Molecular systematics and evolution in New Zealand: Applications to cryptic skink species

Abstract

The polymerase chain reaction (PCR) and DNA sequencing are being used for the study of the origin and evolution of New Zealand plants and animals. Here we describe methods for rapidly obtaining DNA sequences from small amounts of fresh and preserved tissue, and apply them to the problem ofresolving relationships among skinks of the Austrnlasian genus Leiolopisma. DNA extracted from frozen tail muscle was used for the main part of the study. Part ofthe mitochondrial12S ribosomal RNA gene was isolated using PCR. Direct sequencing of this 400 nucleotide region supports the separation of “L. nigriplantare” into several genetically distinct but morphologically similar species. The sequence data have also identified a potential case of hybridisation between two sympatric species in Otago, L. nigriplantare polychroma and L. maccanni. The data set supports the view that skinks have been in New Zealand very much longer than five million years.

From these results we expect that application of the PCR and other techniques in molecular biology will help solve many questions about the origins and evolution of New Zealand's biota, as well as help identify unique populations for conservation.     

Phylogeography of two New Zealand lizards: McCann's skink (Oligosoma maccanni) and the brown skink (O. zelandicum)

The New Zealand skink fauna has proven to be an ideal taxonomic group in which to examine the impact of climatic and geological processes on the evolution of the New Zealand biota since the Pliocene. Here we examine the phylogeography of McCann's skink (Oligosoma maccanni) in order to gain insight into the relative contribution of Pliocene and Pleistocene processes on patterns of genetic structure in the South Island biota, and investigate the phylogeography of the brown skink (O. zelandicum) to examine whether Cook Strait landbridges facilitated geneflow between the North and South Islands in the late-Pleistocene. We obtained mitochondrial DNA sequence data (ND2 and ND4; 1282bp) from across the range of both species. We examined the phylogeographic patterns evident in each species using Neighbour-Joining, Maximum Likelihood and Bayesian methods. We found substantial phylogeographic structure within O. maccanni, with seven distinct clades identified. Divergences among clades are estimated to have occurred during the Pliocene. Populations in the Otago/Southland region (south of the Waitaki River valley) formed a well-supported lineage within O. maccanni. A substantial genetic break was evident between populations in east and west Otago, either side of the Nevis-Cardrona fault system, while north-south genetic breaks were evident within the Canterbury region. Within-clade divergences in O. maccanni appear to have occurred during the mid- to late-Pleistocene. Shimodaira-Hasegawa topology tests indicated that the 'Garston' skink is not genetically distinct from O. maccanni. There was only relatively minor phylogeographic structure within O. zelandicum, with divergences among populations occurring during the mid- to late-Pleistocene. Our genetic data supports a single colonisation of the North Island by O. zelandicum from the South Island, with the estimated timing of this event (0.46mya) consistent with the initial formation of Cook Strait.     

Sympatric cryptic species in New Zealand Onychophora

Allozyme electrophoresis was used to examine genetic diversity within live-bearing Peripatopsid Onychophora from the North Island of New Zealand. Specimens of two previously described morpho-species that differ in leg number (Peripatoides suteri and P. novaezealandiae) were found to be genetically diverse. P. suteri showed little intraspecific genetic variation but were very distinct from specimens assignable to P. novaezealandiae. Within P. novaezealandiae five genetically differentiated species were identified although none showed any consistent morphological differentiation, thus P. novaezealandiae (Hutton) is a species complex. All of these species occur in sympatry or parapatry (in one instance) with other cryptic species of the P. novaezealandiae group or with P. suteri. Four new species are described on the basis of this genetic evidence, they are P. morgani, P. aurorbis, P. kawekaensis and P. sympatrica. Other genotypes encountered indicate further cryptic species remain unrecognized. Among the North Island species, P. suteri and P. aurorbis sp. nov. are both more closely related to undescribed species from the South Island than to others examined from the North Island. P. sympatrica sp. nov. exists in sympatry with at least three other species in different parts of its range. The complexity of relationships and distributions probably arose through the interaction of low vagility in peripatus and the active geophysical history of the region. How these cryptic species persist in sympatry is not known but may be linked to differences in ecology not evident in their morphology, and/or may indicate recent dispersal from allopatry.     

Mitochondrial DNA sequences support allozyme evidence for cryptic radiation of New Zealand Peripatoides (Onychophora)

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 + Gamma) 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.     

Genetic divergences pre-date Pleistocene glacial cycles in the New Zealand speckled skink, Oligosoma infrapunctatum

Aim To examine the hypothesis raised by Graham S. Hardy that Pleistocene glacial cycles suffice to explain divergence among lineages within the endemic New Zealand speckled skink, Oligosoma infrapunctatum Boulenger. Location Populations were sampled from across the entire range of the species, on the North and South Islands of New Zealand. Methods We sequenced the mitochondrial genes ND2 (550 bp), ND4 + tRNAs (773 bp) and cytochrome b (610 bp) of 45 individuals from 21 locations. Maximum likelihood, maximum parsimony and Bayesian methods were used for phylogenetic reconstruction. The Shimodaira–Hasegawa test was used to examine hypotheses about the taxonomic status of morphologically distinctive populations. Results Our analysis revealed four strongly supported clades within O. infrapunctatum. Clades were largely allopatric, except on the west coast of the South Island, where representatives from all four clades were found. Divergences among lineages within the species were extremely deep, reaching over 5%. Two contrasting phylogeographical patterns are evident within O. infrapunctatum. Main conclusions The deep genetic divisions we found suggest that O. infrapunctatum is a complex of cryptic species which diverged in the Pliocene, contrary to the existing Pleistocene‐based hypothesis. Although Pleistocene glacial cycles do not underlie major divergences within this species, they may be responsible for the shallower phylogeographical patterns that are found within O. infrapunctatum, which include a radiation of haplotypes in the Nelson and Westland regions.     

Lizards in the suburbs: a single-garden study of a small endemic New Zealand skink (Oligosoma aeneum)

Detailed investigations of ecology and life history of lizards in New Zealand/Aotearoa are needed to inform their conservation and management. An early demographic study of copper skinks (Oligosoma aeneum) in a suburban garden was undertaken in Lower Hutt using mark–recapture methods, sampling weekly from March 1971 to December 1973. Oligosoma aeneum were seen on 1372 occasions, with 972 captures of 169 individuals. The maximum snout–vent length was 66 mm, 33% of skinks had complete tails, and colour differences suggested sexual signalling and warrant further study. Most captures occurred over November–March, with fewest over June–August. The estimated spring/summer population size in the garden (± SEM) was 83.6 (± 9.4) skinks, and the annual survival rate (± SEM) was 38.0% (± 16.0%). This is one of the few New Zealand lizard studies that have extended through all months of the year and it adds to limited information on lizard populations in suburban areas.     

Simultaneous polyphenism and cryptic species in an intertidal limpet from New Zealand

The small intertidal limpets known under the name Notoacmea helmsi occupy a wide variety of habitats in New Zealand and exhibit a variety of shell forms. Phylogenetic analyses of DNA sequences from two genes, mitochondrial COI and nuclear ITS1, reveal that this taxon comprises at least five morphologically cryptic species, with at least one of these species, N. scapha, consisting of individuals with two obviously different shell types. One of these forms is an ecophenotypic response to living on eelgrass (Zostera) fronds. Unlike its extinct relative, Lottia alveus, N. scapha is not restricted to this substrate, but individuals living elsewhere are larger and have a different shell shape. Although there is significant overlap in shell form among the different cryptic species, there is some habitat differentiation, with two species predominantly found on exposed shores and three confined to mudflats. One species exhibits distinctive light-avoiding behaviour, the first known case in which behaviour can be used to separate cryptic species in molluscs.     

Improving the preregistration experience: the New Zealand approach.

There is currently much debate about how to improve undergraduate medical education, and in particular on how best to prepare students for clinical responsibility. For 20 years a period of trainee internship has formed part of New Zealand medical students'' undergraduate training, and the model could have much to offer the United Kingdom. Students take their final examinations at the end of the second clinical year; they spend their final year in a series of eight clinical attachments, during each of which they shadow a preregistration house officer or senior house officer. As trainee interns they are paid 60% of a house officer''s salary for their clinical work, which is supervised by the firm''s registrars and consultants under the overall responsibility of the head of the academic department. The order of the attachments is determined on educational, not service, grounds, and trainees have to attend educational sessions and pass assessments on each attachment. The trainee internship, funded jointly by the education and health departments, offers a more seamless transition from student to house officer and aims at improving both general medical education and clinical training.     

A SINE of restricted gene flow across the Alpine Fault: phylogeography of the New Zealand common skink (Oligosoma nigriplantare polychroma)

New Zealand has experienced a complex climatic and geological history since the Pliocene. Thus, identifying the processes most important in having driven the evolution of New Zealand's biota has proven difficult. Here we examine the phylogeography of the New Zealand common skink ( Oligosoma nigriplantare polychroma ) which is distributed throughout much of New Zealand and crosses many putative biogeographical boundaries. Using mitochondrial DNA sequence data, we revealed five geographically distinct lineages that are highly differentiated (pairwise Φ_ST 0.54–0.80). The phylogeographical pattern and inferred age of the lineages suggests Pliocene mountain building along active fault lines promoted their divergence 3.98–5.45 million years ago. A short interspersed nuclear element (SINE) polymorphism in the myosin gene intron ( MYH-2 ) confirmed a pattern of restricted gene flow between lineages on either side of the mountain ranges associated with the Alpine Fault that runs southwest to northeast across the South Island of New Zealand. An analysis of molecular variance confirmed that ~40% of the genetic differentiation in O. n. polychroma is distributed across this major fault line. The straits between the main islands of New Zealand accounted for much less of the variation found within O. n. polychroma , most likely due to the repeated existence of landbridges between islands during periods of the Pleistocene that allowed migration. Overall, our findings reveal the relative roles of different climatic and geological processes, and in particular, demonstrate the importance of the Alpine Fault in the evolution of New Zealand's biota.     

Reproduction and growth of the black shore skink,Leiolopisma suteri (Lacertilia: Scincidae), in north‐eastern New Zealand

Reproduction and growth of a boulder beach population of the nocturnal skink Leiolopisma suteri on Motutapu Island were studied for 19 months. Epididymes contain sperm from April to November, and copulation probably occurs in October or November (spring). Ovulation occurs in late October or early November, and three or four (mean, 3.7) eggs are laid in late December. Incubation takes 3 months. Total development time from ovulation to hatching is 5 months, one of the longest development periods recorded for Scincidae. Growth to sexual maturity is slow, most females depositing their first clutch 33 months after hatching; males mature at 29 months.     

Phylogeographic analysis of the brooding brittle star Amphipholis squamata (Echinodermata) along the coast of New Zealand reveals high cryptic genetic variation and cryptic dispersal potential

Abstract.— Direct development in benthic marine invertebrates is usually associated with narrow geographical range, low rates of colonization, and low levels of gene flow. Paradoxically, the small brittle star Amphipholis squamata broods its larvae to a crawl-away juvenile stage, yet has a cosmopolitan distribution. Using sequence and restriction-fragment-length-polymorphisms (RFLP) analyses of nuclear and mitochondrial DNA from 16 coastal populations throughout New Zealand, we tested whether the species is indeed a poor disperser, as may be expected from its brooding habit. We predicted that local and regional populations would be genetically structured according to isolation by distance. We also suspected that this ubiquitous "species" is composed of a variety of cryptic taxa in different geographic areas, as has been discovered in an increasing number of marine invertebrates. We found evidence of four genetically divergent and reproductively isolated lineages that can exist in syntopy. Lineages vary in abundance, haplotype diversity, and geographic distribution. The partitioning of genetic variation within the most common lineage, as well as the geographic distribution of the four lineages, suggest a north/south split. This pattern is consistent with known New Zealand marine biogeographic zones and appears to be linked to the regime of oceanic circulation, which is characterized by subtropical, southward-moving water masses in the north, and sub-Antarctic, northward-moving water in the south. We conclude that the dispersal ability of A. squamata is regionally restricted but with sporadic long-distance dispersal, which serves to increase local genetic variation. Our results support the idea that dispersal occurs through passive transport by drifting or rafting on macroalgae, which A. squamata commonly inhabits, and emphasize that poor dispersal ability is not necessarily a corollary of direct development.     

White clover cryptic virus‐1 in New Zealand and eastern Australia

The distribution and abundance of cryptic viruses (Partitiviridae) in natural and agricultural systems have received little attention. White clover cryptic virus‐1 (WCCV‐1) was detected infecting white clover plants and seed growing in New Zealand, but was not detected in field collections and in only two of 114 seedlings grown from seed collected in eastern Australia using enzyme‐linked immunosorbent assay and RT‐PCR. WCCV‐1 was detected at high incidences (14–48%) in the cultivars Huia, Kopu II and Tahora and at low incidences (0–7%) in Aran, Haifa, Ladino, Siral and Sustain white clovers. WCCV‐1 remained viable in seed stored for 50 years. The results are compared with another group of obligate seed‐transmitted pasture microbes: the fungal endophytes of perennial grasses.     

A new skink species (Oligosoma taumakae sp. nov.; Reptilia: Scincidae) from the Open Bay Islands,New Zealand

Abstract

We describe a new skink species (Oligosoma taumakae sp. nov.) from the Open Bay Islands, New Zealand. This species is diagnosed on the basis of several morphological characteristics, and its specific status is supported by mitochondrial sequence data (ND2, ND4). The new species appears to be most closely related to O. acrinasum, O. infra‐punctatum, O. otagense and O. waimatense. The new taxon appears to be rare and endemic to the island of Taumaka in the Open Bay Islands (off the west coast of the South Island). Predation by a flightless rail (weka, Gallirallus australis), native to New Zealand but introduced to the Open Bay Islands, is a major conservation concern.     

Phylogenetic analysis of New Zealand earthworms (Oligochaeta: Megascolecidae) reveals ancient clades and cryptic taxonomic diversity

We have constructed the first ever phylogeny for the New Zealand earthworm fauna (Megascolecinae and Acanthodrilinae) including representatives from other major continental regions. Bayesian and maximum likelihood phylogenetic trees were constructed from 427 base pairs from the mitochondrial large subunit (16S) rRNA gene and 661 base pairs from the nuclear large subunit (28S) rRNA gene. Within the Acanthodrilinae we were able to identify a number of well-supported clades that were restricted to continental landmasses. Estimates of nodal support for these major clades were generally high, but relationships among clades were poorly resolved. The phylogenetic analyses revealed several independent lineages in New Zealand, some of which had a comparable phylogenetic depth to monophyletic groups sampled from Madagascar, Africa, North America and Australia. These results are consistent with at least some of these clades having inhabited New Zealand since rifting from Gondwana in the Late Cretaceous. Within the New Zealand Acanthodrilinae, major clades tended to be restricted to specific regions of New Zealand, with the central North Island and Cook Strait representing major biogeographic boundaries. Our field surveys of New Zealand and subsequent identification has also revealed extensive cryptic taxonomic diversity with approximately 48 new species sampled in addition to the 199 species recognized by previous authors. Our results indicate that further survey and taxonomic work is required to establish a foundation for future biogeographic and ecological research on this vitally important component of the New Zealand biota.