Abstract
New Caledonia is a megadiverse tropical island in the southwest Pacific, however, inhabited by only one species of amphibian, Litoria aurea (Hylidae). We used both molecular (CO1 and ND4 gene sequencing) and morphometric data to explore its geographical origin and timing of colonisation. We tested whether this species arrived through transoceanic dispersal before human arrival in the island, or recently through anthropogenic introduction. We found a weak phylogeographical structure within this species, and lower haplotype diversity in New Zealand, New Caledonia and Wallis compared to Australia. No significant genetic differentiation was found between pairs of populations in New Caledonia and Wallis, or between pairs of population from these two islands. We observed a high level of morphometric differentiation between Australian and island populations, and a low level of morphometric differentiation between island populations. Our results support an Australian origin for insular frogs. The possibility of a trans-marine dispersal from Australia to New Caledonia and/or Wallis in-between the Eocene and the Pleistocene cannot be favoured, given the low level of genetic differentiation. Our results are consistent with a recent human introduction, most likely during European times. Our data support the historical absence of amphibians in the old island New Caledonia, and is consistent with the new biogeographical paradigm that this island was totally re-colonized after emergence in Eocene. More studies are necessary to explain the success of this frog in oceanic islands, where it is widespread and abundant, compared to Australia, where it is declining.
Introduction
Species diversity is unevenly distributed across the globe, with terrestrial diversity concentrated in a few restricted biodiversity hotspots (Gaston, 2000). Understanding the origin of species richness in Earth’s biodiversity hotspots constitutes one of the most significant intellectual challenges to ecologists and biogeographers, and is vital to develop effective conservation strategies. New Caledonia is a large and megadiverse tropical island in the southwest Pacific (fig. 1), situated 1220 km from Australia and 1700 km from New Zealand. The origin of this hotspot of biodiversity has traditionally been traced back to the fragmentation of Gondwana, specifically the separation of Australia around 80 Ma (Raven and Axelrod, 1972; Morat et al., 1986; Jaffré, 1992; Chazeau, 1993; de Laubenfels, 1996; Lowry, 1998). This assumption of an old origin of the local biota was based on both the antiquity of New Caledonia’s geological basement and the presence of many so-called relict groups (Raven and Axelrod, 1972; Raven, 1979; Morat et al., 1986; Grandcolas et al., 2014). This widely accepted Gondwanan paradigm and continental characterization of New Caledonia was recently questioned in the light of geological and phylogenetic evidence (Murienne et al., 2005, 2008; Grandcolas et al., 2008). These studies showed clear geological evidence for Palaeocene marine transgression, suggesting that the biota was much more recent, and emphasized the absence (or presence of few local representatives) of certain widely distributed groups. For example, New Caledonia lacks most groups of mammals except a few bats and rodents, whole groups of insects or other arthropods (e.g., mantids, velvet worms), as well as snakes or amphibians (Darwin, 1859; Darlington, 1957; Gressitt, 1960; Carlquist, 1974; Diamond, 1984; Chazeau, 1993; Grandcolas et al., 2008). The implication of this new scenario is that all the New Caledonian biota colonized the island since 37 Ma, the postulated re-emergence time of New Caledonia (end of Eocene; Nattier et al., 2011).
Map showing the geographical origin of the samples used in this study.
Citation: Amphibia-Reptilia 36, 1 (2015) ; 10.1163/15685381-00002978
Amphibians are represented in New Caledonia by only one species of frog, Litoria aurea (Lesson, 1826), the green and golden bell frog, that was assumed recently introduced. Their case is especially interesting since this group is supposed unable to disperse naturally to oceanic islands because of its intolerance to salt water (Gressitt, 1956; Duellman and Trueb, 1985). However, several recent molecular studies documented examples of dispersal of amphibians over a marine barrier, like in the Gulf of Guinea islands (Measey et al., 2007) or in Caribbean (Heinicke, Duellman and Hedges, 2007). Possible mechanisms of colonization of islands by amphibians are unclear.
All the scenarios for Amphibia in New Caledonia remain quite speculative. The timing of colonisation of this island by L. aurea are nearly undocumented. Based on fossil records, this species is present in the west coast of New Caledonia (Mé Auré Cave) since 2300 years BP, where it could have been introduced from Australia by the indigenous people rather than by Europeans, who arrived in New Caledonia 230 years BP (Grant-Mackie, Bauer and Tyler, 2003; Grant-Mackie et al., 2013). Apart from these fossils data, the earliest mentions of L. aurea in New Caledonia are from Nouméa in the late 19th century (Lemire, 1884; eight adult male specimens in the collection of Naturhistorisches Museum Wien with the collection numbers NMW 5971, NMW 18228 and NMW 18891 collected by Haller in 1885; one specimen in the collections of the Natural History Museum of London with the collection number BMNH 1886.3.11.25, a number that indicates that is was collected before March 1886). In contrast, at the beginning of the 20th century it was already distributed over a large part of the island (Sarasin, 1926).
Genetic data are commonly used to infer the geographical origin and colonisation time of a given population. For example, Vörös et al. (2008), based on partial sequences of the mitochondrial cytochrome oxidase I (cox1) gene, showed that L. aurea was introduced into the North Island of New Zealand in the last 140 years 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.
Morphometric data can also be used as an indirect mean to give clues on either the time of colonisation or the size of the initial population. Populations of organisms confined to islands often evolve extensive morphological changes over a short period of time (a few thousand years; Lister, 1989; Montesinos, da Silva and Gomes de Carvalho, 2012). Changes in body size, body shape, behaviour, reproduction and physiology are often referred as “Island Syndrome”, and have been recorded in various vertebrate species like rodents, passerine birds, lizards and even amphibians (Raia et al., 2010; Wu, Li and Murray, 2006; Montesinos, da Silva and Gomes de Carvalho, 2012). Adler and Levins (1994) linked the emergence of the island syndrome to the reduced interspecific competition and predation pressure that, they claimed, are typical of islands, and to the size and degree of isolation of the island itself. Another important factor that may explain why colonizing populations may be likely to differentiate rapidly from their ancestral source population is that founder populations are usually relatively small, which may enhance the likelihood of change by genetic restructuring or drift. However the role of founder effects in driving differentiation is controversial and several recent studies downplay its role in small vertebrates (Losos et al., 2001; Pargams and Ashely, 2001). To better understand the evolutionary history of colonising populations it is interesting not only to compare island to mainland populations, but also island populations’ there between. If significant differences are observed it suggests an old colonisation of the island followed by local differentiation.
The species Litoria aurea is considered endangered in Australia, where it is currently rare and distributed in a patchy way. Its distribution range includes eastern coastal Australia from northern New South Wales to eastern Victoria (White and Pike, 1996), and it was in historical times present in inland locations like Bathurst, west of Blue Mountains. In New Zealand, New Caledonia, Wallis and some of the islands of Vanuatu, including Santos (Tyler, 1979; Gill, 1995), it is considered as an invasive species (Pyke et al., 2002).
In this paper we use molecular and morphological data to compare L. aurea populations from New Caledonia, Australia and New Zealand to: 1) confirm that only one species, L. aurea, is present in New Caledonia; 2) quantify the degree of genetic and morphometric differentiation among island and inland populations, and among island populations; and 3) discuss the conservation paradox that this species is endangered in Australia and invasive in several islands. These results should allow us to conclude whether this species arrived through transoceanic dispersal by itself before human arrival in the island, or recently through anthropogenic introduction. To place this study on a larger context of island colonisation we also included new samples from other oceanic islands closed to New Caledonia, i.e. Wallis and Santo.
Material and methods
Specimens included
One hundred and thirty two new specimens were collected in the field: 99 in New Caledonia, 31 in Wallis and 2 in Santo. For each specimen we took tissue samples in alcohol for genetic analyses and we performed 49 external measurements. External measurements were also taken on series of specimens from Australian Museum (table 1) for which no tissue samples were available.
List of specimens of Litoria aurea considered in this paper. AMS: Australian Museum, Department of Herpetology, Sydney, Australia; MNHN: Museum National d’Histoire Naturelle, Reptiles et Amphibiens, Paris, France.
We also retrieved from Genbank all L. aurea CO1 and ND4 sequences previously published by Burns and Crayn (2006), Burns et al. (2007) and Vörös et al. (2008), coming from Australia and New Zealand (283 individuals for the CO1 gene, and 263 individuals for the ND4 gene). For these specimens the precise locality of collection was not available. Thus we grouped samples according to geographical regions as follows: Australia – Upper North Coast, Lower North Coast, Hunter, Central Coast, Sydney, Illarwarra, Shoalhaven, Southern Tablelands and Northeast Victoria; New Zealand – Coromandel, Northland, Waikato (we followed Burns et al., 2007 for Australia and Vörös et al., 2008 for New Zealand). Figure 1 displays all localities and/or regions of collect on a map.
DNA extraction, amplification, sequencing and phylogenetic analyses
We extracted total genomic DNA using the QIAGEN DNA micro kit following the manufacturer protocol. We chose for sequencing two protein-coding genes, cytochrome oxydase 1 (CO1) and NADH dehydrogenase subunit 4 (ND4). Both genes are known to be informative to elucidate frog phylogeography and numerous L. aurea sequences from Australia and New Zealand were available in Genbank (Burns and Crayn, 2006; Burns et al., 2007; Vörös et al., 2008). We amplified the CO1 gene for the 132 new collected specimens using primers CO1-SmallF and CO1-SmallR (Burns et al., 2007), which amplified an approximate 330 pb product. We amplified the ND4 gene for 130 specimens (the two specimens from Santo could not be amplified) using primers ND4-3 and ND4-1 (Burns et al., 2007), which amplified an approximate 750 bp product. The PCR consisted of 35 cycles: 30 s at 94°C, 60 s at 58°C and 60 s at 72°C. The double-stranded PCR products were purified and sequenced at EUROFINS (http://www.eurofins.fr). Sequences were aligned and trimmed to 324 bps (CO1) and 524 bps (ND4) in order to include in our analyses all ND4 and CO1 sequences available in Genbank.
Several analyses were carried out to test the level of genetic variation and of genetic differentiation between populations from Australia, New Zealand, New Caledonia, Wallis and Santo. Higher genetic variability is expected in source populations, and the level of genetic differentiation between populations should allow us to identify source populations and to tests for a recent colonisation time. We could not concatenate the CO1 and ND4 sequence data because for all the data available in Genbank specimens’ numbers are not provided. Thus we could not infer which CO1 sequence should be combined with which ND4 sequence. Thus results for both genes were treated separately. Relationships among haplotypes were inferred for each gene separately by constructing a network using the median-joining method available in Network v. 4.5.1.6 (Bandelt, Forster and Rohl, 1999). To estimate haplotype richness while controlling for unequal sample sizes (Leberg, 2002) we used rarefaction analysis (Analytic Rarefactation v. 1.4; UGA Stratigraphy Lab website; http://www.uga.edu/~strata/software/). To test for genetic differentiation between pairs of populations we used the Fst test (based on pairwise nucleotide differences) and the exact test of population differentiation (Raymond and Rousset, 1995) available in ARLEQUIN 3.5. We also analysed population structure by analysis of molecular variance (AMOVA) in ARLEQUIN, version 3.5. AMOVA divides the total variance into additive components (i.e. variation within populations, among populations within groups and among groups). A population was defined as all individuals coming from one geographical locality; and groups of populations were defined according to their island or mainland origin (i.e., New Caledonia, Wallis or Australia). Only localities with more than 9 individuals sampled were included in these analyses (i.e. 12 localities). Two AMOVA were performed: considering New Caledonia, Santo and Australia, and one considering only New Caledonia and Santo.
The green and golden bell frog is widespread and abundant in New Caledonia and Wallis. If it was introduce in these island from a small number of migrants, a signal of population expansion is expected in these populations. Thus we inferred the demographic history of these populations using tests of population growth. Fu’s Fs (Fu, 1997) and Ramos-Onsins & Rozas’ R2 (Ramos-Onsins and Rozas, 2002) are among the most powerful statistics to detect demographic expansions (Ramos-Onsins and Rozas, 2002): the behaviour of the R2 test is superior for small sample sizes, whereas Fs is better for large sample sizes. These statistics were estimated using ARLEQUIN 3.11 (Excoffier, Laval and Schneider, 2005) and DNASP 5.10 (Librado and Rozas, 2009), and their significances were assessed using 1000 coalescent simulated resamplings. As suggested in the ARLEQUIN manual, the Fs statistics was considered significant when the P-value was below 0.02.
Morphometrical studies
A total of 49 measurements have been taken on 150 specimens: 98 from New Caledonia (8 populations), 31 from Wallis (4 populations) and 21 from Australia (2 populations; table 1). These measurements were chosen in order to estimate variation in size and shape of overall morphology (head, forelimb, hindlimb and webbing). EL – eye length; EN – distance from the front of the eye to the nostril; FFTF – distance from the maximum incurvation of the web between fourth and fifth toe to the tip of fourth toe; FL – femur length (from vent to knee); FLL – forelimb length (from the elbow to the base of the outer metacarpal tubercle); FOL – foot length (from the base of the inner metatarsal tubercle to the tip of the toe); FTL – fourth toe length (from the base of the first subarticular tubercle); HAL – hand length (from the base of the outer metacarpal tubercle to the tip of the toe); HL – head length (from the back of the mandible to the tip of snout); HW – head width; IBE – distance between the back of the eyes; IFE – distance between the front of the eyes; IMT – length of inner metatarsal tubercle; IN – internasal space; ITL – inner toe length; IUE – minimum distance between upper eyelids; MBE – distance from the back of the mandible to the back of the eye; MFE – distance from the back of the mandible to the front of the eye; MN – distance from the back of the mandible to the nostril; MTFF – distance from the distal edge of the metatarsal tubercle to the maximum incurvation of the web between fourth and fifth toe; PAI-PAIV – width of pads of fingers I to IV; PPI-PPV – width of pads of toes I to V; SL – distance from the front of the eye to the tip of the snout; SN – distance from the nostril to the tip of the snout; SVL – snout-vent length; TFL – third finger length (from the base of the first subarticular tubercle); TFTF – distance from the maximum incurvation of the web between third and fourth toe to the tip of fourth toe; TL – tibia length; TYD – greatest tympanum diameter; TYE – distance from tympanum to the back of the eye; UEW – maximum width of inter upper eyelid; WAI-WAIV – width of fingers I to IV; WFF – webbing between fourth and fifth toe (from the base of the first subarticular tubercle); WI – webbing between third and fourth toe when folded along fourth toe (from the base of the first subarticular tubercle); WII – webbing between forth and fifth toe when folded along fourth toe (from the base of the first subarticular tubercle); WPI-WPV – width of toes I to V; WTF – webbing between third and fourth toe (from the base of the first subarticular tubercle).
Measurements were transformed into their ratios to SVL. Kolmogorov-Smirnov tests were used to check samples for normality. As no significant deviation from normal distribution was observed for all measurements, oneway ANOVA and post-hoc Scheffé test were applied on 49 measurements to compare populations from Australia, New Caledonia and Wallis. To visualise differences between populations, treediagrams were constructed based on the percentage of measurements significantly different between pairs of populations (according to Scheffé post-hoc analysis) using the Unweighted Pair Group Arithmetic Average method (UPGMA). Statistical analyses were performed using SPSS statistical software (Norusis, 1992).
Results
Molecular analyses
Minimum spanning networks confirm that all specimens from New Caledonia, Wallis and Santo belong to the species L. aurea (fig. 2). Weak phylogeogaphical structure is observed within this species, with only 21 haplotypes and 14 variable positions for the CO1 gene (415 individuals, see online supplementary table S1), and 29 haplotypes and 31 variables positions for the ND4 gene (393 individuals, supplementary table S2). Our CO1 network highlights the close affinity between populations from New Caledonia, Santo, Wallis, New Zealand, and the two Australian regions of Sydney and Shoalhaven: haplotype C16 is shared among all these regions; haplotype C11 is found in Wallis, New Caledonia and the two Australian regions, as well as in Illawara, Hunter and Central Coast. Our ND4 network also highlights the close affinity between populations from New Caledonia, Wallis, Sydney, Illarwarra and Shoalhaven: haplotype N10 is shared among all these regions. New Caledonia possesses one unique CO1 haplotype (C10) and three unique ND4 haplotypes (N2, N3 and N4). New Caledonia and Wallis possess one unique ND4 haplotype (N1).
Minimum spanning network depicting relationships among CO1 (top) and ND4 (bottom) Litoria aurea haplotypes. The size of the circle is proportional to the haplotype frequency, and the length of the connecting line is proportional to the number of mutations. Colours refer to distinct Australian or New Zealand regions, and to distinct oceanic islands (New Caledonia, Wallis and Santo).
Citation: Amphibia-Reptilia 36, 1 (2015) ; 10.1163/15685381-00002978
When all populations from Australia or a given island are combined, the expected number of CO1 or ND4 haplotypes is significantly greater in Australia than in New Caledonia, Wallis or New Zealand (fig. 3). No significant difference is observed between New Caledonia, Wallis and New Zealand.
Rarefaction curves plotting the number of individuals sampled against the expected number of mitochondrial haplotypes (calculated using the ANALYTIC RAREFACTATION.1.4 software available at the UGA Stratigraphy Lab website – http://www.uga.edu/~strata/software/). 95% confident limits are shown.
Citation: Amphibia-Reptilia 36, 1 (2015) ; 10.1163/15685381-00002978
In Wallis, no significant genetic differentiation is found between pairs of populations for both genes (for both Fst statistic and exact test of population differentiation; table 2). The same is true for nearly all pairwise comparisons between New Caledonian populations. Most Wallis-New Caledonia comparisons are also not significant. For the CO1 gene Fst values are low (0.030-0.274), and mostly not significant (), between Sydney and New Caledonian or Wallis populations. Fst values are low (0.138-0.323), but mostly significant, between Shoalhaven and New Caledonian or Wallis populations. Fst values between all other Australian and New Caledonian or Wallis populations are high (0.392-1.000) and significant. For the ND4 gene all but one (between Sydney and Sarraméa) pairwise comparisons between Australian and New Caledonian or Wallis populations are significant. Fst values tend to be lower between Sydney (mean = 0.236; range = 0.048-0.517) or Shoalhaven (mean = 0.261, range = 0.068-0.463) and New Caledonian or Wallis populations, than between other Australian and New Caledonian or Wallis populations (mean = 0.747, range = 0.214-1.000).
Two AMOVA analyses were performed to explore how the genetic variance is partionned among individuals within a locality, among localities and among the main regions. When three groups of populations are considered (Australia, New Caledonia and Wallis), 55% of the genetic variance is observed among individuals within sampled sites, 26% is observed among populations within groups and 19% is observed among groups for the CO1 gene (38%, 39% and 23% respectively for the ND4 gene). However when only two groups of populations are considered (New Caledonia and Wallis) 90% of the genetic variance is observed among individuals within sampled sites and 10% is observed among sampling sites within islands (for both genes). There is no partition of the genetic variance between New Caledonia and Wallis.
A significant signal of population expansion was detected for the ND4 gene in Wallis and New Caledonia according to the R2 test (). All Fu’s Fs tests were not significant (), and the R2 test was not significant for the CO1 gene ().
Morphometrical analyses
Morphometric analyses were performed in order to assess the level of morphometric differentiation between mainland and island populations, and between island populations. The frogs from Australia show significant differentiation in 43 measurements from those from New Caledonia and in 39 measurements from those from Wallis. The frogs from New Caledonia and Wallis are significantly different in only 9 measurements (fig. 4, online supplementary table S3). Measurements for which no significant differentiation between populations could be observed concern foot morphology and webbing (ftl, wi, wtf, wff).
Genetic differentiation between Litoria aurea island populations. Fst values are shown (lower left hand matrix based on CO1 sequences, upper right hand matrix based on ND4 sequences). Significant comparisons are shown in bold (). Cases in grey indicate significant comparisons for the exact test of sample differentiation (). A = Australia, NC = New Caledonia.
UPGMA dendrograms based upon the percentage of measurements significantly different between pairs of Litoria aurea populations (according to Scheffé post-hoc analyses). (a) Comparison between Australia, New Caledonia and Wallis populations; (b) comparison between New Caledonian populations; (c) comparison between Wallis populations. AUS, Australia; NC, New Caledonia; W, Wallis; Aka, Aka-aka; Kou, Koumac; Lik, Liku; Kik, Lake Kikila; Nak, Nakutakoin; Nou, Noumea; Poi, Poingam; Pou, Pouembout; Sar, Sarraméa; Thi, Thio.
Citation: Amphibia-Reptilia 36, 1 (2015) ; 10.1163/15685381-00002978
In New Caledonia, no significant difference could be found in any measurement between the populations of Koumac, Nakutakoin, Poingam, Pouembout and Sarramea, and between the populations of Thio and Nouméa (fig. 4, supplementary table S4). Few statistically significant measurements were observed between others localities (2 to 8 measurements significantly different between pairs of localities). Differences between populations mainly concern size (svl), head shape (in, ibe, tyd) and pad morphology (paiii, ppi).
The three populations from Wallis differ from one another by 6 to 7 measurements (pairwise comparisons; fig. 4, supplementary table S5). Differences between populations mainly concern size (svl), head shape (in, en), foreleg (fll, hal, tfl), webbing (mttf, wi, wii) and toe pad morphology (pai, paii, paiii, ppi, wpv).
Discussion
Origin and colonisation time of New Caledonian and Wallis populations
Our molecular and morphometric data clearly indicate that all frogs from New Caledonia, Wallis and Santo belong to the species Litoria aurea. Given the weak phylogeographical structure observed within this species (with several haplotypes shared among Australia, New Zealand, New Caledonia, Wallis and Santo; fig. 2) and the lower haplotype diversity in New Zealand, New Caledonia and Wallis compared to Australia (fig. 3), a colonisation of these islands from Australia is probable. Moreover, given the low level of genetic differentiation between these islands and Australia, the hypothesis of a recent arrival through anthropogenic dispersion is favoured, against an old transoceanic dispersal (between the Eocene and the Pleistocene): if island populations were colonised before the end of the Pleistocene one would expect to find discrete lineages in these islands compared to Australia, given the rapid evolutionary rate of the two mitochondrial gene fragments considered.
No significant genetic differentiation is found between pairs of populations in New Caledonia and Wallis, or between pairs of population from these two islands (Fst statistic, exact test of population differentiation; AMOVA). This result suggests that only one colonisation event is responsible for the colonisation of both islands. This hypothesis is also supported by the low level of morphometric differentiation observed between island populations, compared to the high level of morphometric differentiation observed between Australian and island populations.
The question is now to try to estimate when this event occurred: did it occurred by indigenous people (3000 years BP), or by Europeans (less than 230 years BP)? Our estimates are based on non-recombinant, haploid mitochondrial markers. More precise date estimates could be obtained using coalescent analyses of nuclear loci. Moreover, given the difficulty to estimate precise divergence dates from molecular data, it is inherently difficult to answer this question. However, several lines of evidence can be combined to try to address this difficult issue.
First, we know that the species was introduced from Australia into New Zealand in the late 1800s, supposedly to provide food for ducks and to control mosquito larvae (McCann, 1961; Vörös et al., 2008). Vörös et al. (2008), based on the same portion of the CO1 gene that the one used in present study, found several haplotypes shared by New Zealand and Australian populations, as well as one unique haplotype in New Zealand differentiated from other haplotypes by a single mutational event. We found exactly the same pattern when we compared New Caledonian and Wallis populations to Australian ones. This result suggests a similar time frame for the colonisation of New Caledonia and Wallis compared to New Zealand, even if it not straightforward to distinguish from molecular data events occurring either hundreds or thousands years ago.
Second, our molecular data indicates that L. aurea was most likely introduced from Sydney and Shoalhaven to New Caledonia and Wallis: 1) the two island frog populations share several haplotypes only with Australian populations localised between Shoalhaven and Hunter; 2) the number of shared haplotypes is greater with Sydney and Shoalhaven than with other Australian populations; 3) the Fst values are lower between the island localities and these two Australian localities than with other Australian localities. Haplotype diversity found in New Caledonia and Wallis represent only a subset of the genetic diversity present in Sydney and Shoalhaven. Given that these two oceanic islands were probably colonised by a low number of migrant individuals it is not surprising to find such a result. The region of Sydney corresponds to the main harbour from which intense economic exchange between New Caledonia and Australia occurred from the second half of the 19th century onwards (Schreiner, 1882; Terrier, 2010). These shipments could have intentionally transported frogs, as no accidental transfer of Litoria is documented (Thomson, 1922). In fact frogs have been (Medway and Marshall, 1975; Bishop, 2008), and are still used (Ohler, unpublished data, 2011), to limit mosquito populations around houses. Since the beginning of colonial settlement frogs have accompanied human and have probably been actively introduced in all islands, as it is documented for New Zealand (Thomson, 1922). Since our molecular data fit the most important human and commercial lines of exchanges since the 19th Century it seems likely that colonisation of New Caledonia occurred at that time.
Third, our morphometric analyses show a high degree of morphological differentiation between Australian and island populations, and a low degree of genetic differentiation between island populations. It is not surprising to find a high level of morphological variation between Australian and island populations, either due to founding effect or to insular syndrome: New Zealand populations of L. aurea were found to be larger in body size (McCann, 1961) than Australian populations (van de Mortel and Goldingay, 1998; Goldingay and Newell, 2005) and this difference arose in less than 140 years. The fact that few significant differences are observed between island populations suggests either a recent colonisation of these islands, or strong gene flow between populations preventing the establishment of local differentiation.
These three lines of evidence tend to indicate a recent European anthropogenic origin of the New Caledonian and Wallis frog populations. This conclusion is in conflict with fossil data, which suggest that this species is present in New Caledonia since 2300-1200 years BP (Grant-Mackie et al., 2013 for the first estimation; Hand and Grant-Mackie, 2012 for the second one), but other dates should also be considered. Radiocarbon age determinations on human and quail bones found in the same soil level (f1248) than the frog bones give an age varying from 248 to 3857 years BP (Horrocks, Grant-Mackie and Matisoo-Smith, 2008), but the authors underline the inherent difficulties in obtaining valid dates from some material. An interesting result is that Horrocks, Grant-Mackie and Matisoo-Smith deliberately omitted from their summary table the date of 103.8 ± 0.6% years from frog bones, even if “the Director of the Radiocarbon Dating Lab stated that he did not think the date to be the result of contamination”. According to the authors “these results were set aside and ignored” because “the bones were clearly not of post-1950 age”. Given the uncertainty on fossil age determination, we favour an European origin of L. aurea population, even if we cannot completely reject the possibility of a double introduction (first introduction by indigenous people around 2300 years BP, followed by extinction and second introduction by Europeans).
Another interesting point is the absence of genetic differentiation between New Caledonian and Wallis frog populations. Wallis and New Caledonia could have been colonised either at the same time from the same Australian source population, or one of this island could have been colonised from the other one. No significant difference in haplotype diversity was found between islands, thus we cannot decide from molecular data which one of these two islands would have serve as the source population. After signing a protectorate with the French government, Wallis and Futuna was put under administration of New Caledonia. All but local agricultural products are imported, mostly from New Caledonia, and direct marine relationships to Australia have been limited until recently. These historical data suggest that frog populations could have been transported by boat from New Caledonia to Wallis. Moreover, the first specimen of the species collected from Wallis date from 1986 (Gill, 1995).
Conservation
Litoria aurea was historically distributed in Australia from northern New South Wales to eastern Victoria, with inland populations as far west as Bathurst and Tumut (Pyke and White, 1996; Tyler, 1979). The current distribution is limited to isolated populations at various locations, mostly from coastal lowlands, and inland populations as well as populations from higher altitude seem to have gone extinct (NPWS, 1999). Through the early 1980s, L. aurea was considered common but since had undergone dramatic declines with disappearances reported from 80% of its former range. It is now listed as “Vulnerable” nationally (Environmental Protection and Biodiversity Conservation Act 1999) and Endangered in New South Wales (Threatened Species Conservation Act 1995). While it is now rare in its native habitat, it has successfully dispersed after introduction by human throughout several oceanic islands, establishing large and widespread populations in New Zealand (Pyke et al., 2002), New Caledonia or Wallis (Ohler, personal observation) in areas of similar breeding habitat to those in their native Australia. Given the low number of haplotypes in each island it seems probable that this species has recently undergone a rapid population expansion in these oceanic islands from a low number of founder individuals. However we only detected a significant signal of population expansion with the R2 test in Wallis. This could be due to the low sample sizes (the R2 test is known to be more powerful than the Fs test to detect population expansion from low sample size), or more probably to fact that the two genes studied here evolve too slowly to detect an expansion that would have occurred during the past few hundred years. It would be interesting to test this hypothesis with more variable genetic markers, like microsatellites. Many factors are thought to be responsible for the dramatic decline of this species in Australia, including habitat fragmentation, erosion and sedimentation of soil, insecticides and fertilisers contaminating water systems, the introduction of predatory fish, and alteration of drainage regimes, predation by introduced mammals, such as cats and foxes, changes to water quality at breeding sites, herbicide use, and loss of habitat through the destruction of wetlands or the amphibian chytrid fungus but the relative importance of the various factors is unclear (Hero et al., 2004; Department of Environment and Conservation NSW, 2005; Penman et al., 2008). Among amphibian species considered to be invasive, few cases are known with such contrasting pattern in conservation status (for example Litoria raniformis, a phylogenetically close species). Usually common species tolerant to human habitat are chosen for voluntary introduction (Kraus, 2009), as such species are much more favoured in invasiveness. From ecological studies it was shown that Australian Litoria aurea occur in similar breeding habitats as this species does in New Zealand (Pyke et al., 2002) and there is no indication that they changed their niche. Furthermore the species only declined within its original range in the last 40 years, much after the introduction of the species to New Zealand and New Caledonia, and decline is considered related to local environmental threats and probably not due to genetic factors. The threats act locally and geographic isolation of these populations is complete; moreover the economic development and environmental politics are quite different as the territories belong to different states. It would be very interesting to conduct comparative ecological studies in Australia and New Caledonia, New Zealand or Wallis to understand why this species is declining only in Australia.
In conclusion, our study has permitted to propose an Australian origin for frogs from New Caledonia and Wallis. Populations established in Wallis could have been introduced from populations first established in New Caledonia, or alternatively they could have been colonised simultaneously from the same Australian source populations. The possibility of a spontaneous trans-marine dispersal from Australia to New Caledonia and/or Wallis in-between the Eocene and the Pleistocene cannot be favoured, given the low level of genetic differentiation between these frog populations. Our genetic data alone are not sufficient to distinguish between an introduction around 3000 years ago compared to a post-European introduction. However the combination of genetic, morphometric and bibliographical data seems to favour a recent human introduction, most probably during European times. It is one of the first “invasions” that can be recorded for New Caledonia. Hundreds of introduced species recently arose in New Caledonia, particularly the last 50-60 years, and provoked serious damages to native biota and ecosystems (Pellens and Grandcolas, 2010). Given the recent colonisation of the island it is not surprising that no tendency for microendemism was recorded in this species, contrary to what was observed in many other taxonomic groups (e.g., Bartish et al., 2005; Balke et al., 2007; Murienne et al., 2008; Murienne, Guilbert and Grandcolas, 2009; Bauer et al., 2012; Nattier et al., 2012). The whole picture for L. aurea drawn from the present study reinforces the scenario for primitive natural absence of Amphibia in the old island New Caledonia, and is consistent with the new biogeographical paradigm that this island was totally re-colonized after emergence in Eocene. More studies are necessary to explain the success of this frog in oceanic islands, where it is widespread and abundant, compared to Australia, where it is declining.
Acknowledgements
Field work in New Caledonia has been funded by ANR Biodiversité BIONEOCAL (grant to PG). The “Contrat de développement du territoire 2011-2016” and the “Territoire de Wallis et Futuna” covered travel coasts and fieldwork in Wallis and Futuna. Collection permits No17398/DENV/SCB from Direction de l’Environnement, Province Sud and No60912-2807-2011/JJC from “Direction du développement économique et de l’environnement, Province Nord” allowed access to specimens.
This work was supported by the ‘Action Transversale du Muséum: Taxonomie moléculaire, DNA Barcode & gestion durable des collections’, the ‘Centre National de Séquençage’ (Genoscope) and the ‘Service de Systématique moléculaire’ of the Muséum National d’Histoire Naturelle (UMS 2700, Paris, France). This project was supported by the network “Bibliothèque du Vivant” funded by the CNRS, the Muséum National d’Histoire Naturelle, the INRA and the CEA (Genoscope). Thanks to Patrick Campell from Natural History Museum (London) and Heinz Grillitsch from Naturhistorisches Museum Wien (Austria) who provided data and photograph of specimen from Nouméa in natural history collections. Ross Sadlier from Australian Museum (Sydney) is acknowledged for loan of specimens from Australia.
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Footnotes
Associate Editor: Matthias Stöck.