Delimitation biological species in genus
Acanthoxyla (Phasmidae) using
mitochondrial and nuclear DNA

Gualdrón-Diaz J. C.

1. Introduction

From the broader perspective of evolutionary theory, delimiting species is im-
portant in the context of understanding many evolutionary mechanism and pro-
cesses(Sites & Marshall, 2003). The species boundary will define the limist
within or across which evolutionary processes operate (Barton & Gale , 1993).
Species are also routinely used as fundamental units of analysis in biogeogra-
phy, ecology, macroevolution and conservation biology (Brown et al., 1996) and
a better understanding of these larger scale processes requires that systema-
tists employ methods to delimit objectively and rigorously what species are in

nature.

There have been many proposals for the conceptual and practical handling of
species and among these, the biological species concept (Mayr, 1942) has been
the most thoroughly promoted and most widely accepted. One of the more
frequently stated deficiencies of the BSC is its inapplicability to uniparental
organisms (Foottit, 1997). The parthenogenesis is a common phenomenon in
the Animal Kingdom. It has been estimated that there are over 1000 obligately
parthenogenetic species situated over a broad range of taxa and over 15000
species which reproduce by cyclic parthenogenesis (White, 1978; Bell, 1982).
In stick insects(phasmids) the hybrid speciation and parthenogenesis both
appear to be unusually common phenomena (Bullini, 1994). In New Zealand
thera are parthenogenetic populations of otherwise sexual species of Clitarchus
and Argosarchus (Salmon, 1991). However, genus Acanthoxyla is entirely parthenogenetic. It comprises eight species without males and no closely related bisexual species (Jewell & Brock, 2002). This eight Acanthoxyla species exhibit a degree of morphological diversity not evident in any other New Zealand stick insects genus (Morgan & Trewick, 2005). The aim of this work is to make a nested clade analysis and phylogeography of haplotypes using nuclear and mitochondrial DNA to delineate species Acanthoxyla (Phasmatidae).

2. Materials and Methods

Five different genes of the genera Acanthoxyla and Clitarchus were used. 21
localities of New Zealand and 171 sequences in total of both genera were ana-
lyzed (fig. 1). Three were sequences nuclear genes, elongation factor 1x (EF1x),
phosphoglucose isomerase(PGI) and the large ribosomal subunit (28S) and two
were mitochondrial, cytochrome oxidase subunits I and II (COI-II) listed in
Buckley et al. (2008). The sequences have been deposited in GenBank under
accession EU492930-EU493090; Two operational criteria to delimit specie were
used, nested clade analysis (NCA)and phylogeographic analysis sensu Avise.
The nucleotide sequences were aligned using multiple alignment with Mus-
cle 3.6 software package. The nested clade analysis conducted using the ANECA software, which implements programs TCSv2.1 and GEODIS for analysis(Panchal, 2007). Haplotype networks were reconstructed in TCS v2.1. Then examined the relationship between haplotype/clades and geography through a statistical analysis of permutations, using the program GEODIS. For the phylogeographic analysis sensu Avise (Avise et al, 1987): ML bootstrap analysis (1000 permutations) were realized using the softwate Phyml 3.0 (Guindon & Gascuel, 2003).


3 Results and Discussion

3.1 Nested clade analysis

The cladistic analysis of haplotypes for mitochondrial genes shows 10 clades for
both COI and COII, in terms of nuclear genes, 28S, PGI and EF1x, there are
9, 11 and 7 respectively clades, but none of them considers the fragmentation
allopatric pattern as inferred from the clades formed. Therefore can not assign
the species status of these genera using this method since only fragmentation
allopatric is the important biological implication of the NCA to infer species
status (Templeton 2001).
Patterns inferred from the clades formed were restricted gene flow with iso-
lation by distance, contiguous range expansion and restricted gene flow with
isolation by distance (restricted dispersal by distance in non-sexual species) for
COII genes, EF1x and PGI, respectively. The 28S and COI gene showed Incon-
clusive outcome.

3.2 Phylogeographic analysis sensu Avise

The phylogeographic analysis, with mitochondrial genes (COI-COII) reveals the
presence of two different groups, which have a high value of confidence limits
(fig. 2-3), according to Avise (1995) assert that to infer species status requires a
confidence limit ( 95%) therefore they deserve the status of species. By contrast
with the results obtained using nuclear DNA cladograms show low resolution
and with diverging results obtained with mitochondrial DNA, in the analysis
with the 28S gene, showed levels very low limits of confidence, the evidence
suggests an clagograma unresolved with this type of gene. Similar results were
obtained through EF1x and PGI genes, however these if you showed higher
values of confidence limit (fig. 4-6).

Given the resutlados obtained, it appears that for the nested clade analsis
neither nuclear nor mitochondrial DNA showed species status. As for the re-
sults obtained with the phylogeographic analysis, differences in the phylogenetic relationships of the genus Acanthoxyla haplotypes generated with mitochondrial DNA with respect to nuclear. The mitochondrial DNA results show that only A. inermis and A. geisovii deserve species status.

4. References

Avise, J. C., Arnold, J., Ball, R. M., Bermingham, E., Lamb, T., Neigel, L. E., Reeb, C. A. & Saunders, N. C., 1987. Intraspecific phylogeography: The mitocondrial DNA bridge between population ge­netics and systematics. Annual Review in Ecology and Systematics, 18: 489–522.

Bell, G. 1982. The Masterpiece of Nature. The Evolution and Genetics of Sexuality, University of California Press, Berkeley and Los Angeles

Buckley T.R., Attanayake D., Park D., Ravidran S., Jewell T.R., Normark B.B. 2008. Investigating hybridization in the parthenogenetic New Zealand stick insects Acanthoxyla (Phasmatodea) using single-copy nuclear loci. Molecular Phylogenetics and Evolution 48: 335-349.

Bullini L. 1994. Origin and evolution of animal hybrid species. Trends Ecol. Evol. 9:422-426.

Guindon S. & Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology, 52(5):696-704, 2003.

Jewell T., Brock, P.D. 2002. A review of the New Zealand stick insects: new genera synonymy, keys and a catalogue. J. Orthopteran Res. 11, 189-197.

Mayr, E. 1942. Systematics and the Origin of Species from the Viewpoint of a Zoologist. Columbia University Press: New York.

Panchal, M. (2007), 'The automation of Nested Clade Phylogeographic Analysis', bioinformatics 23, 509-510.

Salmon, J.T., 1991. The Stick Insects of New Zealand. Redd, Auckland, New Zealand.

Sites J. W. Jr., Marshall J. C. 2003. Delimiting species: A Renaissance issue in systematic biology. Trends Ecol. Evol. ;18:462-470.

Templeton AR (2001) Using phylogeographic analyses of gene trees to test species status and processes. Molecular Ecology, 10, 779–791.

White, M.J.D. 1978. Modes of Speciation, W.H, Freeman and Company, San Francisco.

5. Figures



Figure 1. Map of New Zealand showing sampling localities


Figura 2. Maximun likelihood gene trees for the mtDNA data (COII). Numbers above branches are bootstrap value.


Figura 3. Maximun likelihood gene trees for the mtDNA data (COI). Numbers above branches are bootstrap value.


Figura 4. Maximun likelihood gene trees for the 28S data. Numbers above branches are bootstrap value


Figura 5. Maximun likelihood gene trees for the PGI data. Numbers above branches are bootstrap value.


Figura 6. Maximun likelihood gene trees for the EF1 data. Numbers above branches are bootstrap value.