Mitochondrial DNA Sequences Support Allozyme Evidence For

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Molecular Ecology (2000) 9, 269–281

BlackwellMitochondrial Science, Ltd DNA sequences support allozyme evidence for cryptic radiation of New Zealand Peripatoides (Onychophora)

S. A. TREWICK Department of Zoology, University of Otago, Dunedin, PO Box 56, New Zealand

Abstract A combination of single-strand conformation polymorphism analysis (SSCP) and sequencing were used to survey cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) diversity among New Zealand ovoviviparous Onychophora. Most of the sites and individuals had previously been analysed using allozyme electrophoresis. A total of 157 peripatus collected at 54 sites throughout New Zealand were screened yielding 62 different haplotypes. Comparison of 540-bp COI sequences from Peripatoides revealed mean among-clade genetic distances of up to 11.4% using Kimura 2-parameter (K2P) analysis or 17.5% using general time-reversible (GTR + I + Γ) analysis. Phylogenetic analysis revealed eight well-supported clades that were consistent with the allozyme analysis. Five of the six cryptic peripatus species distinguished by allozymes were conﬁrmed by mtDNA analysis. The sixth taxon appeared to be paraphyletic, but genetic and geographical evidence suggested recent speciation. Two additional taxa were evident from the mtDNA data but neither occurred within the areas surveyed using allozymes. Among the peripatus surveyed with both mtDNA and allozymes, only one clear instance of recent introgression was evident, even though several taxa occurred in sympatry. This suggests well-developed mate recognition despite minimal morphological variation and low overall genetic diversity. Keywords: biogeography, cytochrome oxidase I, mtDNA, New Zealand, Onychophora, peripatus Received 11 July 1999; revision received 24 September 1999; accepted 24 September 1999

Introduction restricted to that region, and this distribution, coupled with a presumed low vagility (Sedgwick 1908; Monge-Nájera The Onychophora are at one and the same time highly 1995), has typically been interpreted as arising from distinctive and obscure animals. Their obscurity is largely Gondwanan vicariance (Stevens 1981; Monge-Nájera 1996). the result of rarity, behavioural crypticism and lack of Using molecular techniques, researchers in Australia study. The phylum consists of just two extant families have recently revealed a deeply structured Onychophoran with some hundred described species, and this number fauna (Briscoe & Tait 1995). This consists of extensive was until recently far smaller. Because of their unusual sympatry of multiple genera (Sunnucks et al. 2000) and combination of traits, including a hydrolastic skeleton previously undetected morphological diversity (Reid et al. and clawed unjointed legs, the group is proverbially 1995; Reid 1996), which includes unique sperm transfer known as a ‘missing link’ and a ‘living fossil’ (e.g. Wenzel structures (Tait & Briscoe 1990). 1950; Ghiselin 1984). These descriptions are of course Although they have their common origins in Gondwana, both semantically self-contradictory and biologically New Zealand and Australia are vastly different landmasses. dubious. Onychophora appear to have diverged early New Zealand is substantially smaller, separated from in the arthropod radiation (Ballard et al. 1992; Briggs Gondwana earlier (≈ 80 million years [Myr] vs. ≈ 50 Myr) et al. 1992; Zrzavy et al. 1998) and are represented in the and has had an exceptionally turbulent geological northern hemisphere fossil record among Cambrian history, experiencing a sequence of marine inundations, marine deposits (Ramsköld 1992; Hou & Bergstrom 1995) volcanism and seismic activity (Cooper & Millener 1993). and Tertiary amber (Poinar 1996). No fossils are known During the Oligocene (≈ 25 Myr), much of the landmass from the southern hemisphere but all extant taxa are was totally submerged beneath the sea, and was later subjected to mountain building in the late Pliocene Correspondence: Dr Steve Trewick. Fax: +64-03-4797584; E-mail: (≈ 5 Myr), and extensive glaciation and climate change [email protected] in the Pleistocene (2 Myr). Nevertheless, New Zealand

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displays a distinctive, if depauperate, biota with many unique taxa (e.g. Moa, tuatara and Leiopelmid frogs) as well as typical southern hemisphere taxa, including Onycho- phora. There is also ample evidence of various taxa recently colonizing New Zealand by dispersal so that the modern assemblage has elements typical of both continental and oceanic biotas (Fleming 1979; Daugherty et al. 1993). Thus, the origins and phylogeographical structure of the New Zealand biota provide an interesting model system in which to explore the roles of vicariance and dispersal in the evolution of species and species assemblages. Within New Zealand, three live-bearing (ovoviviparous) species of Onychophora can be distinguished by leg number: Peripatoides indigo Ruhberg (1985), P. novaezealandiae (Hutton 1876) and P. suteri (Dendy 1894), having 14, 15 and 16 pairs of legs, respectively. However, P. novaezealandiae consists of a species complex (Trewick 1998). Four new species (P. morgani, P. aurorbis, P. kawekaensis and P. sympatrica) were described on the basis of allozyme data, although unfortunately no morphological characters distinguishing them were apparent (Trewick 1998). The present work is a survey of mitochondrial sequence variation among the Peripatoides individuals used in that allozyme study, plus additional specimens extending the sample to include most regions of New Zealand. These mitochondrial DNA (mtDNA) data are here used to fur- Fig. 1 Map of New Zealand showing the locations of the ther explore the origins, phylogeographical structure and Peripatoides sampling sites. ecology of the endemic Peripatoides in light of the turbulent geological history of New Zealand. towards the 3′ end of COI using the universal primer C1- N-2195 (Simon et al. 1994) and either of two primers designed for use with peripatus: Perip241r and NotLEUr Materials and methods (Trewick 1999). Standard polymerase chain reactions Specimens of Peripatoides were collected from decaying (PCRs) were performed in 25-µL volumes and products logs and other moist, dark habitats throughout South Island were gel-purified in 2% agarose stained with ethidium and North Island, New Zealand (Fig. 1, Appendix 1). bromide (see Trewick 1999). Bands of expected molecular Of the specimens previously studied using allozyme weight (MW) were excised and the DNA extracted from electrophoresis, those from Whakapapa, Takaka and the agarose using QIAquick spin columns (Qiagen). Purified Pelorus were not available for the present study, but DNA fragments were quantified (by eye) by comparison new material was obtained from Pelorus. DNA was with a MW marker following agarose-gel electrophoresis. extracted from whole tissue derived either from frozen Cycle sequencing used Bigdye chemistry (Perkin Elmer), body sections remaining from allozyme research or, following the manufacturer’s instructions. Sequences were for specimens collected subsequently, from one or two legs aligned manually using seqed, version 1.0.3 (ABI, PE). dissected from frozen or alcohol-preserved specimens. Phylogenetic analyses were performed using paup, ver- Extractions used a simple, solvent-free proteinase K and sion 4.0 (Swofford 1998). The Mantel test was performed salting-out method (Sunnucks & Hales 1996). using genepop, version 3.1b (Rousset 1997). Universal Peripatus individuals were screened for haplotypic primers used were sourced from the insect mtDNA variation using isotopic labelling and single-stranded primer set (John Hobbs; UBC). Sequence data were conformation polymorphism analysis (SSCP) according deposited at GenBank (accession nos: AF188241–188248, to the method described by Trewick (1999). SSCP utilized 188251–188254, 188258–188262, 221447–221497) and voucher mitochondrial primer pairs C1-J-1718: C1-N-2191, and specimens at the Museum of New Zealand (MONZ) and SR-J-14233: SR-N-14588 (Simon et al. 1994), which amplify Otago Museum (OMNZ). Outgroup sequences were obtained short fragments (< 400 bp) of the cytochrome oxidase I from individuals assignable to the egglaying species (COI) gene and small ribosomal subunit (12S), respect- Ooperipatellus viridimaculatus (Dendy) and O. nanus Ruhberg, ively. Haplotypes were sequenced for a longer fragment collected in South Island, New Zealand.

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Table 1 Summary statistics for 540-bp Codon position cytochrome oxidase I (COI) sequences from Peripatoides First Second Third Total

A 0.285 0.197 0.418 0.300 C 0.107 0.207 0.027 0.114 G 0.272 0.151 0.036 0.153 T 0.335 0.445 0.518 0.433 A + T 0.620 0.64 0.94 0.733 Drosophila 0.524 0.537 0.891 Greya 0.676 0.718 0.917 Constant percentage 83.3 (150) 92.2 (166) 24.4 (44) 66.7 Variable percentage 16.7 (30) 7.8 (14) 75.6 (136) 33.3 Informative percentage 12.8 (23) 2.8 (5) 62.2 (112) 26 No. of sites 180 180 180 540

Proportions of A + T nucleotides at ﬁrst, second and third codon positions in two insect taxa are given for comparison. The Drosophila (Diptera) data are from COI Spicer 1995) and Greya (Lepidoptera) from COI and COII (Brown et al. 1994).

Fig. 2 Mean amino acid variability for 10 structural regions of the cytochrome oxidase I (COI) gene (Lunt et al. 1996) spanned by a 540-bp DNA sequence. The regions comprise membrane helices (M), external loops (E) and internal loops (I). Variability is expressed as the average number of amino acids observed per site in a given region. Open and ﬁlled bars represent data from a suite of insect taxa (Lunt et al. 1996) and Peripatoides from the present study, respectively.

the ingroup (Table 1). There was a transition : transversion Results ratio of 2.2 : 1 calculated from the neighbour–joining (NJ) tree, but this was ≈ 3 : 1 for comparisons within clades. Sequence data A χ 2-test indicated no significant variation in base com- Through a combination of SSCP and sequencing, 62 position among sequences (paup, version 4.0). However, COI haplotypes were identified from 157 Peripatoides these sequences were AT-rich (73%), similar to a level collected at 54 sites. Aligned sequences consisted of observed for this gene in Australasian peripatus (Gleeson 540 bp from the 3′ end of COI corresponding to positions et al. 1998) but not as high as that found in some inver- 754–1293 of the insect COI sequence described by tebrates (e.g. > 80% in beetles, Howland & Hewitt 1995). Lunt et al. (1996). These DNA sequences contained The majority of the AT bias was focused on third-codon 140 (26%) parsimony-informative sites (149 including positions, which were almost entirely A + T (94%), as in the outgroup). Approximately 84% of all substitutions other invertebrates (Table 1). The pattern of inferred were at third-codon positions, and 11.5% and 4.5% at amino acid variability among structural regions of COI first- and second-codon positions, respectively. Seventy- was also consistent with that found across a spectrum of five per cent of third-codon positions were variable in insects (Lunt et al. 1996) (Fig. 2).

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Table 2 Average genetic distances within and between taxa

K2P GTR O. viridimac. P. suteri P. aurorbis P. sympat. P. kaweka. P. novaezea. P. morgani ‘Catlins’ ‘Piano’

0.01 0.04 0.09 0 0.07 0.01 O. viridimaculatus P. suteri 3.1 3.6 12.4 20.8 1.75 1.23 1.25 2.66 1.72 P. aurorbis 3.8 4.6 13.3 23.6 8.7 12.1 1.02 0.95 0.73 0.97 P. sympatrica 2.4 2.7 12.8 20.8 7.4 9.7 11.3 16.9 0.36 0.63 0.85 P. kawekaensis 0.8 0.4 12.6 20.5 7.9 10.5 10.5 15.9 6.5 8.9 0.44 0.48 P. novaezealandiae 2.9 3.2 13.2 22.4 7.4 9.7 10.5 16.1 6.0 7.8 6.2 8.2 0.24 P. morgani 1.9 4.6 13.3 21.9 7.6 10.2 10.7 16.5 6.3 8.4 6.7 7.9 3.2 4.3 ‘Catlins’ 2.0 2.2 14.1 24.9 9.2 11.9 7.9 10.5 9.9 13.3 9.9 13.5 9.7 12.8 9.7 13.1 ‘Piano’ 1.7 1.8 15.9 32.4 10.8 15.9 9.7 14.0 10.8 15.6 11.4 17.5 11.1 16.5 11.1 17.3 6.9 8.9 ‘Dunedin’ 2.8 3.2 14.0 28.0 9.1 12.9 8.5 11.9 10.4 15.2 10.5 15.6 11.2 17.5 11.1 16.8 7.7 13.3 9.3 9.1

Nei’s D from allozyme data (Trewick 1998) is presented above the diagonal, Kimura 2-parameter (K2P) and general time-reversible (GTR + I+ Γ) mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) distances are presented below the diagonal. Within-taxon distances are given in the ﬁrst row for Nei’s D, and in the ﬁrst and second columns for K2P and GTR + I + Γ, respectively. O. viridimac., Ooperipatellus viridimaculatus; P. sympat., Peripatoides sympatrica; P. kaweka., P. kawekaensis; P. novaezea., P. novaezealandiae.

Most peripatus collected from a single site shared Cantor (JC) ( Jukes & Cantor 1969), K2P (Kimura 1980), the same COI haplotype. At all sites where sympatric Hasegawa–Kishino–Yang (HKY85) (Hasegawa et al. 1985) species had previously been found using allozymes and general time-reversible (GTR) (Yang 1994) with a com- (Trewick 1998), more than one haplotype was detected. bination of among-site rate variation models: I (invari- Individuals assigned to a particular species from able sites) and Γ (gamma distribution). There was a allozyme data also had mtDNA haplotypes consistent substantial improvement in likelihood scores for models with this (see phylogenetic analysis). This was true incorporating among-site rate variation, with GTR + I + Γ even for those rare peripatus individuals that were having a signiﬁcantly better score (–lnL 3612.344) than other heterozygous or homozygous for allozyme alleles char- models. With the estimated proportion of invariant sites of acteristic of a different species. Thus, at Balls Clearing, 0.524, the α-shape parameter was 0.863. These parameter two P. kawekaensis individuals were heterozygous for estimates were used to recalculate the distance matrix. an AATc allele characteristic of P. sympatrica, and a Pairwise genetic distances among the ingroup taxa third P. sympatrica individual was homozygous for an (Peripatoides) calculated from COI sequences using K2P aconitase (ACON) allele characteristic of P. kawekaensis. and GTR + I + Γ, reached a maximum of 13.4% and 22.4%, At Norsewood, two P. morgani had an aspartate amino- respectively. Mean distance between clades ranged from transferase (AATa) allele typical of P. sympatrica (Trewick 3.2 to 11.4% using the K2P model, and 4.3 to 17.5% using 1998). In all these individuals their mtDNA haplotype the GTR + I + Γ models (Table 2). The highest within-taxon was concordant with the majority of their nuclear mean distance was 3.8% using the K2P model, in the markers. P. aurorbis clade (maximum 6.6%). Mean genetic distances Of the locations not surveyed for allozyme variability, between Ooperipatellus viridimaculatus and Peripatoides only one (Piano Flat, South Island) had two haplotypes. clades ranged from 12.4 to 15.9% using the K2P model, and Sequences from individuals collected at three locations in 20.8 to 32.4% using the GTR + I + Γ models. The greatest North Island (Ngaiotonga, Ball’s Clearing and Opepe), single distance was between O. viridimaculatus and a which were otherwise unambiguous, contained some Piano peripatus (16.3% using the K2P model, 34.2% using sites with ambiguous nucleotides. These suspect nucle- the GTR + I + Γ models). The two outgroup Ooperipatellus otide substitutions were coded as N. differed by 9.7% using the K2P. The greatest linear geographical distance between sample sites with iden- tical haplotypes was ≈ 190 km (P. morgani, Monckton — Genetic distance Tikitapu). Genetic distances were initially calculated using the Kimura 2-parameter (K2P) model. A NJ tree derived Phylogenetic analysis from these distances was then used to test for the most appropriate nucleotide substitution model by Phylogenetic analysis of all haplotypes using NJ with comparing likelihood scores for a suite of models: Jukes– K2P weighting produced the tree shown in Fig. 3.

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Fig. 3 Neighbour–joining (NJ) tree of mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) sequences from New Zealand Peripatoides, with Ooperipatellus as outgroup. Numbers above and below the edges are maximum parsimony (MP) bootstrap values obtained from 500 replicates using: (i) the fast-stepwise addition option of paup, version 4.0, with equal weighting across the entire data set; and (ii) the full heuristic search option with a reduced set of operational taxonomic units (OTUs) and a transversion : transition weighting of 3 : 1. Numbers beside site labels indicate sample sizes. Other symbols indicate that only one haplotype was evident from single-strand conformation polymorphism analysis (SSCP) so that the frequency of alternatives was not known (*); and OTUs scored by allozymes as P. novaezealandiae (z) or P. morgani (m).

Maximum parsimony (MP) bootstrap support was cal- set of haplotypes (Fig. 3). A Maximum likelihood (ML) culated across the entire data set using the fast-search option analysis of 12 taxa, incorporating the above GTR + I + Γ of paup 4.0, with equal weighting, and this yielded a tree statistics, yielded a tree with the same internal topology of similar topology to NJ. Repetition of this analysis as NJ and MP. with transversion : transition weighting 3 : 1 produced a These trees showed strong support for ﬁve of the six tree with the same internal topology but poor resolution species previously indicated by analysis of allozyme data of terminal lineages within clades (bootstrap values are (Fig. 4). The exception was P. morgani, which appeared to presented in Fig. 3). A reduced data set consisting of two be paraphyletic with P. novaezealandiae. Altogether, eight to four haplotypes from each of the clades indicated by clades were well supported by analysis of mtDNA, cor- the full analysis was subjected to a full heuristic search responding to ﬁve described and three undescribed yielding a single shortest MP tree. This was consistent species. Although the relationships among these taxa with the NJ tree and each clade had equivalent or higher were poorly resolved, there was some bootstrap sup- bootstrap support to that found by MP analysis of the full port (> 70%) for two clades consisting of several taxa:

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Fig. 4 Neighbour–joining (NJ) tree using the arc distance of Cavalli-Sforza & Edwards (1967) from 17 loci for 35 populations of Peripatoides. Species names are indicated, except for those samples of undetermined afﬁnity which comprised single individuals and/or were basal to the principal clades (from Trewick 1998). Samples from Pelorus and Takaka, used for allozyme analysis, were not available for mitochondrial DNA (mtDNA) analysis.

P. suteri, P. sympatrica, P. kawekaensis, P. novaezealandiae/P. taxa into two clades that were consistent with the allozyme morgani, which are all found only in North Island, and P. data (Fig. 6). aurorbis, ‘Catlins’, ‘Piano’ and ‘Dunedin’, which occur in Among the peripatus surveyed with allozymes and South Island. P. aurorbis has populations in both main mtDNA there was only one clear instance of gene ﬂow islands (Fig. 5), but mitochondrial lineages within the clade (introgression) between species. One individual from associated with northern and southern populations could Norsewood, which had nuclear markers indicative of not be separated phylogenetically. There was strong sup- P. morgani, had the same COI haplotype as P. kawekaensis port (88–96%) for a clade of exclusively North Island taxa found at Ball’s Clearing and Hutchinson (Fig. 3). that did not include P. suteri. Ambiguous sequence haplotypes encountered in The known range of some of the species detected with individuals from Ngaiotonga, Ball’s Clearing and Opepe allozymes has been extended through the use of DNA clustered within a clade consistent with the allozyme sequencing, but no new taxa are evident in the regions species P. sympatrica. Detailed comparison of haplotype previously surveyed using nuclear markers. Thus, north- sequences in this clade showed that some were unambi- ern P. aurorbis are shown to be present at Waitakere, and guous and bore only one of the alternative nucleotides at the range of the southern population extends south to the sites in question. This indicated that the ambiguous Mount Arthur and Lake Rotoroa. The range of P. sympatrica sequences could be reconciled as mixtures of alternative extends into Northland (Waipoua, Ngaiotonga, Herekino unambiguous haplotypes found in related individuals, and Puketi). and suggested heteroplasmy or nuclear integration in this Although a P. novaezealandiae/P. morgani clade was evid- clade. There was no statistical evidence of base bias or an ent in this analysis, the relationships among populations unexpected transition/transversion ratio among these were not well supported (Fig. 3). MP analysis (with equal sequences, features that would otherwise provide evid- weighting) of the 17 P. novaezealandiae/P. morgani population ence of nuclear copying. Scoring ambiguous substitutions haplotypes produced three equally short trees. These trees as ‘N’ did not prevent resolution of a phylogeny consistent grouped the majority of P. novaezealandiae and P. morgani with the allozyme data.

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Fig. 5 Approximate geographical ranges of Peripatoides taxa based on phylogenetic analysis of mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) sequence data. Sites from which peripatus bearing related haplotypes originate are indicated by approximate minimum area polygons drawn to avoid sites where a particular taxon has not yet been encountered. Some polygons are ﬁlled for clarity. Stars indicate the approximate location of rare peripatus taxa not included in this study; P. indigo, ﬁlled; undescribed species, open.

ture have changed little during the evolutionary history Discussion of these invertebrates. Interspecific sequence and allozyme divergences within Sequence evolution Peripatoides were higher than recorded for many insects A two-dimensional model of the insect COI gene has (Fig. 7). In fact, the genetic diversity of the Peripatoides been proposed that consists of a number of structural is more similar to that found in intergeneric studies of regions associated with membrane-spanning helices, insects. This highlights the extreme level of morphological external loops and internal loops (Lunt et al. 1996). The conservatism of the Onychophora (Ghiselin 1984) and pattern of amino acid variability among these various the difficulty of predicting phylogenetic suitability of genetic regions is constrained by the function of the regions. markers on the basis of morphological variation. Inter- Comparison of among-region amino acid variability of estingly, although there is evidence that these data from insects and Peripatoides revealed a very similar pattern Peripatoides are influenced by saturation (e.g. transition : (Fig. 2). A + T composition of peripatus COI DNA sequence transversion ratio) and this will affect distance calibration, overall, and at third-codon positions in particular, was the phylogenetic signal appears to be relatively strong. A also similar to that found in insects (Table 1), and together similar situation has been found in other studies of inverte- these features suggest that COI composition and struc- brates using this gene (e.g. Caterino & Sperling 1999).

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Fig. 6 Detail of North Island showing consensus support and distribution of Peripatoides novaezealandiae/P. morgani peripatus only. Small circles indicate sampling sites and allozyme genotypes: P. morgani, coarse stipple; P. novaezealandiae, coarse crosshatch; unspecified, open. Minimum area polygons indicate mitochondrial DNA (mtDNA) clustering as shown in the accompanying unweighted MP tree: P. morgani, fine stipple; P. novaezealandiae, fine crosshatch. Asterisks on the most-parsimonious (MP) tree indicate relevant nodes, supported in the three shortest trees.

viously identiﬁed by allozyme analysis, and three (Piano, Catlins, Dunedin) are based on samples from sites not included in that previous study (Trewick 1998). The com- bined evidence supports the notion that these latter, newly identiﬁed lineages also correspond to distinct species. The details of mtDNA diversity encountered in the Catlins and Dunedin areas have been discussed elsewhere (Trewick 1999), but in that analysis the distinctiveness of haplotypes from Piano Flat was not as apparent as it is in the present wider analysis. Further sampling at other locations in the vicinity is required to determine the distribution of this taxon. The species P. suteri, which is distinctive in having 16 rather than 15 pairs of legs, is well supported by mtDNA and allozyme evidence across its range. Collection ana- Fig. 7 Relationship between uncorrected mitochondrial DNA lysis indicates that the species is rare outside the Mount (mtDNA) divergence and allozyme (Nei’s D) divergence from Taranaki area (Dawson and Rotokare), a region where no peripatus and a range of insects. Peripatoides (circles); Gerris, Limnop- other Peripatoides have been reported. Four other North orous Aquarius et al , water striders (Sperling . 1997) (diamonds); Island taxa form a distinct clade according to the mtDNA Anopholes mosquitoes (triangles); Pissodes beetles (Langor & Sperling analyses. Of these, P. kawekaensis appears to have a very 1997) (squares). restricted range, in contrast to P. sympatrica which has the widest and most continuous distribution across central and upper North Island. The allozyme species P. novaezea- Systematics landiae and P. morgani are the least well differentiated by Eight distinct lineages are evident among the ingroup mtDNA, but a consensus of MP mtDNA analysis, allozyme taxa. Five of these are consistent with the grouping pre- evidence and geographical distribution do support the

RADIATION OF NEW ZEALAND ONYCHOPHORA 277

two groups (Fig. 6). Low mtDNA genetic distances, the studied using both methods (Table 2; Fig. 7). Allozyme existence of two lineages at some sites (Monckton, Otari, distances (Nei’s D) were very high, which suggests that Waiohine) and absence of recent introgression, all suggest divergence between the COI sequences is constrained that these taxa have recently speciated or are in the process by saturation so that the two measures cease to be cor- of doing so. If this is so, then minor inconsistencies related, or that Nei’s D is not amenable to this type of between allozyme and mtDNA evidence are expected calibration (Fig. 7). In a study of Jamaican peripatus, and can be explained by stochastic lineage sorting which showed low divergence, Hebert et al. (1991) used (Avise 1994). an allozymic clock calibration that assumed one unit of The near absence of introgression among the peripatus D to be equivalent to 19 Myr (Carlson et al. 1978; Vawter surveyed with nuclear and mtDNA markers demon- et al. 1980). In the present study, this would suggest strates the robustness of the described species (Fig. 3). divergence between the taxa with the highest Nei’s D Evidently, despite a lack of obvious morphological diver- (P. aurorbis and P. novaezealandiae) of > 50 Myr. Maxson sity, the Peripatoides do have effective species recogni- & Maxson (1979) used a rate of one D equivalent to tion systems and it is probable, given the nature of their 14 Myr, and Gardner & Thompson (1999) used the rate of habitat, that these involve pheromones (Eliott et al. 1993). Nei (1987, p. 247), equivalent to one unit of D per 5 Myr. In several instances of sympatry, separate species were Clearly, these various calibrations give a wide range of found not only in the same patches of forest but within time estimates and may, regardless, be largely meaningless the same decaying log. Beyond their requirement for given that the high Nei’s D-values among the Peripatoides moist, dark conditions, with suitable invertebrate prey, appear to be derived from sorting of a small number of the New Zealand Onychophora are tolerant of a wide alternative alleles among the loci surveyed, rather than range of conditions. There is no evidence for species- from mutations unique to particular taxa. The alternative specific habitat requirements, with individuals of various would require the convergent evolution of alleles. taxa being found from sea level to an altitude of at least Several studies of the rate at which mtDNA evolves 1300 m, in decaying logs of native and exotic trees, in have yielded similar estimates for distance divergence of grass tussocks, in a buried rubbish dump, under stones ≈ 2% per Myr (2%, Brown et al. 1979; 2.2–2.6%, Knowlton and scree, and in forest detritus. et al. 1993; 2.3% Brower 1994; Juan et al. 1995, 1996). How- ever, analysis of shrimp taxa across the Panama isthmus has suggested that a rate nearer 1.4% may be applicable Biogeography in at least some instances (Knowlton & Weigt 1998). COI Analysis of mtDNA data supports the allozyme evidence sequences have proved useful for studies of recently for a species-complex within New Zealand Peripatoides. diverged species because they rapidly accumulate phylo- This diversity appears to be most pronounced in North genetic information at that range. Furthermore, although Island where the distributions of taxa are complicated COI sequence data tend to saturate relatively rapidly, and overlapping. At this stage, the southern South Island phylogenetic information is retained at higher evolution- taxa appear to be parapatric. The northern South Island ary levels (Lunt et al. 1996; Caterino & Sperling 1999). taxon, P. aurorbis, is also present in central and northern Although COI accumulates substitutions at ≈ 2% per Myr North Island, and is therefore disjunct across Cook Strait. between closely related taxa, repeat substitutions cause Two further South Island morphologically distinct species this apparent rate to diminish markedly within diver- are very localized and were not available for inclusion gences of less than 15% (Juan et al. 1995). in this analysis (see Fig. 5). Other geographical gaps in In the present study, Peripatoides clades based on COI the present analysis are largely caused by the absence or sequence data differed by up to 11.4% using an often scarcity of ovoviviparous peripatus in those regions (e.g. quoted substitution model (K2P). However, by incor- west and northeast South Island). The west coast and porating a gamma correction for among-site rate variation Alps of South Island appear to be chiefly the domain of and a proportion of invariant sites, the estimated maximum the egglaying Ooperipatellus. In North Island, P. sympatrica genetic distance is 17.5% GTR + I + Γ. These estimates is the most widespread and is represented in most instances imply a probable maximum time since divergence of of sympatry. The distribution of the North Island species between 5.7 and 8.75 million years ago (Ma) using a implies high levels of sympatry, and their actual ranges rate of 2% per Myr. Diversity within clades (highest in will doubtless be broader than so far documented (Fig. 5). P. aurorbis 3.8% using the K2P model; 4.6% using the There is, however, some evidence of an east–west GTR + I + Γ model) apparently dates from ≈ 2 Myr (i.e. separation, with P. aurorbis and P. suteri in the west and Pleistocene). However, a model that accommodates the P. novaezealandiae, P. morgani and P. kawekaensis in the east. effects of saturation by reducing the estimated rate of A Mantel test showed no significant correlation of evolution over time will probably provide a more realistic allozyme and mtDNA genetic distances among the taxa indication of divergence times beyond ≈ 3 Myr ( Juan et al.

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1995; Sandoval et al. 1998). Thus, pairwise distances of unlikely to have the capacity to retain accurate phylo- 11.8% using the K2P model to 17.5% using the GTR + genetic information across that time frame (Howland & I + Γ model (the upper range in the present study of Hewitt 1995). Peripatoides) may well indicate divergence nearer to As much of New Zealands biota has traditionally been 10 Myr (Sandoval et al. 1998). While these estimates have considered to be derived directly by vicariance from to be treated with caution, they suggest that most, if not Gondwana (Fleming 1979; Steven 1981; Cooper & Millener all, of the diversity within the ovoviviparous Onycho- 1993), evidence to the contrary is of considerable interest phora of New Zealand may have arisen relatively (e.g. Linder & Crisp 1995). Further exploration of recently in the geological history of New Zealand, perhaps Onychophora and other invertebrate taxa using a range of during the middle Miocene (15–7 Myr). In New Zealand, different genes may well help to resolve this enigma. the late Miocene to early Pliocene period was marked by marine inundation events that formed a different configuration of islands than that of today. The North Acknowledgements Island area, in particular, was subdivided and marine I am grateful to the Department of Conservation for the provision straits formed across its lower third (Stevens 1981). of relevant collecting permits. I wish to thank Mary Morgan- During the Pliocene (7–2 Myr), the South Island mountain Richards, Jon Waters, Graham Wallis, Andrea Taylor, Paul ranges developed, and in the Pleistocene (< 2 Myr) Sunnucks, Warren Chin, Tony Harris, Ken Mason, Dave Randal, volcanic activity formed mountains in North Island and Malcolm Haddon, Scott Dunavan, Ray Forster, Lyn Forster, Sue Millar, Karen Tutt and colleagues for their contribution to the would have been the cause of extinction events in the sampling. Many thanks to the members of the Evolutionary central region. At this time, North and South Island were Genetics Laboratory for providing feedback on various aspects a single entity, having been separated by the Cook Strait of the work, and an amiable environment. Thanks also to Paul for only ≈ 16 000 years (Lewis et al. 1994). Sunnucks and Alex Wilson who introduced me to the wonders Habitat fragmentation during the late Miocene may of simple SSCP, and David Lunt who provided data on insect have given rise to allopatric peripatus populations, but COI. Johns Hobbs (Nucleic Acid — Protein Service, NAPS Unit, also led to extinction of some taxa. Dispersal between the University of British Columbia, Vancouver, BC, Canada) supplied insect mitochondrial DNA primers, taken from sequences compiled two modern islands would not have been subsequently by Dr B. Crespi (Simon Fraser University, Burnaby, BC, Canada) hampered by sea until after the Pleistocene, and rapid and described on postings to [email protected]. This work was climate fluctuations at this time could have led to popu- supported by a Foundation for Research Science and Technology lation genetic structuring. The absence of deeper molecu- fellowship (contract UOO704). lar diversity and concomitant morphological variation may be the result of high levels of extinction into the References Miocene. Large-scale extinction of New Zealand biota during Oligocene marine inundation has previously been Avise JC (1994) Molecular Markers, Natural History and Evolution. proposed (Cooper & Millener 1993). Alternatively, the Chapman & Hall, New York. molecular evidence may imply an unexpectedly recent Ballard JWO, Olsen GJ, Faith DP, Odgers WA, Rowell DM, arrival of Onychophora in New Zealand. Atkinson PW (1992) Evidence from 12S ribosomal RNA sequences that Onychophorans are modified arthropods. Science, 258, Analysis of COI sequence data from Australian Onycho- 1345–1348. phora plus representatives from New Zealand and Briggs DEG, Fortey RA, Wills MA (1992) Morphological dispar- South America revealed a maximum genetic distance of ity in the Cambrian. Science, 256, 1670–1673. 20.6% (Gleeson et al. 1998). Genetic distance between Briscoe DA, Tait NN (1995) Allozyme evidence for extensive and some Tasmanian and New Zealand Onychophora was as ancient radiations in Australia Onychophora. Zoological Journal low as 10.5%. While it is apparent that these relatively of the Linnean Society, 114, 91–102. low levels are to a greater or lesser extent the result of Brower AVZ (1994) Rapid morphological radiation and conver- gence among races of the butterfly Helioconius erato inferred saturation, greater distances have been reported from from patterns of mitochondrial DNA evolution. Proceedings of other invertebrate taxa for the same gene (e.g. 27% in the National Academy of Sciences of the USA, 91, 6491–6495. Coleoptera, Howland & Hewitt 1995; 30% in Orthoptera, Brown JM, Pellmyr O, Thompson JN, Harrison RG (1994) Phylo- S. A. Trewick, unpublished). This suggests that either the geny of Greya (Lepidoptera: Proxidae), based on nucleotide extant global Onychophoran fauna evolved far more sequence variation in mitochondrial cytochrome oxidase I and recently than Gondwana vicariance would suggest (e.g. II: congruence with morphological data. Molecular Biology and 11 140 Myr split of S. America from other continents) or that Evolution, , 128–141. Brown WM, George M Jr, Wilson AC (1979) Rapid evolution the evolutionary rate and/or capacity for change of of animal mitochondrial DNA. Proceedings of the National COI in Onychophora is, despite the evidence of amino Academy of Sciences of the USA, 76, 1967–1971. acid variability, etc., described above, very different from Carlson JW, Amato GD, Maxson RD (1978) Do albumin clocks that in other invertebrates. Either way, the COI gene is run on time? A reply. Science, 200, 1183–1185.

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Appendix 1 Details of sampling sites: location names, latitude/longitude and other notes of interest

North Island Ngaiotonga reserve (35°18′: 174°15′); Waipoua forest (35°38′: 173°33′); Herekino (35°09′: 173°16′); Puketi forest (35°16′: 173°40′); Waitakere forest (36°54′: 174°32′); Kawau Island [two sites separated by > 2 km] (36°30′: 174°40′); Waiwawa, Coromandel Range (36°59′: 175°37′); Kaueranga, Coromandel Range (37°06′: 175°38′); Forthbranch, Coromandel Range (37°08′: 175°44′); Waitomo Caves reserve (38°15′: 175°06′); Mangatutara, Raukumara Range (37°55′: 177°55′); Lake Tikitapu (38°11′: 176°20′); Opepe historic reserve, Taupo (38°42′: 176°10′); Rangataiki (38°59′: 175°39′); Kakaho, Pureora forest (38°33′: 175°43′); Ball′s Clearing reserve (39°16′: 176°39′); Hutchinson reserve (39°16′: 176°32′); Tangoio, White Pine reserve (39°18′: 176°49′); Oueroa [farm] (40°06′: 176°41′); Mohi Bush reserve (39°35′: 177°05′); Monckton reserve (39°57′: 176°16′); Miller reserve (40°42′: 175°39′); ANZAC reserve, Norsewood (40°52′: 176°13′); Saddle Road (40°17′: 175°49′); Bideford (40°51′: 175°52′); Bideford south [paddock] (40°50′: 175°52′); Perry’s Road, Carterton (41°00′: 175°35′); Pahiatua [paddock] (40°26′: 175°47′); Waiohine reserve (40°59′: 175°23′); Akatarawa (40°57′: 175°06′); Otari plant museum (41°6′: 174°45′); Waiwawa, Coromandel Range (36°59′: 175°37′); Whakapapa village (39°12′: 175°32′); Dawson Falls (39°19′: 174°06′); Lake Rotokare (39°27′: 174°24′). South Island Takaka scenic reserve (40°45′: 172°55′); Pelorus Bridge (41°05′: 173°30′); Cobb valley (41°05′: 172°43′); Mnt Arthur (41°11′: 172°44′); Pyramid Pk (41°11′: 172°40′); Lake Rotoroa (41°47′: 172°30′); Gunn’s Bush (44°39′: 170°57′); Peel Forest (44°53′: 171°15′); Trotter′s Gorge (45°24′: 170°47′); Kakanui Mnts [in scree] (45°56′: 170°28′); Caversham Valley (45°53′: 170°28′); Saddle Hill (45°54′: 169°21′); Taieri Mouth (46°03′: 170°11′); Outram (45°50′: 170°14′); Maungatua (45°53′: 170°08′); Piano Flat (45°33′: 169°01′); Tom’s Creek (45°54′: 169°29′); Hokonui (46°04′: 168°50′); Matai Falls (46°30′: 169°29′); Haldane (46°34′: 169°00′).