Phylogeography and ecological niche modelling of the New Zealand stick insect Clitarchus hookeri (White) support survival in multiple coastal refugia

Aim Increasing our understanding of the effects of the Last Glacial Maximum (LGM) and determining the location of refugia requires studies on widely distributed species with dense sampling of populations. We have reconstructed the biogeographic history of Clitarchus hookeri (White), a widespread species of New Zealand stick insect that exhibits geographic parthenogenesis, using phylogeographic analysis and ecological niche modelling. Location New Zealand. Methods We used DNA sequence data from the mitochondrial cytochrome c oxidase subunit I gene to reconstruct phylogenetic relationships among haplotypes from C. hookeri and two undescribed Clitarchus species. We also used distribution data from our own field surveys and museum records to reconstruct the geographic distribution of C. hookeri during the present and the LGM, using ecological niche modelling. Results The ecological niche models showed that the geographic distribution of C. hookeri has expanded dramatically since the LGM. Our model predicted large areas of suitable LGM habitat in upper North Island, and small patches along the east coast of South Island. The phylogeographic analysis shows that populations in the northern half of North Island contain much higher levels of genetic variation than those from southern North Island and South Island, and is congruent with the ecological niche model. The distribution of bisexual populations is also non-random, with males completely absent from South Island and very rare in southern North Island. Main conclusions During the LGM C. hookeri was most likely restricted to several refugia in upper North Island and one or more smaller refugia along the east coast of South Island. The unisexual populations predominate in post-glacial landscapes and are clearly favoured in the recolonization of such areas. Our study exemplifies the utility of integrating ecological niche modelling and phylogeographic analysis.     

Investigating hybridization in the parthenogenetic New Zealand stick insect Acanthoxyla (Phasmatodea) using single-copy nuclear loci

The New Zealand stick insect genus Acanthoxyla Uvarov is extremely unusual among higher taxa of animals in that all known species are obligate parthenogens. We have used a combination of the mitochondrial DNA genes cytochrome oxidase subunits I and II, 28S nuclear ribosomal RNA, and the two single-copy nuclear genes elongation factor 1alpha and phosphoglucose isomerase to test hypotheses on the role of hybridization in the evolution of this genus. Alleles at the single-copy nuclear loci in three sampled species of Acanthoxyla were resolved by cloning the PCR products. Analysis of multilocus genotypes shows that most sampled individuals of Acanthoxyla possess three alleles at the single-copy nuclear loci, which we have interpreted to indicate triploidy. Because most of the alleles from Acanthoxyla form a monophyletic group, including sets of alleles possessed by the putative triploids, we have inferred that the extant parthenogenetic lineages formed via hybridization between species of Acanthoxyla, at least one of which must have been sexual. More recently, there have been multiple introgression events from the related species Clitarchus hookeri White, although C. hookeri does not appear to be involved with the origin of parthenogenesis in Acanthoxyla. Our study demonstrates the utility of cloning alleles from multiple single-copy nuclear genes for resolving the origins of parthenogenetic lineages.     

An enigmatic new stick insect from the Philippine Islands (Insecta: Phasmatodea)

A new genus and species of stick insect is described and figured from Mount Halcon, on the Philippine island of Mindoro. Conlephasma enigma gen. et sp. n. is a stout, flightless, and apparently ground-dwelling species with vivid integumental colors. When disturbed, specimens spray a defensive secretion from the prothoracic exocrine glands. The systematic position of Conlephasma within Euphasmatodea is unclear. The elongated galealobulus and the trichome area located laterally in the galea, represent unusual apomorphic characters of the maxilla that could indicate affinities with Necrosciinae or Pseudophasmatinae. All tibiae exhibit the anareolate condition. Euplantulae are of two types: those of tarsomeres I–IV feature a nubby microstructure, whilst the one on the ventral side of the pretarsus is smooth. Males are characterized by the presence of a well-developed vomer on the tenth abdominal segment. A distinctive and apomorphic trait of female terminalia is represented by the elongated tenth abdominal tergum. Conlephasma can represent an interesting taxon for studies on the evolution of the stick and leaf insects.     

Identifying glacial refugia in a geographic parthenogen using palaeoclimate modelling and phylogeography: the New Zealand stick insect Argosarchus horridus (White)

We have used phylogeographic analysis of mitochondrial DNA (COI and COII genes) and ecological niche modelling (ENM) to reconstruct the population history of Argosarchus horridus (White), a widespread species of New Zealand stick insect. These data were used to address outstanding questions on the role of glacial refugia in determining the distribution and genetic structure of New Zealand species. Phylogeographic analysis shows a general pattern of high diversity in upper North Island and reduced diversity in lower North Island and South Island. The ENM indicates that during the last glacial maximum, A. horridus was largely restricted to refugia around coastal areas of North Island. The ENM also suggests refugia on the northeast coast of South Island and southeast coast of North Island and this prediction is verified by phylogeographic analysis, which shows a clade restricted to this region. Argosarchus horridus is also most likely a geographic parthenogen where males are much rarer at higher latitudes. The higher levels of genetic variation in northern, bisexual populations suggest southern and largely unisexual populations originated from southwardly expanding parthenogenetic lineages. Bayesian skyline analysis also provides support for a recent population size increase consistent with a large increase in geographic distribution in the late Pleistocene. These results exemplify the utility of integrating ENM and phylogeographic analysis in testing hypotheses on the origin of geographic parthenogenesis and effects of Pleistocene environmental change on biodiversity.     

Genetic divergence of three freshwater isopod species from southern New Zealand

Aim We examined the biogeography of three freshwater isopod species (Austridotea annectens, A. lacustris, A. benhami), and tested the hypotheses that genetic differences would: (1) exist between geographic locations; and (2) correspond to known geological events (e.g. appearance of islands leading to the availability of habitat). Location Southern New Zealand, including South Island, Stewart Island, Campbell Island and Chatham Islands. Methods We examined specimens throughout the known species range from 12 populations of A. lacustris, five populations of A. annectens, and three populations of A. benhami, using mitochondrial DNA (cytochrome c oxidase I) sequence analyses. Results We resolved three main clades corresponding to the three species, with 16% sequence divergence between A. annectens and A. benhami, and 31% divergence between these species and A. lacustris. Divergence within A. benhami was < 2.0%. However, divergence within A. lacustris reached up to 10% with four main groupings: (1) Chatham Islands; (2) Campbell Island; (3) Fiordland; and (4) east coast South Island and Stewart Island. Divergence within A. annectens reached up to 4.4%, with two main groupings: (1) Chatham Islands and (2) east coast South Island and Stewart Island. Patterns of genetic divergence were most likely the result of geographical isolation among A. lacustris and A. annectens populations. In particular, the divergence of A. lacustris and A. annectens on Chatham Islands may correspond to the availability of this habitat c. 4 Ma, whereas the divergence of A. lacustris on the much older Campbell Island and in Fiordland may indicate either a rare founder event or a change in ocean circulation that resulted in their isolation from a once more widespread gene pool. Main conclusions The three New Zealand species of Austridotea are genetically distinct, with up to 31% divergence between species. Genetic variability was highest between populations of the two most widely distributed species, and divergence was greatest on islands distant from mainland New Zealand and in the discrete Fiordland region. The magnitude of genetic divergence of isopods on the Auckland and Chatham Islands is consistent with these populations having been founded in the Pliocene via oceanic dispersal from mainland New Zealand.     

Phylogenetic estimation of Mytilidae in the East China Sea inferred from mitochondrial genes and nuclear DNA sequence variation

We performed a phylogenetic estimation of the family Mytilidae in the East China Sea based on nuclear internal transcribed spacer (ITS) genes and two mitochondrial genes (COI and 16S RNA). Analysis of five mytilid species based on each of the three genes resulted in mostly congruent trees, although there were some discrepancies in the classification of these species. We combine the results obtained from the three separate analyses to provide a phylogenetic estimation of Mytilidae. We found that the Mytilidae was divided into two major lineages: in one clade, Mytilus galloprovincialis was grouped with Mytilus coruscus; in the second clade, Septifer bilocularis was placed at the basal position in an individual clade, and Perna viridis and Musculista senhousia were recovered as a monophyletic group. Although these finding provide important insights into the taxonomic relationships among the Mytilidae, many aspects of Mytilidae phylogeny remain unresolved. Further analysis based on more molecular information and extensive taxon sampling is necessary to elucidate the phylogenetic relationships among the major lineages within the Mytilidae.     

External macro- and micromorphology of the male of the stick insect Hermarchus leytensis (Insecta: Phasmatodea) with phylogenetic considerations

External morphological features of adult males are described in the stick insect Hermarchus leytensis from the Philippines, a species belonging to the little-known euphasmatodean lineage Stephanacridini. Mouthparts are characterized by some likely specialized features: (1) a dentate dorsal cutting edge on the mandibles; (2) distinct differences in size and shape between the galeae; (3) absence of an apical field of trichomes on the galeae; and (4) lacinial setae not protruding from the mesal margin, which features three bearing-like protuberances. The latter character state represents a very unusual condition, not known in other phasmatodeans or even in polyneopteran insects. A distinctive characteristic of attachment devices is that each euplantula is divided into two separated pads with a smooth microstructure, as it also occurs in some members of the clade Schizodecema. Male terminalia exhibit character states previously unknown in Stephanacridini, including (1) a symmetrical type of vomer and (2) claspers equipped with ∼70 very minute ventral teeth on each thorn pad. Potential implications for the systematic placement of H. leytensis are discussed. The results also underline the importance of microanatomical investigations as source of substantial characters for future analyses on phasmatodean systematics.     

Crossing the Tasman Sea: Inferring the introduction history of Litoria aurea and Litoria raniformis (Anura: Hylidae) from Australia into New Zealand

Abstract The amphibian fauna of New Zealand consists of three native species (Leiopelma spp.), and three Litoria species introduced from Australia in the last 140 years. We conducted a molecular phylogeographical study that aimed to identify the Australian origins of two species, Litoria aurea and Litoria raniformis. We used partial sequences of the mitochondrial cytochrome oxidase I (cox1) gene from 59 specimens sampled from across the range of both species to identify the probable source populations for the New Zealand introductions, and to describe the current genetic diversity among New Zealand Litoria populations. Our genetic data suggest that L. aurea was introduced into the North Island of New Zealand from two regions in Australia, once from the northern part of coastal New South Wales and once from the southern part of coastal New South Wales. Our data indicate that L. raniformis introductions originated from the Melbourne region of southern Victoria and once established in the South Island of New Zealand, the species subsequently spread throughout both islands. In addition, we found a distinct haplotype in L. raniformis from Tasmania that strongly suggests, contrary to earlier reports, that this species was not introduced into New Zealand from Tasmania. Finally, we identified two very distinctive mitochondrial lineages of L. raniformis within the mainland Australia distribution, which may be previously unrecognized species.     

Mitochondrial phylogeography of the Dalmatian wall lizard, Podarcis melisellensis (Lacertidae)

A 903 bp section of the mitochondrial cytochrome b gene was sequenced from 73 specimens of Podarcis melisellensis collected at 52 localities distributed over the major part of the species’ range. In addition, parts of the 12S (about 470 bp) and 16S rRNA (about 500 bp) genes were analysed for 11 representative samples leading to a congruent phylogeny. Our study includes representatives of all 20 subspecies recognized today. The phylogenetic analysis of the sequence data revealed three main clades: mainland with nearby islands, Vis archipelago, and Lastovo archipelago. The degree of mitochondrial DNA divergence among these clades suggests a separation of the respective population groups during the earliest Pleistocene. The phylogenetic pattern observed within the species is in sharp contrast to the actual taxonomic division into subspecies. A correlation between genetic diversity of P. melisellensis populations and paleogeography of the regions they inhabit is discussed.     

Independent suboesophageal neuronal innervation of the defense gland and longitudinal muscles in the stick insect (Peruphasma schultei) prothorax

This study investigates the neuroanatomy of the defense gland and a related muscle in the stick insect Peruphasma schultei with axonal tracing and histological sections. The gland is innervated by three neurons through the Nervus anterior of the suboesophageal ganglion (SOG), the ipsilateral neuron (ILN), the contralateral neuron (CLN) and the prothoracic intersegmental neuron (PIN). The ILN has a large soma which is typical for motoneurons that cause fast contraction of large muscles and its dendrites are located in motor-sensory and sensory neuropile areas of the SOG. The CLN might be involved in the coordination of bilateral or unilateral discharge as its neurites are closely associated to the ILN of the contralateral gland. Close to the ejaculatory duct of the gland lies a dorsal longitudinal neck muscle, musculus pronoto-occipitalis (Idlm2), which is likely indirectly involved in gland discharge by controlling neck movements and, therefore, the direction of discharge. This muscle is innervated by three ventral median neurons (VMN). Thus, three neuron types (ILN, CLN, and PIN) innervate the gland muscle directly, and the VMNs could aid secretion indirectly. The cytoanatomy of motorneurons innervating the defense gland and neck muscle are discussed regarding the structure and functions of the neuropile in the SOG. As a basis for the neuroanatomical study on the defense gland we assembled a map of the SOG in Phasmatodea.     

Phylogeographic history of the yellow-necked fieldmouse (Apodemus flavicollis) in Europe and in the Near and Middle East

The exact location of glacial refugia and the patterns of postglacial range expansion of European mammals are not yet completely elucidated. Therefore, further detailed studies covering a large part of the Western Palearctic region are still needed. In this order, we sequenced 972 bp of the mitochondrial DNA cytochrome b (mtDNA cyt b) from 124 yellow-necked fieldmice (Apodemus flavicollis) collected from 53 European localities. The aims of the study were to answer the following questions: Did the Mediterranean peninsulas act as the main refuge for yellow-necked fieldmouse or did the species also survive in more easterly refugia (the Caucasus or the southern Ural) and in Central Europe? What is the role of Turkey and Near East regions as Quaternary glacial refuges for this species and as a source for postglacial recolonisers of the Western Palearctic region? The results provide a clear picture of the impact of the quaternary glaciations on the genetic and geographic structure of the fieldmouse. This species survived the ice ages in two main refuges, the first one in the Italo-Balkan region; the second one in Turkey and the Near East regions. It is from the Balkan refuge that it recolonised all European regions at the end of the last glaciation. The Turkish and Near East populations are distinct from the European ones and they did not recolonise the Palearctic region probably because: (i) they were blocked by the Black Sea and the Caucasus, (ii) the long term presence of fieldmice populations in the Balkans prevented their expansion. These are genetically differentiated from the European and Russian ones and could be described as a particular subspecies. This result emphasises the importance of Turkey and the Near and Middle East regions as a refuge for Palearctic mammals.     

Biogeography and phylogeny of the New Zealand cicada genera (Hemiptera: Cicadidae) based on nuclear and mitochondrial DNA data

Aim Determine the geographical and temporal origins of New Zealand cicadas. Location New Zealand, eastern Australia and New Caledonia. Methods DNA sequences from 14 species of cicadas from New Zealand, Australia, and New Caledonia were examined. A total of 4628 bp were analysed from whole genome extraction of four mitochondrial genes (cytochrome oxidase subunits I and II, and ribosomal 12S and 16S subunits) and one nuclear gene (elongation factor‐1 alpha). These DNA sequences were aligned and analysed using standard phylogenetic methods based primarily on the maximum likelihood optimality criterion. Dates of divergences between clades were determined using several molecular clock methods. Results New Zealand cicadas form two well‐defined clades. One clade groups with Australian taxa, the other with New Caledonian taxa. The molecular clock analyses indicate that New Zealand genera diverged from the Australian and New Caledonian genera within the last 11.6 Myr. Main conclusions New Zealand was likely colonized by two or more invasions. One NZ lineage has its closest relatives in Australia and the other in New Caledonia. These invasions occurred well after New Zealand became isolated from other land masses, therefore cicadas must have crossed large bodies of water to reach New Zealand.     

Species boundaries in Malagasy snakes of the genus Madagascarophis (Serpentes: Colubridae sensu lato) assessed by nuclear and mitochondrial markers

We studied mitochondrial divergence in 27 individuals of colubrid snakes of the genus Madagascarophis Mertens from most of its distribution area in Madagascar. Combined analyses of 16S rRNA and cytochrome b sequences identified six major clades which only partly agreed with previously proposed classifications. Analysis of nuclear DNA sequences of the c-mos gene as well as of ISSR fingerprints revealed consistent differences only among three clades which we consider as distinct species: a widespread Madagascarophis colubrinus (Schlegel), with M. citrinus (Boettger) as a junior synonym, a southern M. meridionalis Domergue, and a presumably undescribed species from the extreme north of Madagascar. The species M. ocellatus Domergue was not available for our study. Within M. colubrinus there are two populations from the north-west, each showing two divergent haplotypes with pairwise divergences of up to 5.2% in the cytochrome b gene. Maximum divergence in this gene within M. colubrinus was 7.1%. These high values emphasise that caution needs to be applied before genetic distance values are used for species delimitation. Phylogeographically, most of the genetic variation in M. colubrinus is found in northern Madagascar, indicating that the species might have originated in this region. Later one haplotype clade colonised western and eastern Madagascar, with a putative secondary introgression into north-western populations.     

New species of Euptyctima (Acari,Oribatida) from New Zealand

Abstract

Oribotritia contortula n. sp., O. contraria n. sp., O. teretis n. sp., Rhysotritia bifurcata n. sp., Microtritia contraria n. sp., M. glabrata n. sp., Hoplophthiracarus bisulcus n. sp., Austrophthiracarus pulchellus n. sp., and Notophthiracarus claviger n. sp. are described from New Zealand.

The genera of Oribotritia Jacot, Rhysotritia Märkel & Meyer, Hoplophthiracarus Jacot, and Austrophthiracarus Balogh & Mahunka are new records for New Zealand.     

Molecular phylogeny of the Chinese ranids inferred from nuclear and mitochondrial DNA sequences

The phylogeny of representative species of Chinese ranids was reconstructed using two nuclear (tyrosinase and rhodopsin) and two mitochondrial (12S rRNA, 16S rRNA) DNA fragments. Maximum parsimony, Bayesian, and maximum likelihood analyses were employed. In comparison with the results from nuclear and mitochondrial data, we used nuclear gene data as our preferred phylogenetic hypothesis. We proposed two families (Ranidae, Dicroglossidae) for Chinese ranids, with the exception of genus Ingerana. Within Dicroglossidae, four tribes were supported including Dicroglossini, Paini, Limnonectini, and Occidozygini. A broader sampling strategy and evidence from additional molecular markers are required to decisively evaluate the evolutionary history of Chinese ranids.     

Plant microfossil analysis of coprolites of the critically endangered kakapo (Strigops habroptilus) parrot from New Zealand

We present here the results of the analysis of pollen, spores, starch and other microscopic plant material in 52 coprolites of the critically endangered kakapo (Strigops habroptilus), a New Zealand endemic parrot. The six sampling sites (caves, rock overhangs) are in widely separate geographic regions in the South Island, across an altitudinal range encompassing lowland, montane and sub-alpine environments. Radiometric testing gives average ages of discrete coprolite groups ranging from modern to 2514 ± 43 ¹⁴C yr BP (Wk-19164). A wide variety of plant taxa is identified in the coprolites, comprising cones, flowers, leaves and fronds of ~ 30 podocarps, dicotyledonous trees, shrubs and lianas, grasses, ferns, lycopods, mosses and liverworts. This supports previous observations that kakapo are versatile feeders, using a broad spectrum of foods that may only be available for short periods and intermittent years. Many of the food taxa we identify are consistent with the studies of modern kakapo diet. We also identify several new foods, potentially of value in developing food supplements to increase breeding frequency of the current kakapo population. The most commonly identified plant parts in coprolites were fern fronds (monoletes, Hymenophyllum, Cyathea smithii), followed by podocarp cones and leaves. Lack of starch in the coprolites suggests that the lycopod and fern rhizomes reported in modern kakapo diet were not commonly eaten. The data provide initial botanical evidence of diet from coprolites of an extant bird.     

The phylogenetic placement and biogeographical origins of the New Zealand stick insects (Phasmatodea)

The Lanceocercata are a clade of stick insects (Phasmatodea) that have undergone an impressive evolutionary radiation in Australia, New Caledonia, the Mascarene Islands and areas of the Pacific. Previous research showed that this clade also contained at least two of the nine New Zealand stick insect genera. We have constructed a phylogeny of the Lanceocercata using 2277 bp of mitochondrial and nuclear DNA sequence data to determine whether all nine New Zealand genera are indeed Lanceocercata and whether the New Zealand fauna is monophyletic. DNA sequence data were obtained from mitochondrial cytochrome oxidase subunits I and II and the nuclear large subunit ribosomal RNA and histone subunit 3. These data were subjected to Bayesian phylogenetic inference under a partitioned model and maximum parsimony. The resulting trees show that all the New Zealand genera are nested within a large New Caledonian radiation. The New Zealand genera do not form a monophyletic group, with the genus Spinotectarchus Salmon forming an independent lineage from the remaining eight genera. We analysed Lanceocercata apomorphies to confirm the molecular placement of the New Zealand genera and to identify characters that confirm the polyphyly of the fauna. Molecular dating analyses under a relaxed clock coupled with a Bayesian extension to dispersal‐vicariance analysis was used to reconstruct the biogeographical history for the Lanceocercata. These analyses show that Lanceocercata and their sister group, the Stephanacridini, probably diverged from their South American relatives, the Cladomorphinae, as a result of the separation of Australia, Antarctica and South America. The radiation of the New Caledonian and New Zealand clade began 41.06 million years ago (mya, 29.05–55.40 mya), which corresponds to a period of uplift in New Caledonia. The main New Zealand lineage and Spinotectarchus split from their New Caledonian sister groups 33.72 (23.9–45.62 mya) and 29.9 mya (19.79–41.16 mya) and began to radiate during the late Oligocene and early Miocene, probably in response to a reduction in land area and subsequent uplift in the late Oligocene and early Miocene. We discuss briefly shared host plant patterns between New Zealand and New Caledonia. Because Acrophylla sensu Brock & Hasenpusch is polyphyletic, we have removed Vetilia Stål from synonymy with Acrophylla Gray.     

Diversification of New Zealand weta (Orthoptera: Ensifera: Anostostomatidae) and their relationships in Australasia

New Zealand taxa from the Orthopteran family Anostostomatidae have been shown to consist of three broad groups, Hemiandrus (ground weta), Anisoura/Motuweta (tusked weta) and Hemideina-Deinacrida (tree-giant weta). The family is also present in Australia and New Caledonia, the nearest large land masses to New Zealand. All genera are endemic to their respective countries except Hemiandrus that occurs in New Zealand and Australia. We used nuclear and mitochondrial DNA sequence data to study within genera and among species-level genetic diversity within New Zealand and to examine phylogenetic relationships of taxa in Australasia. We found the Anostostomatidae to be monophyletic within Ensifera, and justifiably distinguished from the Stenopelmatidae among which they were formerly placed. However, the New Zealand Anostostomatidae are not monophyletic with respect to Australian and New Caledonian species in our analyses. Two of the New Zealand groups have closer allies in Australia and one in New Caledonia. We carried out maximum-likelihood and Bayesian analyses to reveal several well supported subgroupings. Our analysis included the most extensive sampling to date of Hemiandrus species and indicate that Australian and New Zealand Hemiandrus are not monophyletic. We used molecular dating approaches to test the plausibility of alternative biogeographic hypotheses for the origin of the New Zealand anostostomatid fauna and found support for divergence of the main clades at, or shortly after, Gondwanan break-up, and dispersal across the Tasman much more recently.     

Phylogeography of the New Zealand cicada Maoricicada campbelli based on mitochondrial DNA sequences: ancient clades associated with cenozoic environmental change

New Zealand's isolation, its well-studied rapidly changing landscape, and its many examples of rampant speciation make it an excellent location for studying the process of genetic differentiation. Using 1520 base pairs of mitochondrial DNA from the cytochrome oxidase subunit I, ATPase subunits 6 and 8 and tRNA(Asp) genes, we detected two well-differentiated, parapatrically distributed clades within the widespread New Zealand cicada species Maoricicada campbelli that may prove to represent two species. The situation that we uncovered is unusual in that an ancient lineage with low genetic diversity is surrounded on three sides by two recently diverged lineages. Using a relaxed molecular clock model coupled with Bayesian statistics, we dated the earliest divergence within M. campbelli at 2.3 +/- 0.55 million years. Our data suggest that geological and climatological events of the late Pliocene divided a once-widespread species into northern and southern components and that near the middle of the Pleistocene the northern lineage began moving south eventually reaching the southern clade. The southern clade seems to have moved northward to only a limited extent. We discovered five potential zones of secondary contact through mountain passes that will be examined in future work. We predict that, as in North American periodical cicadas, contact between these highly differentiated lineages will exist but will not involve gene flow.