Isolation and characterization of microsatellite loci from the Australasian sea snake,Aipysurus laevis

We developed 13 microsatellite loci for the olive sea snake, Aipysurus laevis, using both enriched and unenriched genomic DNA libraries. Eleven codominant loci, that reliably amplified, were used to screen 32 individuals across the geographic range of A. laevis. Four loci had four or more alleles (maximum 12), whereas the other seven had either two or three. All but one locus was in Hardy–Weinberg equilibrium. These loci will provide useful markers to investigate population genetic structure for the olive sea snake.     

Does foraging mode influence life history traits? A comparative study of growth,maturation and survival of two species of sympatric snakes from south‐eastern Australia

Abstract Theory predicts that compared with active searchers, ambush foragers should have lower rates of energy intake, slower growth, and higher survival rates. We tested these predictions with data on two species of sympatric, saurophagous, small‐bodied, viviparous elapid snakes: the broad‐headed snake, Hoplocephalus bungaroides, and the small‐eyed snake, Rhinoplocephalus nigrescens. Demographic parameters and growth curves for both species were estimated from a long‐term (9 years) mark‐recapture study in Morton National Park, south‐eastern Australia. The ambush predator (H. bungaroides) displayed slower juvenile growth and later maturation (5 years for males, 6 years for females) than did the active forager (R. nigrescens, 3 years). Litter sizes were similar in both species, but reproductive frequency was higher in R. nigrescens (90–100%) than in H. bungaroides (50%). Juvenile survival was lower in the active searcher (31%) than in the ambush forager (55%), but adult survivorship was similar (74% vs 82%). Our results support the hypothesis that ambush foragers display ‘slow’ life history traits, but additional phylogenetically independent comparisons are needed to evaluate the generality of this pattern.     

Molecular and morphological assessment of Australia’s most endangered snake, <Emphasis Type="Italic">Hoplocephalus bungaroides</Emphasis>, reveals two evolutionarily significant units for conservation

The Broad-headed snake Hoplocephalus bungaroides is one of Australia’s most endangered vertebrates. Extant populations of H. bungaroides are restricted to several geographically isolated reserves to the north, west, and south of Sydney. We analysed mitochondrial DNA from 184 specimens drawn from across the geographic range of the Broad-headed snake. Phylogenetic analysis demonstrated that H. bungaroides comprises two divergent mitochondrial lineages with a “northern” clade comprising populations west and north of Sydney and a “southern” clade comprising animals in Morton National Park. The two clades differ by an uncorrected genetic distance of 1.7%, which implies a divergence dating to approximately 755,000–850,000 years ago. We complemented our molecular data set with a detailed analysis of morphological variation both between and within the genetic clades. The two H. bungaroides genetic clades are morphologically indistinguishable and show little sexual dimorphism. Our results demonstrate that the populations north and south of this biogeographic split function as two distinct populations with no recent gene flow. There is no reason for separate taxonomic recognition of these two clades, but they do represent distinct evolutionarily significant units (ESUs) that require separate conservation management. In addition, within the northern ESU, populations from Royal National Park, Blue Mountains National Park, Wollemi National Park, and the Sydney Water Catchment supply areas should be considered as separate management units to conserve both evolutionary and ecological processes.     

Novel microsatellite loci identified from the Australian eastern small‐eyed snake (Elapidae: Rhinocephalus nigrescens) and cross species amplification in the related genus Suta

A total of 15 microsatellite primers pairs were developed for the Australian small‐eyed snake Rhinoplocephalus nigrescens. Five primers were used to screen 93 individuals of R. nigrescens and were also tested against eight species of the closely related genus Suta. Allelic diversity in R. nigrescens was high in three loci (12–27) and there was high heterozygosity (0.58–0.82). Observed heterozygosity did not deviate from Hardy–Weinberg expectations for the five loci tested. These primers will be useful in studies of population genetics and mating systems of small‐eyed snakes and related species.     

Differential Effects of an Intense Wildfire on Survival of Sympatric Snakes

Abstract: We analyzed 16 years of mark-recapture data to investigate whether a wildfire influenced survival of an arboreal ambush-forager (broad-headed snake [Hoplocephalus bungaroides]) and a terrestrial active forager (small-eyed snake Cryptophis nigrescens). We predicted that wildfire would cause direct mortality and reduce subsequent survival of both snake species. Contrary to this prediction, wildfire did not affect abundance of broad-headed snakes, but abundance of small-eyed snakes decreased by 48% after the wildfire. Estimated annual survival of small-eyed snakes was 37% lower after fire (s=0.47, SE=0.07) than before fire (s=0.74, SE=0.05). Prescribed burning may be a suitable tool for creating open habitat mosaics for the endangered broad-headed snake.     

Isolation and characterization of 14 polymorphic microsatellite loci in the ringneck snake <Emphasis Type="Italic">Diadophis punctatus</Emphasis> (Colubridae: Dipsadinae)

We constructed a genomic library and developed 14 polymorphic loci to assess species boundaries between previously described mtDNA lineages and to examine the genetic variability between populations of the ringneck snake Diadophis punctatus species complex. Polymorphism was investigated for these markers in 34 individuals from populations in southeastern Louisiana and southern Florida. The number of alleles ranged from 5 to 29, expected heterozygosity ranged from 0.816 to 0.962 and observed heterozygosity ranged from 0.452 to 0.889. These new loci provide the tools necessary to examine species boundaries, population genetics within species and to determine the conservation status of imperiled populations.     

What makes a species vulnerable to extinction? Comparative life-history traits of two sympatric snakes

Although it is well known that species vary in their vulnerability to extinction, the reasons are poorly understood. Theory predicts that long-lived species with slow life histories (small litters, slow growth, late maturation) should be at greater risk than short-lived species with high potential rates of increase. This hypothesis was tested by comparing life-history traits of two species of sympatric, elapid snakes: the endangered broad-headed snake, Hoplocephalus bungaroides, and common small-eyed snake, Cryptophis nigrescens. From 1992 to 2000 a mark–recapture study of both species was undertaken in Morton National Park, south-eastern Australia, and this information was used to construct transition matrices for each species. The endangered H. bungaroides was found to mature late (6 years of age), had a high juvenile (54.7%) and adult (81.6%) survival rate, and a long generation length (10.4 years). In striking contrast, the common C. nigrescens matured early (within 3 years), had a lower juvenile (30.4%) and adult (74.4%) survival rate (but higher recruitment rate), and a substantially shorter generation length (5.9 years). Elasticity analyses revealed that H. bungaroides was considerably more sensitive to survival past the age of 2 years (68.6%) than C. nigrescens (37.4%). These results provide support for the hypothesis that species with slow life histories are more vulnerable to extinction.     

Actions of snake neurotoxins on an insect nicotinic cholinergic synapse

Here we examine the actions of six snake neurotoxins (α-cobratoxin from Naja naja siamensis, erabutoxin-a and b from Laticauda semifasciata; CM12 from N. haje annulifera, toxin III 4 from Notechis scutatus and a long toxin from N. haje) on nicotinic acetylcholine receptors in the cercal afferent, giant interneurone 2 synapse of the cockroach, Periplaneta americana. All toxins tested reduced responses to directly-applied ACh as well as EPSPs evoked by electrical stimulation of nerve XI with similar time courses, suggesting that their action is postsynaptic. Thus, these nicotinic receptors in a well-characterized insect synapse are senstive to both long and short chain neurotoxins. This considerably expands the range of snake toxins that block insect nicotinic acetylcholine receptors and may enable further pharmacological distinctions between nAChR subtypes.     

Isolation and characterization of microsatellite loci in the black rat snake (Elaphe obsoleta)

We obtained molecular markers useful for population level studies of the black rat snake (Elaphe obsoleta) by screening genomic DNA libraries enriched for dinucleotide, tetranucleotide, and pentanucleotide microsatellite repeats. Following sequencing of the positive clones, 11 pairs of primers were designed for polymorphic loci and their variability assessed in > 350 individuals from four populations in North America. The loci had between 9 and 40 alleles and observed heterozygosities ranged from 0.071 to 0.87. Some of these pairs of primers also successfully amplified DNA from two other snake species.     

Small‐scale field experiments provide important insights to restore the rock habitat of Australia's most endangered snake

Habitat disturbance poses a major threat to biodiversity worldwide. The broad‐headed snake (Hoplocephalus bungaroides) has become Australia's most endangered snake due to a dependence on rock habitats that are subject to high rates of human‐induced degradation. The permanence of degradation can only be reversed by outcrop restoration. We constructed experimental outcrops near (<150 m) and far (>500 m) from roads and walking trails (access points). We tested two hypotheses over a 14‐year period: that outcrop restoration can restore habitat quality, and that the frequency of outcrop degradation is influenced by distance from access points. We confirmed that habitat value was restored: lizard prey of the broad‐headed snake was more abundant in constructed compared to natural outcrops; broad‐headed snakes were detected equally in constructed and natural outcrops. Disturbance to constructed outcrops occurred more often and was more severe at near compared to far outcrops. The probability of occupancy by broad‐headed snakes was 0.75 ± 0.13 in 29 far outcrops compared to 0.41 ± 0.11 in 35 near outcrops, suggesting a response to disturbance and more frequent poaching at near outcrops. Habitat restoration for the broad‐headed snake should have its greatest value at locations far from access points. Restricting access should be a principal strategy to manage rock outcrop ecosystems. Outcrop disturbance is not unique to Australia. These findings have relevance to management of rock outcrops worldwide. Habitat restoration is an expensive conservation tool for endangered species, particularly if unsuccessful. Small‐scale restoration experiments that aim to refine procedures should precede large‐scale restoration.     

Isolation of highly polymorphic microsatellite loci from the temperate damselfish Parma microlepis

Microsatellites were isolated from the damselfish Parma microlepis (Günther 1862) (Pomacentridae) and screened for 100 individuals. Seven of the eight loci tested were highly polymorphic, having 14–43 alleles with average heterozygosities between 0.86 and 0.97. These loci should be informative for studies on population genetics of this species.     

Population ecology of the velvet gecko,Oedura lesueurii in south eastern Australia: Implications for the persistence of an endangered snake

Abstract Ecological specialization, such as major dependence upon a single‐prey species, can render a predator taxon vulnerable to extinction. In such cases, understanding the population dynamics of that prey type is important for conserving the predator that relies upon it. In eastern Australia, the endangered broad‐headed snake Hoplocephalus bungaroides feeds largely on velvet geckos (Oedura lesueurii). We studied growth, longevity and reproduction in a population of velvet geckos in Morton National Park in south‐eastern Australia. We marked 458 individual geckos over a 3‐year period (1992–1995) and made yearly visits to field sites from 1995–2006 to recapture marked individuals. Female geckos grew larger than males, and produced their first clutch at age 4 years. Males can mature at 2 years, but male–male combat for females probably forces males to delay reproduction until age 3 years. Females lay a single clutch of two eggs in communal nests in November, and up to 22 females deposited eggs in a single nest. Egg hatching success was high (100%), and juveniles had high survival (76%) during their first 6 months of life. Velvet geckos are long‐lived, and the mean age of marked animals recaptured after 1995 was 6.1 years (males) and 8.4 years (females). Older females (7.5–9.5 years) were all gravid when last recaptured. Like other temperate‐climate gekkonids, O. lesueurii has a ‘slow’ life history, and population viability could be threatened by any factors that increase egg or adult mortality. Two such factors – the removal of ‘bush rocks’ for urban gardens, and the overgrowth of rock outcrops by vegetation – could render small gecko populations vulnerable to extinction. In turn, the reliance of predatory broad‐headed snakes on this slow‐growing lizard species may increase its vulnerability to extinction.     

Effects of the Australian 2019–2020 megafires on a population of endangered broad-headed snakes Hoplocephalus bungaroides

The 2019–2020 Australian megafires were unprecedented in their intensity and extent. These wildfires may have caused high mortality of adult broad-headed snakes Hoplocephalus bungaroides which shelter inside tree hollows during summer. We evaluated the impacts of two high-intensity wildfires (2002 Touga Fire and 2020 Morton Fire) on a broad-headed snake population in Morton National Park, south-eastern Australia. We analysed a 29-year mark–recapture data set to estimate survival rates of adults in years with and without wildfires, and with and without human disturbance to rock outcrops. To examine the short-term effects of fire on occupancy, we analysed presence–absence data collected from 25 sites during 2019 and 2020. Estimates of occupancy were higher for 2020 (0.93 ± 0.09) than for 2019 (0.66 ± 0.14), while detection rates were constant (0.40 ± 0.06). Over the period 1992–2020, the best supported Cormack–Jolly–Seber model was one in which adult survival rates were high and stable (0.81 ± 0.04), but were 23% lower in years when humans disturbed rocks (0.63 ± 0.08). A model in which adult mortality was 20% higher in years with human disturbance and 14% higher in years with wildfires was also supported. Estimates of abundance revealed that the population declined by 34% after the Touga Fire, and 26% after the Morton Fire. Over the 29-year study, the population has declined by 60%. Our results highlight how mortality events from wildfires need to be evaluated in the context of other threatening processes. For this population, the removal of snakes and associated habitat disturbance poses a more serious threat to population viability than infrequent wildfires.     

Structure of presynaptic toxins: Crystallization and preliminary x-ray diffraction data on Notechis II-5, a presynaptic toxin phospholipase

The presynaptic neutrotoxin-phospholipase, Notechis II-5 from the venom of NotechisScutatus scutatus (Australian tiger snake) has been crystallized in a form suited for x-ray diffraction analysis. The crystals belong to the orthorhombic space group P2₁ 2₁,2₁, with unit cell dimensions, a=146.1,b =43.5 and c =39.0 A. There are two molecules of Notechis II-5 in the asymmetric unit. The molecular weight is about 13,500. Notechis II-5 is highly homologous to Notexin, another presynaptic toxin from the venom of the Australian tiger snake, to bovine and porcine pancreatic phospholipases A and other venom phospholipases.     

Development of microsatellite markers for an extremely limited distributed rare diving beetle species,Acilius kishii,and a widely distributed species,Acilius japonicus (Coleoptera: Dytiscidae)

Acilius kishii Nakane, 1963 (Coleoptera: Dytiscidae) is an endangered diving beetle species distributed in only one location, Lake Yashaga‐Ike, Honshu Island, Japan. Acilius japonicus, which is related to A. kishii, is distributed widely in northern Honshu Island and Hokkaido Island in Japan. In this study, we identified 14 microsatellite loci for A. kishii and A. japonicus, including both polymorphic and monomorphic loci, using the next‐generation sequencing method. We observed that 5 and 10 loci showed polymorphisms in 31 and 32 individuals of A. kishii and A. japonicus, respectively. The observed and expected heterozygosities were 0.00–1.00 and 0.00–0.74, respectively. These microsatellite loci could be useful for future conservation genetic studies, including monitoring of genetic diversity and extinction risk of A. kishii.     

Microsatellite markers characterized in the barn owl (Tyto alba) and of high utility in other owls (Strigiformes: AVES)

We have identified 15 polymorphic microsatellite loci for the barn owl (Tyto alba), five from testing published owl loci and 10 from testing non‐owl loci, including loci known to be of high utility in passerines and shorebirds. All 15 loci were sequenced in barn owl, and new primer sets were designed for eight loci. The 15 polymorphic loci displayed two to 26 alleles in 56–58 barn owls. When tested in 10 other owl species (n = 1–6 individuals), between four and nine loci were polymorphic per species. These loci are suitable for studies of population structure and parentage in owls.     

Polymorphic microsatellite loci in the European subterranean termite,Reticulitermes santonensis Feytaud

We report on the identification and characterization of two dinucleotide, two trinucleotide and eight tetranucleotide microsatellite DNA loci isolated from the European subterranean termite Reticulitermes santonensis. We tested the loci on 51–92 individuals from 46 colonies from different regions of France. Eleven loci were polymorphic with 2–8 alleles per locus and low observed heterozygosities (0.10–0.48). We also tested the loci on 17–20 individuals from 10 colonies in the closely related North American species R. flavipes and found significantly more alleles (2–9 alleles per locus) and higher observed heterozygosities (0.15–0.80) than in R. santonensis. The lower observed heterozygosities in R. santonensis are consistent with higher levels of inbreeding in these colonies due to the presence of numerous inbred replacement reproductives.     

Microsatellite primers for Ficus racemosa and Ficus rubiginosa

We developed 11 polymorphic microsatellite loci each for the figs Ficus (Sycomorus) racemosa and Ficus (Urostigma) rubiginosa from AG‐ and TG‐enriched genomic libraries. These 22 loci were investigated for cross‐species amplification and polymorphism in 17–21 F. racemosa and 16–24 F. rubiginosa individuals from Townsville, Australia. Observed heterozygosities range from 0.12 to 0.90 in F. racemosa and from 0.25 to 1.0 in F. rubiginosa.     

Population genetics of the mangrove salt marsh snake, <Emphasis Type="Italic">Nerodia clarkii compressicauda</Emphasis>, in a linear,fragmented habitat

The mangrove salt marsh snake (Nerodia clarkii compressicauda) occupies a unique and disappearing habitat in much of coastal southern Florida. Given extensive habitat fragmentation and high predation pressure in open spaces, it seems likely that populations of N. c. compressicauda consist of isolated groups of related individuals. To assess the degree of population subdivision in this species we genotyped a total of 125 individuals from seven locations along the Florida coast at four microsatellite loci. Overall heterozygosity was moderate (57.7%) and somewhat lower than that seen in other snake species. Population subdivision was particularly pronounced with 19 of 21 sample pair-wise Φ_ST values significantly different from zero and ranging from 0.064 to 0.343 (P ≤ 0.05). About 11 of 39 alleles were private alleles that also tended to be in high frequency in the populations where they occurred (average frequency ~27%). The correlation of genetic and geographic distances was highly significant and positive (r ² = 0.8733 and P < 0.001) with Φ_ST increasing by ~0.01 for every 10 km of separation. Overall, salt marsh snake populations appear to be fractured into isolated neighborhoods on the order of 50–80 km. In spite of its apparent local abundance, we believe that N. c. compressicauda is in need of conservation protection. The combination of extremely low dispersal, narrow habitat requirements, and most importantly, extensive habitat alteration resulting from coastal real estate development may mean that N. c. compressicauda is highly susceptible to population extirpation and potentially extinction.     

Isolation and characterization of fourteen polymorphic microsatellite markers in the viperine snake Natrix maura

Nineteen polymorphic microsatellite loci were identified and developed for Natrix maura. Polymorphism was assessed for 120 individuals sampled across four sampling sites from the French Pyrenees Mountains. The number of alleles per locus ranged from 3 to 15, and expected heterozygosity per locus ranged from 0.227 to 0.863. We tested for deviation from Hardy–Weinberg equilibrium and linkage disequilibrium and assessed the presence of null alleles for all loci, resulting in a selection of 14 high‐quality polymorphic markers. These markers will be extremely useful in identifying fine‐scale genetic structures and providing insight into conservation management plans of this species.