Appendix ES-7 Hawaiian Tree Snail Propagation Summary

Hawaiian Tree Snail Conservation Laboratory Pacific Biosciences Research Center

PI: Brenden Holland Annual Report – October 2015

TREE SNAIL PROPAGATION SUMMARY

The UH Tree Snail Conservation Lab currently houses and cares for about 400 snails in 10 endemic Hawaiian achatinelline species, all of which are listed as federally endangered. The tree snails are housed in 28 cages of three different sizes, maintained in environmental chambers. Conditions in chambers are intended to mimic natural conditions of mid-elevation Hawaiian rain forest. Chambers have temperature and light control, on a 12 hour cycle. Temperatures are held at 20 or 21°C for during daylight, and 16°C or 17 during the night. Sprinkler timers are set to water cages each 8 hours, 6 days per week. There has historically been a one day no water period, again to mimic natural conditions. Tasks for lab personnel include weekly scheduled cage changes, removal of old leaves and branches and replacing with fresh leaves of native tree and plant species. We also count births, measure newborn snails and remove, measure and preserve any dead individuals, and note percent cultured fungus consumed. Following removal of old leaves, cages are cleaned with hot water and detergent, sterilized with ethanol, air-dried, and snails are replaced along with fresh foliage. Members of our group hike Oahu trails weekly to collect fresh leaves, providing food for the snails in the form of surface growing arboreal fungus from leaves and tree bark. In addition culture medium is autoclaved weekly, and 45 plates are poured and inoculated with lab stock fungus. Cultured fungus has been used as a dietary supplement in the lab for a number of years.

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Table A. Population status summary for the Waianae species, Achatinella mustelina from most recent cycle (period ending July 1, 2015).

Population source # of snails Juvenile Subadult Adult

Peacock Flats 3 1 2 0

Bornhorst 1 0 1 0

Ekahanui Honouliuli 11 11 0 0

Palikea Gulch 2 0 0 2

Makaha 1 1 0 0

Schofield West 7 6 1 0

Makaleha 11 9 1 1

Totals 36 28 5 3

Table B. Population status summary for the Koolau species, Achatinella lila from most recent cycle (period ending July 1, 2015).

# of Species Cage Births Deaths snails Juvenile Subadult Adult A. lila Pop 1 1 0 25 9 11 5 Pop 2 1 0 38 16 16 6 Pop 3 0 0 35 20 5 10 Pop 4 0 0 33 20 6 7 Pop 5 3 2 35 22 5 8 Pop 6 0 0 25 14 5 6 Pop 7 0 1 15 12 0 3 Pop 8 2 0 21 14 3 4 Totals 7 3 227 127 51 49

There is a long-standing plan to release a subsample of the total 227 Achatinella lila currently housed in the lab, into a recently constructed predator exclusion fence in the central Koolau Mountains at a site called Poamoho. This translocation effort has been delayed due first to climatic conditions leading to complete destruction, due to high wind velocity, of the first steel walled structure. The structure was then completely redesigned

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and replaced with a wooden version by OANRP staff. Currently the fence structure itself is intake and completed, but the persistence of predatory rodents (rats) in the interior, and uncertainty as to whether rats are able to gain access from outside of the fence has delayed this action.

Chamber temperature calibrations During this period we tested all internal chamber temperatures using Hobo data- loggers and analog thermometers. We found that three of the chambers were operating a few degrees (average 2.3° C) below the control panel settings. Therefore we have adjusted panel setting to maintain internal temperatures within day/night target ranges. It is our hope that keeping more precise chamber temperatures might help stabilize reproductive rates and survival for captive species.

Figure A. Electronic and analog means to monitor temperatures in environmental chambers.

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New Tree Snail Population Data Entry System Together with SEPP staff, USFWS, and intern Ryan Pe’a, we have implemented a new data entry system. The new system uses a Weekly Master Update, and will improve data recording consistency, legibility, ability to share, and will be contiguous.

HAWAIIAN SNAIL PREDATOR STUDIES This section of the draft report includes summaries of ongoing and recently completed efforts aimed at obtaining management relevant biological data during this funding cycle. The projects summarized share the objective of understanding and ultimately controlling invasive predators that are known or suspected to impact endangered species on Army lands, including two projects (sections D & E) that were recently submitted for peer- reviewed publication.

Recent OANRP-funded studies conducted in our lab over the past few years have resulted a number of scientific publications concerning:

1.) The first detection of impacts on endemic fauna, including the Oahu tree snail Achatinella mustelina (Holland, Costello & Montgomery 2010).

2.) Impact projections, estimates and overall threat assessment via gut content analyses and ingestion frequency (Chiaverano & Holland 2014).

3.) We have characterized movement behavior and establishment of home range in various forest habitats (Chiaverano, Wright & Holland 2014) using radio-transmitter tracking studies in the field.

4.) We discovered consistent, statistically significant differences in head morphology (size) and invertebrate prey utilization among different Hawaiian islands correlated with differences in bite force due to diet differences and rainfall (Van Kleeck, Chiaverano & Holland in press, summarized below).

For the predatory invasive wrinkled frog, we have submitted our findings for publication (summarized in section D below).

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A) Chameleon dissection results:

We euthanized and dissected 21 adult field collected Jackson’s chameleons during this period, all cleared from the Puu Hapapa site, outside of the snail exclosure in the native forest. The most significant result was one large male was found to have consumed 16 native helicarionid snails, all present in its gut, of various sizes, presumably all were the same species Philonesia harmanni. Jackson’s chameleons continue to pose an immediate threat to the persistence of native Hawaiian invertebrates on Army managed lands. No additional A. mustelina have been observed in chameleon stomachs.

B) Jackson’s Chameleon control feasibility trial based on comparison to Brown Tree Snake management on Guam Ecosystem damage by the introduction of the Brown Tree Snake (BTS) (Boiga irregularis) in the 1950’s on the island of Guam (US Territory) has resulted in the devastation of the island wildlife, particularly the native and naturalized avifauna. In fact 11 of the 18 native birds have been extirpated and all of the remaining taxa severely depleted (by over 90%), and 12 resident species have been extirpated. Following introduction of very few specimens (1998) the BTS spread rapidly, within a few years reaching densities of 12,000 per square mile, resulting in thousands of hospitalizations due to bites in the past two decades, and regular, electrical outages caused by BTS (about 1.5 hrs every other day) (Burnett et al 2008). Several decades and millions of dollars have been invested in attempted control, and have proven largely unsuccessful. Targeted management efforts to date have mainly consisted of baited minnow traps that use live mice, and are placed along residential fence lines and around utilities and power generating stations. There has been a concerted effort both on Guam and at Honolulu International Airport, to prevent transfer of this devastating invasive reptile to the Hawaiian Islands.

Although Hawaii has no invasive snakes, at the present time, there are 26 established predatory invasive reptiles and amphibians in the islands. The Jackson’s chameleon

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(Trioceros jacksoni xantholophus) is the most ecologically damaging species of invasive herpetofauna in Hawaii, for which the threat and impact has been characterized to date. Relative to the density recorded for BTS, of 12,000 snakes per mi2, Jackson’s chameleons have been observed at a density of six times this, or 72,000 per mi2 (45 per 0.4 acres = 72,000 per mi2). Our concern is that predatory activity of Jackson’s chameleons on Oahu could cause the extinction of tree snails in areas where the species overlap in habitat, as has occurred on Guam with the invasive BTS and native birds. In a sense, the Jackson’s chameleon could be considered Hawaii’s BTS, and it warrants immediate control efforts.

Recent innovative field trials on the island of Guam, aimed at control of BTS conducted by the USGS and USDA, appear to be having the desired impact, namely a reduction of the population density of snakes. In this trial the commercially available analgesic Tylenol®, or generic name acetaminophen, is delivered via placement in the esophagus of a dead mouse, at a dosage of 80 mg/kg. This dose has been tested in the laboratory and shown to be lethal to all size classes, even the largest adult snakes, with little or no secondary or non-target impacts. We are interested in investigating the possibility of adopting this strategy for Jackson’s chameleons in Hawaii. We have recently received IACUC protocol approval to test acetaminophen on chameleons in the laboratory.

Once approval was received for lab testing, we started trials immediately, and preliminary results are definitive: this product is toxic to Jackson’s chameleons at same dosage as used for BTS in Guam (80 mg acetaminophen/kg body weight). We will continue to test the same dosages per body weight as were done with BTS lab trials. Once lethal dose for chameleons is optimized we have various ideas in terms of how to deploy the pill fragments in the field, which will also be evaluated in the laboratory setting prior to scaling up for field trials. However this work will require a small amount of funding plus permits. Part of the regulatory requirement to field deployment of acetaminophen addition to IACUC protocol approval, will be meeting the EPA labeling regulations, since acetaminophen is not labeled as a reptile control product.

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C) Improving Chameleon Detection Methods

There is a need to devise an improved method of field detection of Jackson’s chameleons in the field. In collaboration with HECO we have been experimenting with a thermal imaging device, called a Fluke, which is the property of HECO, and we are working with their Environmental staff. We have tried this equipment in the lab and in the field and are continuing to investigate whether this technology imparts a detection advantage when searching for chameleons in the forest canopy by eye. Thus far this technology seems to hold some promise under certain environmental conditions. Trials will continue (Figure C-1).

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Figure C-1. Thermal images recorded with Fluke technology in the laboratory, when chameleon body temperature differs even by less than one degree from ambient or background temp, this technology is a substantial help in detecting chameleons in the tree. The question we now have is, how frequently does this situation occur, where chameleon and background differ. Device was provided and tested courtesy of HECO Environmental office. Trials are ongoing.

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D) Prey-associated head-size variation in an invasive lizard (Trioceros jacksoni xantholophus) in the Hawaiian Islands

(this study was conducted during the funding period, and has been accepted for publication in the Biological Journal of the Linnean Society, the following is a summary, OANRP support was acknowledged)

Biological invasions are recognized as a primary driver of large–scale changes in global ecosystems. This study addresses ecomorphological variation in head size within and among populations of an ecologically destructive invasive predator, and evaluates the potential roles of environmental components in phenotypic differentiation. We used four size-corrected measurements of head morphology in Jackson’s chameleons (Trioceros jacksonii xantholophus)(n=319) collected from three Hawaiian Islands to assess phenotypic variation among and within islands. Head size (PC1) was compared among islands using ANOVA, and its association with factors such as rainfall and exploited prey hardness was assessed by Pearson correlation analysis and Mann-Whitney U-tests. Differences in prey exploitation among islands were tested using Chi-square analysis, revealing head size differences among islands (mean difference >5%), and these differences were found to be correlated with variation in hardness of prey consumed. These results suggest that morphological differences among introduced island populations of the Jackson’s chameleon may be due to ecomorphological adaptation to differences in exploited prey hardness, whether due to prey choice or availability.

Spatial distribution of different environmental factors results in distinct selective pressures on functionally important anatomical characters, such as head size in reptiles. We proposed that, following several dozen generations of reproductive isolation in different environments, head differentiation may be evident among island populations. In order to address this hypothesis we collected morphological data using four size- standardized skull measurements in chameleons from three islands (Hawaii, Maui and Oahu) and tested for correlation among these measurements and environmental factors, such as rainfall and exploited arthropod prey hardness (based on prey type) from gut content data. Our objectives were to 1) quantify the extent of intra-specific skull size variation within and among multiple populations of T.j. xantholophus from three islands

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and 2) conduct and evaluate correlation analyses of morphometric patterns and potential environmental drivers of phenotypic variation in this species.

Results Head morphology comparison among islands - ANOVA detected no significant

differences in SVL among islands (ANOVA: F(2,312)= 0.46, p= 0.63), but indicated significant differences in PC1 among islands (ANOVA: F(2,312)= 5.7, p= 0.02). Chameleons from Oahu had significantly smaller heads than those of their counterparts from Hawaii (Tukey’s, p < 0.001), while no significant differences in head size were detected between Maui and other islands (Tukey’s, p > 0.05). Environmental effects - Chameleon diet composition in terms of prey hardness varied significant among islands (χ2= 7.69, df = 2, p=0.02). Individuals from Hawaii showed an exploitation pattern with the highest percentage of hard prey items (62.5%), while in the diet of chameleons from Oahu, the lowest percentage of hard prey items was observed (37.3%). Intermediate percentage values (52.5%) were found in individuals from Maui. Significant differences in PC1 were observed between low and high rainfall sites on both Maui (Z = -2.89, p< 0.005) and Oahu (Z= -2.08, p< 0.05). In various reptile taxa, prey hardness is an important factor driving head size variation including chameleons. In this study, larger heads of T.j. xantholophus were associated with a significantly higher proportion of hard prey consumed based on gut content analysis, suggesting that inter-island geographic variation in head size could be due to local adaptation to differences in composition of prey exploited (i.e, hard versus soft prey items). Geographically isolated populations are likely to diverge morphologically from one another given time as they approach local fitness optima, especially in the presence of distinct environmental and prey assemblage differences. In cases where gene flow is absent, divergent selection has been shown to drive local adaptation over short ecological time-scales. Relative head size of Jackson’s chameleons was significantly smaller at high rainfall locations on Oahu and Maui. Although rainfall is not likely to directly impinge on morphological variation, ecological factors correlated with rainfall, such as resource availability and quality, may drive variation in functional features such as head size,

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among others. For example, prey type and availability have been shown to vary with precipitation levels, and food abundance has been shown to be higher with higher rainfall in a seasonal tropical ecosystem. Arthropods are the main food source for Jackson’s chameleons in the Hawaiian Islands, and these taxa tend to have thickened cuticles in more arid environments to counteract desiccation, while in wetter environments arthropods had thinner cuticles, and softer body types. Higher rainfall has been shown to translate to increased availability of softer prey, requiring relatively lower bite force and smaller predator head size. In fact Measey et al. (2011) showed that Cape dwarf chameleons exhibited a preference for smaller, softer arthropod prey where available.

Figure D-1. Dotted lines show measurements recorded from the heads of each individual T. j. xantholophus: head length (HL), head height (HH), jaw length (JL) and head width (HW). Male chameleon depicted (three prominent horns shown).

The results of this study suggest that local adaptation to environmental conditions, i.e. exploitation of different dietary resources, whether due to availability or selective preference, may be driving evolutionary divergence in head size of Jackson’s chameleon populations on different islands. It is possible that the observed differences among islands are a result of neutral variation due, for example, to multiple founder events and genetic drift, where small numbers of chameleons likely established each sampled population, and sampling bias has resulted in the phenotypes exhibited by those small populations.

Strict enforcement of regulations prohibiting inter-island transport has effectively eliminated the possibility of interisland gene flow. Assuming that the larger head ecomorph and its concomitant enhanced bite force is an optimal phenotype under the lower precipitation/harder prey habitat scenario observed on the island of Hawaii, this phenotype likely evolved on a relatively short ecological time scale. Therefore, we may have documented insipient adaptive divergence of Oahu and Hawaii chameleon populations.

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Successful establishment of anthropogenically released non-native taxa is one factor that is dramatically altering our island ecosystems, but it can also can provide opportunities to investigate the pace and process of micro-evolutionary change. In such instances where lineages with reduced genetic diversity are placed into varied, novel environments, the primary drivers, whether plasticity or adaptation, can be addressed and potentially elucidated. In further studies, integrated genomic, ecomorphological and behavioral approaches will be useful in elucidating relative genetic versus environmental contributions to evolutionary change in established non-native populations. Such studies that take place during contemporary invasion and range expansion events can detect biological change as it plays out, on ecological time scales, and therefore hold potential to provide real-time insights into the ability of species to adapt in the face of changing global ecosystems, and may provide important biological data that can be informative for management and control of invasive pests.

Figure D-2. Distribution of Trioceros jacksonii xantholophus in Hawaii, based on museum collections, live specimens captured for this study. a.) Map of known distributions main islands. Collection localities are indicated by black dots on the islands b.) Oahu, c.) Maui, and d.) Hawaii. This species exhibits sexual dimorphism in horn morphology, where e.) females lack, and f.) males have horns. Star symbol on map b.) indicates the approximate original location of release of T.j. xantholophus in 1972.

(References and reprints available on request)

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E) Characterization of impacts of the wrinkled frog, Glandirana rugosa, on Hawaii’s native invertebrate fauna (this study was conducted during the funding period, and has been submitted for publication to Biological Invasions, the following is a summary, OANRP support was acknowledged)

Introduction Invertebrates constitute the most diverse animal lineages on Pacific Islands, and have experienced the most significant extinction rates. Recent losses of biodiversity, particularly in the form of gastropod extinctions in the Hawaiian Islands have been driven largely by ecosystem changes brought about by direct predation by introduced predators. Although Hawaii notably lacks native terrestrial reptiles and amphibians, anthropogenic releases of herpetofauna have resulted in the establishment of frogs, toads, turtles and lizards, among which are some of the most conspicuous faunal groups in the islands today (e.g. coqui frog, green day gecko, brown anole). Many of these taxa overlap in their distributions with native Hawaiian forests. However, despite establishment of more than two-dozen predatory reptile species in Hawaii, ecological impacts remain unknown for 24 of 26 species.

In this investigation, we conducted surveys, collected specimens and used museum collections of the wrinkled frog, Glandirana rugosa, an established species intentionally released in the late 19th century, from three main Hawaiian Islands (Kauai, Maui, Oahu). The significance of this species distribution lies in the fact that it overlaps in several locations with endemic snails. We conducted comparative gut content analyses from two islands in an effort to assess impacts and enable prioritization of management decisions. Our results suggest that diet composition in the Hawaiian Islands is significantly different from that in its native Japan, where the dominant taxonomic groups by volume were Coleoptera (beetles), Lepidoptera (moths, butterflies) and Formicidae (ants). Invasive frogs in Hawaii exploited mostly Dermaptera (earwigs), Amphipoda (landhoppers) and Hemiptera (true bugs). In Hawaii this species also exploited endemic insects (~5% total volume, 7 genera) and snails (14 snails in 3 endemic genera).

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The Japanese wrinkled frog, Glandirana rugosa (Jiang and Zhou 2005), was intentionally released on Oahu from Japan intended for agricultural pest control in 1896 by entomologists employed by the Republic of Hawaii with a small number of additional releases between 1900 and 1940 (Bryan 1932; Funasaki et al. 1988). In their native range, G. rugosa inhabits rice paddies and feeds on a variety of invertebrates, including snails and insects (Hirai and Matsui 2000). However, despite having been established across the Hawaiian Islands for nearly 120 years, no previous effort has been made to characterize the threat posed by G. rugosa to native ecosystems. Additionally, since the range G. rugosa and a number of endemic terrestrial invertebrates, including numerous endangered species on Oahu, overlap (Englund 2002; Englund et al., 2003; Englund and Arakaki, 2004; Preston et al. 2007) assessment of the threat posed is warranted. The objectives of this study were to: 1) examine diet of G. rugosa and begin to characterize threats to native invertebrate fauna in a comparative framework by examining diet in its native range; 2) investigate seasonality of reproduction; 3) assess uniformity of exploited taxa among male versus female frogs; 4) report observed recent range expansions using GPS positions of collections of this predatory species.

Methods

Data collection

Locality, gender, reproductive status and body size information were documented from the collections in the Bishop Museum from three islands (12 sites Oahu, 5 Kauai, 6 Maui) and from specimens collected by hand in the Koolau Mountains on Oahu (5 sites)(Figure 1). Stomach contents were microscopically examined from nine sites on Oahu and two on Maui, and prey were identified to family, and genus / species where possible. Most dietary items were intact individual prey, but for those that were disarticulated due to mastication and or digestive processes, prey items were identified by diagnostic morphological features of hard body parts (such as wing venation in Diptera). Length, width, and height of prey items were measured to the nearest 0.01 mm from either specimens removed form the stomach if whole, or averaged from 3-5 identified preserved specimens (Kraus and Preston 2012). Prey volumes were calculated using the ellipsoid equation V=4/3π (abc) where a, b and c are the body axes (Kraus and Preston,

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2012; Kraus et al., 2012). In addition, snout-vent- length (SVL) of all specimens was measured using digital calipers to the nearest 0.01mm and exact collection locality was recorded.

Data analysis

To test whether frogs show gender-correlated differences in exploited prey size, total prey volume was calculated for each individual and differences in average prey volume between males and females were analyzed using Mann-Whitney U tests. Additionally, the relationship between total prey volume and SVL was analyzed using Linear regression, to determine the relationship between prey size and frog body size.

Figure E-1. Map of collection localities; a) Hawaiian Islands, inset photo of Glandirana rugosa, b) Kauai, c) Oahu and d) Maui. Closed circles are sites where gut contents were collected, open circles represent collection localities where only size and reproductive data were collected. Numbers (1-4) on Oahu map shown in c indicate sites where wrinkled frogs had not previously been documented.

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Results

Locality, gender, reproductive status and body size data were collected from 102 individuals, 41 live caught and 61 preserved specimens, from three islands: Kauai (n = 7), Maui (n = 27), and Oahu (n = 68). A total of 447 prey items were identified from a subset of 52 individuals from nine locations on Oahu and two on Maui (Figure E-1). Female biased sexual size (SVL) dimorphism is known for this species (Khonsue et al. 2001): average female body size (n=41) was 45.64± 0.93 mm, and average male body size (n=46) is 37.2 ± 0.66 mm. However, despite body size differences, no differences were detected in total prey volume between males and females (Mann-Whitney U test: Z= 0.041; df= 46; p= 0.97) and there was no relationship between SVL and total prey 2 volume (Linear regression: R = 0.015; F(1,47)=.69; p= 0.41). Preliminary reproductive status analysis suggests that there is seasonality in the cycle, where the peak number of gravid females was observed in January (rainy season) and the highest observed numbers of juvenile frogs were observed in July (dry season).

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Table E-1. Summary of introduced reptile and amphibians in the Hawaiian Islands, including date of release and current status. Abbreviations for current status are: “Rel.” = Released but not established; “Est” = Established; “Era”= Eradicated following release; “Unkn” = Unknown status following release. Totals by group are: Anura = 14; Squamata = 22; Testudines = 7. Total 43 species, 4 are of uncertain status, 26 are established. Note also that the focus of this study, the wrinkled frog Glandirana rugosa, along with Bufo americanus are the earliest documented herpetofaunal releases in Hawaii, although B. americanus did not become established, leaving G. rugosa as the longest standing herpetofaunal introduction. Historical release date and current status, i.e. whether established, eradicated, or unknown, for all herpetofaunal taxa (total 43 species) was compiled and summarized for the Hawaiian Islands (Table E-1). Of the two anuran species that were intentionally released as attempted biocontrol in the late 1800’s, Bufo americanus and G. rugosa, only the wrinkled frog remains, although additional releases of this species are also documented as occurring subsequent to the initial release (Bryan 1932). Total releases by group are 14 species of frogs and toads (Anura), 22 lizards (Squamata), and seven turtles (Testudines). Of the total 43 species released in Hawaii, four are currently of uncertain

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status, in some cases still periodically observed and occasionally documented but not unambiguously naturalized. A total of 26 herpetofaunal taxa are currently established in the Hawaiian Islands.

Frogs examined for exploited prey species analysis were collected at elevations ranging from 100-800 m (Table 2) in Hawaii. In terms of comparison of predatory behavior between introduced and native ranges, the three most common prey item categories in frogs collected in the Hawaiian Islands by number consisted of Amphipoda (22.15%), Hemiptera (13.20%) and Hymenoptera (11.63%), whereas dominant prey groups by counts in Japan were Hymenoptera (59.07%), Diptera (13.60%) and Coleoptera (12.23%) (Hirai & Matsui 2000). Analysis of prey composition by volume for the Hawaii samples revealed that the top groups were Dermaptera (59.42%), Amphipoda (10.92%) and Hemiptera (9.59%). Dominant prey groups by volume for Japan were Coleoptera (28.23%), Lepidoptera (21.27%) and Hymenoptera (12.00%). The only group shared was Hymenoptera, the third most common prey item by number of specimens in Hawaii, and the top prey group by number and third category by volume in Japan. In its native range, G. rugosa consumed a lot of ants, as these individuals are very small in volume, yet still occupied 12% of the relative volume of prey but only 1% by volume in Hawaii.

Identified native fauna comprised ~4% of prey items by counts, and 5% of total prey volume and were found in frogs from all collection localities, including both native and non-native forest sites on both islands we sampled. However it should be noted that these values are likely to be underestimates due to the challenge associated with positively identifying gut contents to species, coupled with the fact that we only recorded those species for which we were most confident. Yet, 52 and 77% of prey items by count and volume, respectively, belong to families with diverse endemic lineages (Table E-4).

There were 17 collection localities on the island of Oahu, we collected G. rugosa from five localities, four of which were new recorded sites, suggesting ongoing range expansion (Figure E-1).

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Table E-2. Summary of stomach contents by family from 52 individuals of Glandirana rugosa from Oahu and Maui.

Discussion

This study is the first to examine Glandirana rugosa impact in Hawaii despite its establishment in the islands for about 120 years. The presence of small snails was recently documented in the diet of coqui frogs in Hawaii (Beard 2009), although taxonomic composition of these snails not reported, 12 species were categorized as “possibly endemic”. The results of this investigation reveal the first confirmed case of an invasive amphibian preying on endemic land snails.

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Table E-3. Summary of diet contents by order, comparing relative abundance and volume of prey items in introduced range (Hawaiian Islands, n=52) and native range (Japan, n=139; Hirai and Matsui 2000). The three most dominant exploited prey items by number and volume from native and introduced ranges are in bold. Prey items present in gut contents of frogs in their native range but not in introduced range, and are not included in this table are: Collembola, Decapoda, Odonata, Plecoptera, Protura, Thysanoptera and Tricoptera.

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Though G. rugosa was released as an attempted biological control for taro pests, known pest species of any group comprised only ~1% of G. rugosa’s diet by volume, consisting of invasive ants, which do not feed on taro. Compared to this tiny fraction of total prey volume, nearly 6% was native endemic invertebrates. The introduction of G. rugosa to control pests has been a dramatic failure.

Amphipods, which comprised the largest prey item by number, play an important ecological role in leaf litter decomposition in the Hawaiian forest ecosystem, and comprise diverse endemic lineages, with 11 species on Oahu (Hurley 1959). Additionally, arthropod abundance surveys from nearby sites of similar elevation, rainfall, and forest composition suggest that amphipods are the most commonly available prey item in the leaf litter (Van Kleeck et al. unpub) as reflected in the number observed in stomach contents. The order Dermaptera, the earwigs, was most dominant prey group by volume observed in G. rugosa diet, and there are ten native Hawaiian species (one indigenous and nine endemic) (Hawaiian Arthropod Checklist 2004). Although the endemic dermapteran species are not common, the frog’s ongoing range expansion (Figure E-1) into native forest observed during this study, will likely increase overlap and interaction of this frog with these and other important endemic species.

Frogs are known as generalist predators that can reach extremely high densities (Beard 2009). Of particular concern is the new observation that Hawaii’s endemic land snails, a group that on the whole is in dire conservation state, were specifically targeted by this frog. Of 21 snails observed in frog guts, 14 individuals (67%) were members of three genera with endemic species on Oahu (Elasmias, Tornatellides, and Philonesia). Geographic spread by the wrinkled frog from low-elevation agricultural land where it was released in taro patches, into native watersheds up to 800 m elevation warrants concern in terms of impacts on rare and threatened native invertebrate fauna which have experienced unprecedented declines due the introduction of predatory invasive species (Solem 1991) in recent decades (Holland 2009; Holland et al. 2008).

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Snails comprised 5% and 6% of dietary items in number and in volume respectively, in this study (Table E-2). Gastropods were also documented in the diet of G. rugosa in the native range (Hirai and Matsui 2000), though at far lower frequencies, and in invasive coqui frogs in Hawaii (Beard 2009). Small land snails are a documented prey item in other ranid frogs as well (e.g. Tyler and Hoestenback 1979). Of the 10 families with endemic Hawaiian land snail species, one genus (Achatinella) the Oahu tree snails, has been afforded endangered status under the U.S. Endangered Species Act (USFWS 1981). Partly because Hawaiian land snail endemism exists at multiple hierarchical levels, including species, genus, subfamily and family levels (Solem 1990), this fauna makes attractive scientific models for the study of biogeography and evolutionary radiations (Cowie and Holland 2008; Holland and Cowie 2009). However the Hawaiian land snail fauna has suffered extinction rates estimated at 65-90% (Solem 1990; Cowie 2001) because of collection, habitat loss, and in recent decades predation by invasive species. Rats, Rattus rattus, and the rosy wolf snail, Euglandina rosea, (Hadfield 1986) have traditionally been considered the primary threats to Hawaiian snails, with the Jackson’s chameleon recently entering the realm of conservation concern (Holland et al. 2010; Chiaverano and Holland 2014). These invasive predators have devastated multiple species, and the threat of extinction persists for extant snail taxa, failing intervention. This study reveals the second herpetofaunal species documented as preying on endemic Hawaiian land snails. However, the wrinkled frog has been shown to occupy primarily riparian leaf litter rather than arboreal habitat, so it is unlikely that this species poses a threat to the endangered tree snails. But leaf litter dwelling snail lineages such as the extremely rare Amastridae and Endodontidae share overlapping habitat with G. rugosa. Frogs hold the potential to alter ecosystems due to their high population densities and generalist feeding habits. Although ranid frogs are generalist predators, and their diet is known to vary with habitat (e.g. Elliot and Karunakaran 1974; Tyler and Hoestenbach 1979), in its native range, G. rugosa exhibits a similar level of preference for ants as other known ant specialists (Hirai and Matsui 2000). Ants were consumed at relatively high frequency by frogs in Hawaii (11% by count), and all ant lineages are invasive in the islands, but without prey availability data from the collection sites, it is uncertain whether G. rugosa displays selectivity for these species.

22 Appendix ES-7 Hawaiian Tree Snail Propagation Summary

Predatory behavior patterns did not vary with frog body size, developmental stage, or with sex suggesting that frogs of both genders and all life stages are potential predators to native arthropod species. Although Tinker (1938) stated that all life stages can be found throughout the year in Hawaii, during this study more juveniles were collected between May and September and gravid females from November to February, suggesting possible seasonality in reproduction. Ranid frogs exhibit high levels of variation length and period of metamorphosis, as a result of varying climates and food availability (Riha and Berven 1991; Merilä et al. 2000). In fact, in our studies, G. rugosa completes metamorphosis in as little as seven weeks in Hawaii (pers observ), as opposed to 12 to 52 weeks in their native range, with longest delays seen where over-wintering is common (Khonsue et al. 2001). Despite its association with agricultural areas such as rice paddies in its native range (Hirai and Matsui 2000), in Hawaii G. rugosa is currently undergoing range expansion into native forest sites and areas of conservation concern (pers observ).

Dramatic differences between predatory patterns observed in frogs from Japan versus Hawaii may reflect differences in prey availability in these different habitats based on forest complexity, plant community structure and invertebrate communities therein. On the other hand, we are beginning to see that adaptation of invasive herpetofauna to Hawaii’s novel and highly diverse microhabitats can be rapid (Van Kleeck et al. in press). It is possible that the accelerated rates of metamorphosis and differences in predatory patterns observed between native and introduced ranges of this frog reflect changes due to adaptation of development rate and prey preference, though further studies will be needed to confirm this possibility.

Impact and threat assessment studies of invasive herpetofaunal species in Hawaii remain few relative to the numerous (26) known established predatory amphibians and reptiles in the islands. Likewise there is a need for additional studies of native and endemic invertebrate diversity, ecosystem services, and changes in areas where new predatory species are established. Studies of declines in native biodiversity are useful, but do not address indirect effects of loss of invertebrate taxa in terms of ecosystem processes such as pollination, leaf litter breakdown, and nutrient cycling. Only once changes in native community structure, alteration of ecosystems and ecology of invasive

23 Appendix ES-7 Hawaiian Tree Snail Propagation Summary

species are reconciled, can we begin to effectively address the loss of native biodiversity by counteracting the spread and controlling the activity of nonnative lineages. Further in- depth assessment of impacts on endemic ecosystems are warranted to improve our ability to manage and ultimately restore diverse island ecosystems.

Acknowledgements

For help with collection of specimens we thank Vince Costello at the Oahu Army Natural Resource Program. We thank Luc LeBlanc at the University of Hawaii Insect Museum, Thomas Arthur Hooper Smith, University of Hawaii Department of Zoology, and Jesse Eiben University of Hawaii, Hilo, for aid in identification. We also thank the National Parks Service and Molly Hagemann at the Bernice P. Bishop Museum for use of museum collections. Collections were conducted under Hawaii Department of Land and Natural Resources collection Permit #EX 12-22.

(References and reprints available on request)

24 Appendix ES-7 Hawaiian Tree Snail Propagation Summary

Table E-4. Summary of number and volume of arthropods comprising stomach contents, in comparison to the species diversity (total number) of endemic and introduced taxa in each of the families that occur in the Hawaiian Islands. Known Hawaiian endemic lineages are in bold.

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