Molecular Diversity of Dunedin Peripatus (Onychophora: Peripatopsidae)

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Molecular Diversity of Dunedin Peripatus (Onychophora: Peripatopsidae) New Zealand Journal of Zoology, 1999, Vol. 26: 381-393 381 0301-4223/2604-0381 $7.00/0 © The Royal Society of New Zealand 1999 Molecular diversity of Dunedin peripatus (Onychophora: Peripatopsidae) S. A. TREWICK Keywords New Zealand; mtDNA; Peripatoides; Department of Zoology invertebrate; population structure; habitat; genetics; University of Otago PCR primers; mitochondrial rearrangement; cyto- P. O. Box 56 chrome oxidase; COI; SSCP Dunedin, New Zealand email: [email protected] INTRODUCTION Abstract Onychophora tend to be morphologi- The living Onychophora are morphologically cally conservative. Several studies using molecular conservative although they can be variable in size techniques have revealed the existence of cryptic and colour within populations. It is of no surprise species and population structuring. The application therefore that early morphology-based taxonomy of allozyme electrophoresis to New Zealand also tended to be conservative and to underestimate peripatus that were thought to belong to a single the true diversity of the group. Molecular studies and widespread species (Peripatoides novaezealandiae) the use of electron microscopy have revealed the has revealed several undetected species, including existence of many cryptic species and genera, most a taxon specific to Dunedin (southern South Island, notably in Australia (Briscoe & Tait 1995; Reid New Zealand). However, almost nothing is known 1996) although the first genetic study was on about the geographic range and variability of this Jamaican peripatus (Hebert et al. 1991). The species, nor indeed whether it comprises one or more Australian onychophoran fauna appears to be both cryptic taxa. I used analysis of cytochrome oxidase diverse, including some 80% of the described global I mtDNA sequences to explore these aspects of fauna (Briscoe & Tait 1995; Reid 1996), and locally peripatus found in the vicinity of Dunedin. Eighteen rich with some areas having as many as five genera different haplotypes were detected in 47 individu- (Sunnucks et al. in press). Contrary to the usual rule als from 21 locations. The sequence of a 540 bp COI of conservatism, some of these taxa possess very fragment contained 64 parsimony informative sites distinctive morphology in the form of mating-related and nucleotide diversity of up to 11% among ingroup structures on the head resolved using electron taxa. Phylogenetic analyses, and genetic distance by microscopy (Tait & Briscoe 1990; Reid 1996). geographic distance correlation, indicated probable In comparison, the onychophoran fauna of New species-level divisions within the sample. Two prin- Zealand appears to be generally less diverse and lo- cipal groups with a boundary on the east coast of cally depauperate. Until recently, only five species New Zealand near the mouth of the Taieri River can of peripatus were recognised in New Zealand be defined, and these are denoted "Dunedin" and (Ruhberg 1985), belonging to two genera; the "Catlins" peripatus. There is a third, possibly distinct egglaying Ooperipatellus and the livebearing lineage at Piano Flat. A rearrangement of the mito- Peripatoides. Of these, Peripatoides novaezealan- chondrial genome, relating to the position of the diae (Hutton) was apparently the most numerous and tLEU rna gene, was detected in these and other New widespread (Ruhberg 1985). Hutton's (1876) de- Zealand peripatus and may be present in all scription was based on specimens from Wellington, Onychophora. Nelson and Dunedin. However, analyses of allozyme data have indicated the existence of a species com- plex (involving at least five taxa) in Norm Island and a separate taxon in Dunedin (south South Island) Z99006 (Tait & Briscoe 1995; Trewick 1998). While the Received 3 March 1999; accepted 4 June 1999 North Island P. novaezealandiae species complex 382 New Zealand Journal of Zoology, 1999, Vol. 26 has so far revealed no gross morphological differ- Onychophora for instance, a group noted for their ences among sister species (Trewick 1998), Tait & morphological conservatism, genetic methods have Briscoe (1995) noted that a combination of morpho- revealed extensive cryptic diversity (Briscoe & Tait logical characteristics (15 pairs of legs, midventral 1995), stimulated revision of morphology based tax- openings of anal glands in males, and lack of crural onomy (Reid 1996) and provided insights into the glands) in the Dunedin taxon distinguished it from origin of diversity within, and biogeography of, spe- other described genera. Allozyme data revealed no cies (Hebert et al. 1991; Gleeson et al. 1998). close relatives among the limited number of New Previous genetic studies of New Zealand Onycho- Zealand and Australian peripatus studied (Tait & phora have used allozyme variation (Tait & Briscoe Briscoe 1995). A survey of Australasian Onycho- 1995; Trewick 1998). These nuclear loci can have phora using mitochondrial DNA, that supported many different alleles (arising from changes in the monophyly of the New Zealand Peripatoides, also sequence of amino acids comprising a particular included specimens from one location in Dunedin protein), and as such are especially useful for (Gleeson et al. 1998). observing gene flow among populations of sexually At present, however, Dunedin peripatus have been reproducing organisms (Murphy et al. 1996). In represented in studies only by specimens from two cases of sympatry, the lack of exchange of alleles sites (Caversham and Leith valleys), both of which are between sexually reproducing individuals demon- close to the city centre (Tait & Briscoe 1995, Gleeson strates the presence of distinct species, even if those et al. 1998). Before any interpretation of nationwide species are not outwardly distinguishable (King & phylogeographic patterns can be made, a more Hanner 1998; Trewick 1998). The other most complete understanding of local diversity and frequently used genetic marker in the 1990's, geographic range is required. Any exploration of mitochondrial DNA, is however, generally non- diversity is likely to raise questions of species recombining (but see Wallis 1999) and in most definition, sympatry and conservation status. organisms is maternally inherited. Although this There are advantages in a morphology based tax- means mtDNA is not appropriate for tests of genetic onomy (e.g., presumed ability to undertake identifi- exchange on its own, it is a powerful indicator of cation in the field and without specialist equipment), historical subdivision, and extensive datasets enable but genetic approaches also have much to offer sys- among-taxa comparisons of diversity and structure tematists, biogeographers and conservationists alike. (Avise 1994). MtDNA sequence data is particularly This is because (a) information is derived, by defi- useful for interspecific phylogenetic analysis. By nition, from the material that is transmitted between using faster-evolving genes and sampling individuals individuals during reproduction, and (b) these mol- throughout the range of a presumed species it is also ecules evolve in a more or less predictable manner feasible to explore intraspecific phylogeography across taxa. (Riddle 1996). The use of mtDNA also has the Perceived conflicts between genetic and morpho- benefit of requiring less sample tissue than allozyme logical approaches arise because morphology has electrophoresis so that the morphological integrity traditionally been a prerequisite of taxonomy, and of specimens can more easily be maintained. This at a fine scale at least, taxonomy can proceed with- is particularly helpful in the study of small or soft out a phylogenetic basis. Genetic analyses assume bodied organisms such as peripatus. an evolutionary process in order to interpret the his- I surveyed mtDNA sequence variation in Dunedin torical relationships of genes and therefore their peripatus in order to identify their geographic range, "hosts". Whilst this is in principle also true of intraspecific variability, possible existence of cryptic cladistic methods, an absence of suitably variable species, and conservation status. The Dunedin morphological characters, or problems of homology, peripatus is already perceived to be of special status can be overwhelming. Many phylogenetic analyses to the extent that New Zealand's first peripatus of genetic data produce gene trees, or their approxi- reserve has been gazetted for it by the city council. mation. It is the job of molecular biologists to infer Whether this action is justified, or the location well how well these trees estimate organismal phylogeny, chosen, has remained untested. By documenting and an extensive array of analytical tools have been diversity of peripatus within the Dunedin region we developed to do this. In a sense this is a reversal of will be better placed to explore the benefit of such the situation that arises from morphology-based reserves, and the status of the Otago and Southland analyses which, naturally, tend to underestimate bio- regions with respect to the wider biogeography of genetic diversity (Avise 1994). In the study of onychophorans in New Zealand. Trewick—Molecular diversity of Dunedin peripatus 383 METHODS and NotLEUr (see Results for details). PCR reactions were performed in 25 jal volumes and products gel- Peripatus specimens were collected in the environs purified in 2% agarose stained with ethidium of Dunedin City and up to 220 km from it (Fig. 1). bromide. Bands of expected molecular weight were This collecting area is probably close to the range excised
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