected to molecular analysis, including barcoding and genetic distance assessments (Spelda et al. 2011). However, inter-generic genetic variations and the thresholds for designating species remain only scarcely touched upon despite, according to Enghoff et al. (2011), molecular data demonstrating that they could resolve inter-generic relationships in millipedes.

DNA sequence data could clarify the validity of species, flag undescribed taxa and augment taxonomy based on male genitalia. Given the poor dispersal abilities of the Diplopoda and their preference for moist micro-habitats, which are patchy, they tend to be local endemics, i.e. geographically isolated in small areas (Hopkin & Read 1992). As such, we expect strong inter-generic and inter-specific genetic differentiation among millipedes.

The aim of this study was to carry out a phylogenetic analysis based on mitochondrial CO1 sequences for seven taxa, morphologically determined to be members of the family Spirostreptidae, and outgroups. The objectives were as follows: (1) to assess the CO1 genetic distance thresholds characterising a range of taxonomic levels (intra-specific to inter-order) within the order Spriostreptida, and between the orders Spirostreptida, Julida and Callipodida within the Helminthomorpha; (2) to assess whether morphologybased taxonomic groups within the Spirostreptidae, comprising members of the genera Archispirostreptus, Bicoxidens, Cacuminostreptus, Doratogonus, Orthoporoides, Plagiotaphrus and Spirostreptus, are supported by molecular data; and (3) to assess whether the family Spirostreptidae (represented by the above genera) is monophyletic.

MATERIAL AND METHODS

Total genomic DNA was extracted from males. Fresh specimens of B. flavicollis Attems, 1928, B. aridis Mwabvu, 2009, S. kruegeri (Attems, 1928), S. sebae Brandt, 1833, D. uncinatus (Attems, 1914), Doratogonus sp. 1, A. tumuliporus (Karsch, 1881), A. gigas (Peters, 1855), P. sulcifer Attems, 1914 and C. mazowensis Mwabvu, 2010 were collected in Zimbabwe; and S. kruegeri, D. cristulatus (Porat, 1872) and O. pyrhocephalus (Lock, 1865) were taken from South Africa. All specimens were collected by hand, after which the material was preserved in 100 % ethanol. Specimens that were collected from South Africa are housed in the KwaZulu-Natal Museum, Pietermaritzburg, South Africa, and those from Zimbabwe are in the Natural History Museum, Bulawayo, Zimbabwe. Specimens of A. gigas (Peters, 1855) were borrowed from the Royal Museum for Central Africa, Tervuren, Belgium.

Legs removed from the midbody rings of individual specimens were ground using a mortar and pestle. DNA was then extracted following the manufacturer's instructions using the Qiagen DNeasy Blood and Tissue Kit (Qiagen). Polymerase chain reactions targeting part of the mitochondrial CO1 gene were performed after optimisation using gradient PCR. PCR amplifications were performed in a total volume of 25 μl containing 12.5 μl EconoTaq plus green mastermix, 0.25 μl of each primer, 1 μl of DNA and 11 μl of H20. The thermal cycling conditions were 94°C for 2 min, 35 cycles of 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 1 min and a final extension of 72°C for 10 min. Primers for the amplification were as used by Lavrov et al. (2002) and Bond and Sierwald (2002). As PCR yields were low, amplicons were cloned prior to sequencing on an ABI 3730 capillary sequencer at Inqaba Biotechnical Industries (Pty) Ltd (Hatfield, Pretoria, South Africa).

Raw sequences were edited in BioEdit version 7 (Hall 1999), and aligned using the Clustal W algorithm in BioEdit, and by inspection. The CO1 alignments were trimmed to a common length of 520 nucleotides. A saturation plot of transitions and transversions versus genetic distance, and Xia's test of substitution saturation (Xia et al. 2003; Xia & Lemey 2009) were carried out in DAMBE (Xia & Xie 2001) to evaluate the level of saturation and establish the usefulness of the dataset as an indicator of phylogeny.

Phylogenetic relationships among the taxa were analysed using Bayesian, maximum parsimony and neighbour-joining analyses. Modeltest (Posada & Crandall 1998) was used to select the model of nucleotide substitution which best fitted the sequence dataset. The general time-reversible (GTR) model was selected using the Akaike Information Criterion (AIC), and was used in Bayesian, neighbour-joining and data-saturation analyses. Neighbour-joining and maximum parsimony analyses were implemented in PAUP (Swofford 2002). Genetic distances were presented as distance matrices and as a neighbour-joining tree, which was bootstrapped using 1 000 pseudo-replicates. For parsimony analysis, the random additions sequence option (n = 100) for discrete, unordered characters was used. The shortest tree was obtained using the heuristic search option under the tree bisection-reconnection (TBR) branch-swapping option. The degree of support for each node of the resulting tree was estimated using bootstrap re-sampling analysis (1 000 pseudo-replicates; Felsenstein 1985). Bayesian analysis was implemented in Mr Bayes 3.0 (Huelsenbeck & Ronquist 2001) using flat priors. For all analyses, four Markov chains were run for 5 million generations each, until the standard deviation of the split frequencies was less than 0.01. The first 500 000 trees were discarded as burn-in. A preliminary run was carried out in order to determine that the burn-in value was sufficiently high to discard all less likely trees generated before the likelihoods had stabilised. Using the methods described above, we also created a tree based on amino-acid sequences, to see if deep node support was greater than in the tree based on nucleotide sequences.

Nucleotide sequence data were submitted to GenBank. The GenBank accession numbers are KF219542 (A. gigas), KF219543 (A. tumuliporus 1), KF219544 (A. tumuliporus 2), KF219545 (B. aridis), KF219546 (B. flavicollis 1), KF219547 (B. flavicollis 2), KF219548 (C. mazowensis), KF219549 (D. cristulatus 1), KF219550 (Doratogonus sp. 1), KF219551 (D. uncinatus), KF219552 (D. cristulatus 2), KF219553 (O. pyrhocephalus), KF219554 (P. sulcifer 1), KF219555 (P. sulcifer 2), KF219556 (S. kruegeri), and KF219557 (S. sebae) (Table 1). Outgroup sequences of taxa belonging to the Orders Callipodida, Julida, and Spirostreptida (Harpagophoridae: Thyropygus sp.) were obtained from the National Center for Biotechnology Information (NCBI) GenBank database.

TABLE 1

Taxa used for phylogenetic analysis, collection information, museum voucher and GenBank accession numbers. NMSA or NM or NM-Myr — KwaZulu-Natal Museum, Pietermaritzburg, South Africa; NMZ — Natural History Museum, Bulawayo, Zimbabwe; MRAC — Royal Museum of Central Africa, Belgium.

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RESULTS

The results of Xia's test of substitution saturation (Xia et al. 2003; Xia & Lemey 2009) showed that ISS (0.3219) was significantly less than ISSC (0.7166) (p = 0.0000), consistent with little saturation in the data and indicating that it is useful for phylogenetics. The saturation plot (Fig. 2) showed no saturation in transversions and some saturation in transitions at GTR genetic distances greater than 17 %.

In order to enable comparisons to be made with genetic distances reported in the literature, we report genetic distances among different taxonomic levels as p-distances. Genetic p-distances within the Spirostredptidae were: inter-specific mean 15.33 % (range 14.09 % –17.02 %); and inter-generic mean 18.43 % (6.83 % –26.81 %) (Table 2, Fig. 1). The mean genetic distance between the orders Spirostreptida, Julida and Callipodida was 24.16 % (18.56 % –30.77 %). In general, genetic distance increased with taxonomic divergence.

TABLE 2

Pairwise genetic p-distances (%) between spirostreptid species and outgroups based on analysis of 520 nucleotides of the mitochondrial cytochrome c oxidase 1 gene. Abbreviations: Ag — A. gigas, At — A. tumuliporus, Ba — B. aridis, BfA — B. flavicollis A, BfC — B. flavicollis C, Dc — D. cristulatus, D — Doratogonus sp. 1, Du — D. uncinatus, Op — O. pyrhocephalus, Ps — P. sulcifer, Sk — S. kruegeri, Ss — S. sebae, Cm — C. mazowensis, Th — Thyropygus sp ,Ju — Julida, Ca — Callipodida.

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Bayesian inference (BI), maximum parsimony (MP) and neighbour-joining (NJ) analyses of the CO1 nucleotide alignment produced congruent trees. These trees were also congruent with the trees based on amino acid sequences, which are not presented here. The Bayesian tree is presented, with bootstrap values from all three analysis methods indicated at the nodes, in the order (BI posterior probability/ MP bootstrap % /NJ bootstrap %) (Fig. 3). Members of the three orders formed monophyletic clades; the Spirostreptida (Clade A; 1.00 / 80 % / 89 %) and Julida (Clade B; 1.00 / 88 % / 92 %) were strongly supported, and the Callipodida relatively weakly supported (Clade C; 0.96 / 81 % /52 %). Within clade A, duplicate representatives of D. cristulatus, A. tumuliporus and P. sulcifer formed very strongly supported clades (1.00 / 100 % / 100 %). A single member of the Harpagophoridae, a Thyropygus species, was nested within the strongly supported clade (A) containing all representatives of the Spirostreptidae and showed a very weakly supported (0.85 / 51 % / 68 %) relationship with Archispirostreptus gigas. Members of the genus Archispirostreptus (tumuliporus and gigas) did not form a monophyletic clade, as would be expected. Although all members of Doratogonus (cristulatus, uncinatus and sp. 1) were part of the same well-supported clade (A1), a representative of Bicoxidens flavicollis was also nested within this clade. Although there was very weak support (0.61 / — / 60 %) for an association between Bicoxidens aridis and a representative of B. flavicollis, not all representatives of this genus were part of the same clade. Further, although members of the genus Spirostreptus (sebae and krugeri) were part of the same unsupported clade, this also included a representative of Cacuminostreptus mazowensis.

Fig. 1.

Summary of intra-specific, inter-specific and inter-generic genetic distances within representatives of the family Spirostreptidae and between members of the orders Spirostreptida, Julida and Callipodida. The dotted line represents the mean value of a category.

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Fig. 2.

Saturation plot of a dataset comprising 520 nucleotides of the mitochondrial cytochrome c oxidase 1 gene created in DAMBE (Xia & Xie 2001). The GTR model was used to calculate genetic distance in substitutions per site; s — transition, v — transversion.

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Fig. 3.

Bayesian inference tree based on an analysis of 520 nucleotides of the mitochondrial cytochrome c oxidase 1 gene showing relationships between Spirostreptida species and outgroups (Julida and Callipodida species). Numbers adjacent to taxon names are GenBank accession numbers, and indicate sequences that were downloaded from the NCBI Genbank. This tree was congruent in structure with maximum parsimony and neighbour-joining analyses of the same dataset. Nodal support values are indicated as (posterior probability / maximum parsimony bootstrap / neighbour-joining bootstrap).

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DISCUSSION

The overall aim of this study was to use CO1 sequences to recover a phylogeny for seven spirostreptid millipede taxa from southern Africa whose taxonomic position had previously been determined by morphological analyses, and to compare the relationships revealed by morphological and molecular methods. The CO1 gene is generally regarded as sufficiently divergent to allow identification and discovery of new species in a variety of taxa, including beetles (Funk 1999), neotropical skipper butterflies (Hebert et al. 2004) and amphibians (Vences et al. 2005). As there is little phylogenetic information available for spirostreptid millipedes, we aimed to evaluate the threshold genetic distances distinguishing different taxonomic levels. We found that genetic distance between taxa generally increased with taxonomic divergence, as would be expected based on a molecular clock hypothesis (Zuckerkandl & Pauling 1962).

The mean genetic distance between the orders Spirostreptida, Julida and Callipodida was 24.16 % (range 18.56 %–30.77 %). Taxa belonging to the same order clustered together in the CO1 tree, providing support for the validity of these orders according to the phylogenetic species concept (Cracraft 1987). The mean inter-specific distances recovered were very high at 15.33 % (14.09 %–17.02 %) relative to those observed in other types of organisms. For example, 74 % of avian sister species are separated by CO1 distances of less than 2.7 % (Moritz & Cicero 2004), and 99 % of all marine nematode species are separated by >5 % (Derycke et al. 2010). This high level of divergence between morphologically identified spirostreptid millipede species could indicate that changes in genital morphology occur rather slowly relative to CO1 sequence substitution, and may underestimate species diversity. Phylogenetic analyses failed to recover support for the genera Archispirostreptus, Bicoxidens, Doratogonus and Spirostreptus. Representatives of Archispirostreptus and Bicoxidens respectively did not form monophyletic clades, as would be expected of a genus under the phylogenetic species concept (Cracraft 1987). Although members of the genera Doratogonus and Spirostreptus were all included in the same clade, both the Doratogonus and Spirostreptus clades included a member of another genus, and were not therefore monophyletic for members of the genus. Further, samples identified as the same species (Bicoxidens flavicollis) were part of two different clades, one of which was well supported and otherwise contained members of Doratogonus.

Based on the selected genera (Archispirostreptus, Bicoxidens, Cacuminostreptus, Doratogonus, Orthoporoides, Plagiotaphrus, Spirostreptus), the CO1 sequences illustrated that representatives of the family Spirostreptidae are all members of the same clade, although the presence within this clade of a sample of Thyropygus, sourced by a Blast search of the Genbank for sequences most similar to the Spirostreptidae study samples, is puzzling. Our phylogenetic analyses yielded a weakly supported sister group relationship between Thyropygus sp. and Archispirostreptus gigas, although genital morphologybased taxonomy places the two genera in different families, Harpagophoridae and Spirostreptidae, respectively.

At this stage, it is premature to question relationships based on genital morphology because of poor taxon sampling and, according to Sole-Cava and Wörheide (2007), possible identification errors in the Genbank database, because sequences are not from holotypes. Archispirostreptus gigas and A. tumuliporus do not form a subclade within the Spirostreptidae clade, even though based on gonopod morphology they are congeneric (Mwabvu et al. 2010); this was unexpected. Whether A. gigas and A. tumuliporus belong in different genera requires further scrutiny; possibly these taxonomic assignments are incorrect. At present it is unclear whether genital morphologic variation is correlated with genetic change in millipedes. Hence, there is a need to increase taxon sampling and use more than one gene sequence to assess intra-generic genetic divergence. C values for Diplopoda range from 0.31 pg to 1.4 pg ( www.genomesize.com, Animal Genome Size Database, accessed 29 January 2015). Based on the mean, the fraction of the genome represented by the CO1 alignment analysed in this study is 5.6 x 10-5 %. Based on this, it would appear advisable to employ a multi-gene system in molecular taxonomy, although this would not significantly increase the proportion of the genome analysed. In addition, Meier et al. (2006) observed that 21 % of dipteran species that they studied did not have unique CO1 sequences, which resulted in low identification success. Thus, in order to establish the degree of genetic divergence in spirostreptid millipedes, wider taxon sampling and the use of more genes to discriminate species are necessary.

Although the genetic distances among species are high, we found little evidence of saturation in the dataset, confirming that these data are suitable for conducting phylogenetic analyses. It is possible that some of the paraphyletic associations observed may be due to long branch attraction artifacts; however, if this were the case, we would expect a discordance in the structure of trees created using the parsimony method, which is most prone to grouping long branches together, and using Bayesian and neighbour-joining analyses. A lack of discordance when different analytical methods are used can be taken as evidence that all are inferring the same true tree (Bergsten 2005). Although it has been suggested that the CO1 locus lacks a phylogenetic signal at deep nodes in millipedes (Brewer et al. 2013), we obtained good support for the deep nodes (representing different orders) in our analyses. Our analyses were based on amino-acid sequences, which are more conservative than nucleotide sequences owing to the redundancy in the genetic code, and they revealed no increased nodal support at any level, and produced trees with congruent structures in which the same nodes were supported.

In summary, we found that genetic distance between taxa generally increased with taxonomic divergence. We recovered varying levels of concordance between morphological and CO1 sequence data. The orders Spirostreptida, Julida and Callipodida were completely resolved. Members of the family Spirostreptidae were all part of the same clade, but were not monophyletic, as a member of a sister family, the Harpagophoridae, was nested within this clade. Most genera within the Spirostreptidae were not supported, and were part of paraphyletic associations. Further, samples identified as the same species (B. flavicollis) were part of two different clades. This demonstrates that in millipedes, genitalia may be reliable indices of taxon validity at higher taxonomic levels (Order), but that the correlation between morphological and molecular (CO1) data is not unequivocal at family, genus or species level. All species-level taxa were characterised by unique CO1 sequences separated by high genetic distances (mean inter-specific distance 14.1 %), probably because genital morphology underestimates true diversity. We further provide CO1 sequence data for seven species of millipedes within the Spirostreptidae.

ACKNOWLEDGEMENTS

The research was funded by the University of KwaZulu-Natal and the National Research Foundation's Focus Area Grants. We wish to thank Miss Theshnie Naidoo for training the first author in molecular laboratory techniques.

REFERENCES

1.

Ács, Z. , Challis, R.J. , Bihari P. , Blaxter, M. , Hayward, A. , Melika G. , Csóka, G. , Pénzes, Z. , Pujade- Villar, J. , Nieves-Aldrey, J-L. , Schönrogge, K. & Stone, G.N. 2010. Phylogeny and DNA barcoding of inquiline oak gallwasps (Hymenoptera: Cynipidae) of the Western Palaearctic. Molecular Phylogenetics and Evolution 55: 210–225. Google Scholar

2.

Adams, D.C. , Berns, C.M. , Kozak, K.H. & Wiens, J.J. 2009. Are rates of species diversification correlated with rates of morphological evolution? Proceedings of the Royal Society B 276 (1668): 2729–2738, https://doi.org/10.1098/rspb.2009.0543 Google Scholar

3.

Bergsten, J. 2005. A review of long-branch attraction. Cladistics 21: 163–193. Google Scholar

4.

Bond, J.E. , Beamer, B.A. , Hedin, M.C. & Sierwald, P. 2003. Gradual evolution of male genitalia in sibling species complex of millipedes (Diplopoda: Spiroboloda: Rhinocricidae: Anadenobolus). Invertebrate Systematics 17: 711–717. Google Scholar

5.

Bond, J.E. & Sierwald, P. 2002. Cryptic speciation in the Anadenobolus excisus millipede species complex on the island of Jamaica. Evolution 56 (6): 1123–1135. Google Scholar

6.

Boyer, S.L. , Karaman, I. & Giribet, G. 2005. The genus Cyphophthalmus (Arachnida, Opiliones, Cyphophthalmi) in Europe: a phylogenetic approach to Balkan Peninsula biogeography. Molecular Phylogenetics & Evolution 36: 554–567. Google Scholar

7.

Brewer, M.S. , Spruill, C.L. , Nandita, S.R. & Bond, J.E. 2012. Phylogenetics of the millipede genus Brachycybe Wood, 1864 (Diplopoda: Platydesmida: Andrognathidae): patterns of deep evolutionary history and recent speciation. Molecular Phylogenetics & Evolution 64: 232–242. Google Scholar

8.

Brewer, M.S. , Swafford, L. , Spruill, C.L. & Bond, J.E. 2013. Arthropod phylogenetics in light of three novel millipede (Myriapoda: Diplopoda) mitochondrial genomes with comments on the appropriateness of mitochondrial genome sequence data for inferring deep level relationships. PLOS ONE 8 (7): e68005. https://doi.org/10.1371/journal.pone.0068005 Google Scholar

9.

Cracraft, J. 1987. Species concepts and the ontology of evolution. Biology and Philosophy 2 (3): 329–346. Google Scholar

10.

Derkarabetian, S. , Ledford, J. & Hedin, M. 2011. Genetic diversification without obvious genitalic morphological divergence in harvestmen (Opiliones, Laniatores, Sclerobunus robustus) from montane sky islands of western North America. Molecular Phylogenetics & Evolution 61: 844–853. Google Scholar

11.

Derycke, S. , Vanaverbeke, J. , Rigaux, A. , Backeljau, T. & Moens, T. 2010. Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes. PLoS ONE 5 (10): e13716. https://doi.org/10.1371/journal.pone.0013716 Google Scholar

12.

Elias, M. , Hill, R.I. , Willmott, K.R. , Dasmahapatra, K.K. , Brower, A.V.Z. , Mallet, J. & Jiggins, C.D. 2007. Limited performance of DNA barcoding in a diverse community of tropical butterflies. Proceedings of the Royal Society B 274: 2881–2889. Google Scholar

13.

Enghoff, H. , Petersen, G. & Seberg, O. 2011. Phylogenetic relationships in the millipede family Julidae. Cladistics 27: 606–616. Google Scholar

14.

Enghoff, H. , Petersen, G. & Seberg, O. 2013. The aberrant millipede genus Pteridoiulus and its position in a revised molecular phylogeny of the family Julidae (Diplopoda: Julida). Invertebrate Systematics 27: 515–529. Google Scholar

15.

Escalante, P. , Ibarra-Vazquez, A. & Rosas-Escobar, P. 2010. Tropical montane nymphalids in Mexico: DNA barcodes reveal greater diversity. Mitochondrial DNA 21: 30–37. Google Scholar

16.

Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using bootstrap. Evolution 39: 783–791. Google Scholar

17.

Funk, D.J. 1999. Molecular systematic of cytochrome oxidase I and 16S from Neochlamisus leaf beetles and the importance of sampling. Molecular Biology & Evolution 16 (1): 67–82. Google Scholar

18.

Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98. Google Scholar

19.

Hamer, M. 2000. Review of the millipede genus Doratogonus, with description of fifteen new species from Southern Africa (Diplopoda, Spirostreptida, Spirostreptidae). Annals of the Natal Museum 41: 1–76. Google Scholar

20.

Hamer, M. 2009. Three new species of Doratogonus from South Africa (Diplopoda: Spirostreptida: Spirostreptidae). In : Roble, S.M. & Mitchell, J.C. , eds, A lifetime of contributions to myriapodology and the natural history of Virginia. A festschrift in honor of Richard L. Hoffman's 80th birthday. Virginia Museum of Natural History Special Publication No. 16 , Martinsville, USA, pp. 187–194. Google Scholar

21.

Hebert, P.D.N. , Penton, E.H. , Burns, J.M. , Janzen, D.H. & Hallwachs, W. 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences 101 (41): 14812–14817. Google Scholar

22.

Hebert, P.D.N. , deWaard, J.R. & Landry, J-F. 2010. DNA barcodes for 1/1000 of the animal kingdom. Biology Letters 6: 359–362. Google Scholar

23.

Hoffman, R.L. 2008. Two new genera of spirostreptid millipeds from central Africa, with revised terminology for male gonopod structures (Diplopoda: Spirostreptida). Tropical Zoology 21 (2): 167–186. Google Scholar

24.

Hopkin, S.J. & Read, H.J. 1992. The Biology of Millipedes. New York: Oxford University Press. Google Scholar

25.

Huber, B.A. , Rheims, C.A. & Brescovit, A.D. 2005. Speciation without changes in genital shape: a case study on Brazilian pholcid spiders (Araneae: Pholcidae). Zoologische Anzeiger 243: 273–279. Google Scholar

26.

Huelsenbeck, J.P. & Ronquist, F. 2001. MRBAYES: a Bayesian inference of phylogeny. Bioinformatics 17: 754–755. Google Scholar

27.

Hunt, B. , Strugnell, J. , Bednarsek, N. , Linse, K. , Nelson, R.J. , Pakhomov, E. , Seibel, B. , Steinke, D. & Würzberg, L. 2010. Poles apart: the “bipolar” pteropod species Limacina helicina is genetically distinct between the Arctic and Antarctic oceans. PLoS ONE 5 (3): e9835. https://doi.org/10.1371/journal. pone.0009835 Google Scholar

28.

Krabbe, E. 1982. Systematik der Spirostreptidae (Diplopoda, Spirostreptomorpha). Abhandlungen des Naturwissenschaftlichen Vereins in Hamburg. Hamburg: Verlag Paul Parey. Google Scholar

29.

Lavrov, D.V. , Boore, J.L. & Brown, W.M. 2002. Complete mtDNA sequences of two millipedes suggest a new model for mitochondrial gene rearrangements: duplication and nonrandom loss. Molecular Biology & Evolution 19 (2): 163–169. Google Scholar

30.

Marek, P.E. & Bond, J.E. 2007. A reassessment of apheloriine millipede phylogeny: additional taxa, Bayesian inference, and direct optimization (Polydesmida: Xystodesmidae). Zootaxa 1610: 27–39. Google Scholar

31.

Meier, R. , Shiyang, K. , Vaidya, G. & Ng, P.K. 2006. DNA barcoding and taxonomy in Diptera: a tale of high intraspecific variability and low identification success. Systematic Biology 55 (5): 715–728. Google Scholar

32.

Moritz, C. & Cicero, C. 2004. DNA barcoding: promise and pitfalls. PLoS Biology 2 (10): 28–30. Google Scholar

33.

Mwabvu, T. , Hamer, M. , Slotow, R. & Barraclough, D. 2010. A revision of the taxonomy and distribution of Archispirostreptus Silvestri 1895 (Diplopoda, Spirostreptida, Spirostreptidae), and description of a new spirostreptid genus with three new species. Zootaxa 2567: 1–49. Google Scholar

34.

Posada, D. & Crandall, K.A. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818. Google Scholar

35.

Sierwald, P. & Bond, J.E. 2007. Current status of the myriapod class Diplopoda (Millipedes): taxonomic diversity and phylogeny. Annual Review of Entomology 52: 401–20. Google Scholar

36.

Spelda, J. , Reip, H.S. , Oliveira-Biener, U. & Melzer, R. 2011. Barcoding Fauna Bavarica: Myriapoda: a contribution to DNA sequence-based identifications of centipedes and millipedes (Chilopoda, Diplopoda). ZooKeys 156: 123–139. Google Scholar

37.

Sole-Cava, A.M. & Wӧrheide, G. 2007. The perils and merits (or the Good, the Bad and the Ugly) of DNA barcoding of sponges — a controversial discussion. In : Custodio, M.R. , Lobo-Hajdu, G. , Muricy, G. , eds, Porifera, Research: Biodiversity, Innovation and Sustainability. Serie livros 28. Rio de Janeiro: Museu Nacional, , pp. 603–612. Google Scholar

38.

Swofford, D.L. 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods), Version 4. Sinauer Associates, Sunderland, Massachusetts. Google Scholar

39.

Vences, M. , Thomas, M. , Van der Meijden, A. , Chiari, Y. & Vieites, D.R. 2005. Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians. ( http://www.frontiersinzoology.com/content/2/1/5; accessed 10/6/2007) Google Scholar

40.

Wesener, T. 2012. Nearctomeris, a new genus of Pill Millipedes from North America, with a comparison of genetic distances of American Pill Millipede Genera (Glomerida, Glomeridae). Zootaxa 3258: 58–68. Google Scholar

41.

Witt, J.D.S. , Threloff, D.L. & Hebert, P.D.N. 2006. DNA barcoding reveals extraordinary cryptic diversity in an amphipod genus: implications for desert spring conservation. Molecular Ecology 15: 3073–3082. Google Scholar

42.

Xia, X. & Xie, Z. 2001. DAMBE: Software package for data analysis in molecular biology and evolution. The Journal of Heredity 92 (4): 371–373. Google Scholar

43.

Xia, X. , Xie, Z. , Salemi, M. , Chen, L. & Wang, Y. 2003. An index of substitution saturation and its application. Molecular Phylogenetics and Evolution 26: 1–7. Google Scholar

44.

Xia, X. & Lemey, P. 2009. Assessing substitution saturation with DAMBE. In : Lemey, P. , Salemi, M. & Vandamme, A-M. , eds, The Phylogenetic Handbook: A Practical Approach to DNA and Protein Phylogeny. 2nd edition. Cambridge: Cambridge University Press, pp. 615–630. Google Scholar

45.

Zuckerkandl, E. & Pauling, L.B. 1962. Molecular disease, evolution, and genic heterogeneity. In : Kasha, M. & Pullman, B. , eds, Horizons in Biochemistry. New York: Academic Press, pp. 189–225. Google Scholar
T. Mwabvu, J. Lamb, R. Slotow, M. Hamer, and D. Barraclough "Do cytochrome c oxidase 1 Gene Sequences Differentiate Species of Spirostreptid Millipedes (Diplopoda: Spirostreptida: Spirostreptidae)?," African Invertebrates 56(3), 651-661, (18 December 2015). https://doi.org/10.5733/afin.056.0311
Published: 18 December 2015
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