Gut check: predatory ecology of the invasive wrinkled ( rugosa) in Hawai´i

By Melissa J. Van Kleeck and Brenden S. Holland*

Abstract Invertebrates constitute the most diverse Pacific island lineages, and have correspondingly suffered the most significant extinction rates. Losses of native invertebrate lineages have been driven largely by ecosystem changes brought about by loss of habitat and direct predation by introduced species. Although Hawaii notably lacks native terrestrial reptiles and , both intentional and unintentional anthropogenic releases of herpetofauna have resulted in the establishment of more than two dozen species of , toads, turtles, lizards, and a snake. Despite well-known presence of nonnative predatory species in Hawaii, ecological impacts remain unstudied for a majority of these species. In this study, we evaluated the diet of the , Glandirana rugosa, an intentional biocontrol release in the Hawaiian Islands in the late 19th century. We collected live frogs on Oahu and used museum collections from both Oahu and Maui to determine exploited diet composition. These data were then compared to a published dietary analysis from the native range in Japan. We compiled and summarized field and museum distribution data from Oahu, Maui, and Kauai to document the current range of this species. Gut content analyses suggest that diet composition in the Hawaiian Islands is significantly different from that that in its native Japan. In the native range, 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 (~4% total volume, 7 genera) and snails (14 snails in 3 endemic genera). Our results suggest the need for more in-depth assessment of ecological impacts of G. rugosa and other established herpetofauna in Hawaii in order to improve our ability to prevent and manage ecological damage and ultimately restore diverse island ecosystems.

*Corresponding Author E-mail: [email protected]

Pacific Science, vol. 72, no. 2 November 8, 2017 (Early view) Introduction

Oceanic islands are isolated land-forms typified by diverse microhabitats that harbor rapidly evolving lineages of flora and fauna with unprecedented endemism. The islands comprising the Hawaiian Archipelago are among the most isolated on Earth, where incomparable biological diversity of relatively few colonizing lineages has evolved, leading to several centuries of scientific attention due to the spectacular terrestrial radiations (Gillespie et al. 1994;

Cowie and Holland 2008; Medeiros and Gillespie 2011). Island assemblages are also of interest by nature of the fact that major lineages present in continental tropical ecosystems are often missing (for example land reptiles and mammals), rendering the native island communities disharmonic. However, habitat alteration and the introduction of non-native species, both intentional and unintentional, have led to unprecedented island extinction rates (Solem 1990;

Cowie 2001; Boyer 2008; Regnier et al. 2015). Over 40 species of reptiles and amphibians have been introduced to Hawaii (Kraus 2008), both unintentionally through horticulture and other anthropogenic transportation, and intentionally for biological control, food, or aesthetic purposes.

Twenty-six of these species are established in the islands, though scientific attention has been devoted, and only recently, to a minority of these, namely the Jackson’s chameleon, Trioceros jacksonii xantholophus (Kraus and Preston 2012); Kraus et al. 2012; Chiaverano et al. 2014; Van

Kleeck et al. 2015) and the coqui and greenhouse frogs, Eleutherodactylus coqui and E. planirostris (Kraus et al. 1999; Beard and Pitt 2005; Woolbright et al. 2006; Beard 2007; Olson and Beard, 2012; Olson et al. 2012) . Due to their opportunistic predatory foraging behaviors, and ongoing dispersal and range expansion via anthropogenic means as well as their own vagility, introduced herpetofaunal lineages have dynamic though poorly characterized distributions. Recent studies suggest that predatory ecology within the native range of a species may not necessarily predict actual impacts in introduced environments due to differences in prey

2 availability or even adaptations to novel environmental conditions (e.g. Beard, 2007; Tillberg et al 2007; de Amorim et al. 2017). Therefore, understanding distribution patterns and predatory impacts constitutes an important first step towards assessment of potential control mechanisms to protect unique Hawaiian biodiversity.

The Japanese wrinkled frog, Glandirana rugosa (Temminck and Schlegal 1838), was intentionally released on Oahu from Japan for agricultural pest control in 1896 by entomologists employed by the Republic of Hawaii (Bryan 1932; Funasaki et al. 1988). There were a few additional releases between 1900 and 1940. 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.

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 examine the diet of G. rugosa and begin to assess threat posed to native invertebrate fauna, while also comparing exploited taxa between male vs. female frogs, and total volume of prey by body size. A comparative examination of diet in the introduced and native ranges was also conducted using results from this study and data from Hirai and Matsui (2000).

MATERIALS AND METHODS

Data collection.—Locality, sex, reproductive status and body size information were documented from the collections in the Bernice P. Bishop Museum from three islands (12 sites on Oahu, 5 on

Kauai, 6 on Maui) and from specimens collected by hand in the Koolau Mountains on Oahu (5 sites; Figure 1). Sex was determined by the presence of nuptial pads on males, and reproductive

3 status was determined by the presence of egg sacs in females. Snout-vent- length (SVL) of all frogs was measured using digital calipers to the nearest 1 mm and is reported as an average ± standard error (SE). Field collected individuals were euthanized according to IACUC protocol

15-2148. Stomach contents were microscopically examined from nine sites on Oahu and two on

Maui, and prey were identified to family, and / species where possible. Most dietary items were intact individual prey, but for those that were disarticulated due to 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 using digital calipers to the nearest 0.1 mm from intact specimens removed from the stomach, or averaged from 3-5 identified preserved specimens (Kraus and Preston 2012). Prey volumes were calculated using the prolate spheroid equation V=4/3π (L/2)(W/2)2 according to Hirai and Matsui

(2000) (see Dunham, 1983 for more details). Diet contents of Hawaii G. rugosa were then compared to literature findings from their native range from Hirai and Matsui (2000).

Data analysis.—To test whether frogs show sex-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, linear regression was used to determine the relationship between total prey volume and SVL. All statistical analyses were performed in JMP version 12.1.0 (SAS©).

RESULTS

Locality, sex, 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). From a total of 17 collection localities on the island of Oahu, we collected G.

4 rugosa from five localities, four of which were previously undocumented sites, suggesting ongoing range expansion (Fig. 1).

A total of 447 prey items were identified from 52 individuals from nine locations on

Oahu and two on Maui (Figure 1; Table 1). Female biased sexual size (SVL) dimorphism is known for this species (Khonsue et al., 2001): average female body size (n=41) was 45.6 0.9 mm (SE), and average male body size (n=46) is 37.2  0.7 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 is no relationship between SVL and

2 total prey volume (linear regression: R = 0.015; F(1,47)=.69; p= 0.41).

Frogs examined for exploited prey species analysis were collected at elevations ranging from 100-800 m (Fig. 1) in Hawaii. The three most common prey item categories in frogs collected in the Hawaiian Islands by number consisted of Amphipoda (22.2%), Hemiptera

(13.2%) and Hymenoptera (11.6%). Analysis of prey composition by volume for the Hawaii samples revealed that the top groups were Dermaptera (61.9%), Amphipoda (8.4%) and

Hemiptera (9.9%). Identified native fauna comprised ~4% of prey items both by counts and 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 (Table 2). 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 high levels of endemism (Table 2). Comparisons of diet between introduced and native ranges can be found in Table 3.

5 DISCUSSION

This study is the first to examine the diet of Glandirana rugosa in Hawaii, despite its intentional release and establishment in the islands for well over a century (Table 1). The presence of small snails was also documented in the diet of coqui frogs in Hawaii (Beard 2007), where 12 species were categorized as “possibly endemic”. Although this previous investigation suggested that coqui frogs may feed on native gastropod lineages (Beard 2007), the results of our investigation reveal the first confirmed case of an invasive that was intentionally released preying on endemic Hawaiian land snails.

Frogs are known as generalist predators that can reach extremely high densities (Beard

2007). Of particular concern here is the observation of endemic land snails, a group that on the whole is of dire conservation concern, in the diet of an intentionally released frog in Hawaii. Of

21 snails observed in Oahu frog guts, 14 individuals (67%) were endemic Hawaiian species in three genera (Elasmias sp., Tornatellides sp., and sp.). 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 1990) in recent decades (Holland et al. 2008,

2010).

Snails comprised ~5% of dietary items by count and volume in this study (Table 2).

Gastropods were also documented in the diet of G. rugosa in the native range (Hirai and Matsui

2000), though at lower frequencies (~1.2%; Table 3), and small land snails are a known prey item in other ranid frogs as well (e.g. Tyler and Hoestenback 1979). Of the 10 families with endemic Hawaiian land snail species, the achatinellid tree snail genera Achatinella, Partulina and

Newcombia have been afforded endangered status under the U.S. Endangered Species Act

6 (USFWS 1981; USFWS 2013). 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 including rats, Rattus rattus, the rosy wolf snail,

Euglandina rosea (Hadfield 1986), and most recently, the Jackson’s chameleon (Holland et al.,

2010; Chiaverano and Holland 2014). 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 confirmed depredating 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 arboreal taxa such as the endangered tree snails, which tend to occupy forest canopy. But leaf litter dwelling snail lineages such as the extremely rare Amastridae and Endodontidae are of conservation concern due to habitat that overlaps with G. rugosa.

Amphipods, which comprised the largest prey item by number in this study, have a diverse though poorly studied endemic radiation in Hawaii, with 11 species on Oahu (Hurley

1959). However, recent studies have shown that the few introduced species may be more abundant than the natives (Richardson 1992). Amphipods likely play an important role in leaf litter decomposition, and like isopods, may also enhance microbial diversity. Crowther et al.

(2013) discovered that by grazing soil fungi, isopods removed mycelia of dominant

Basidomycetes, and thus enhanced diversity of the functional profile of the fungal assemblage in forest ecosystems in the UK. Arthropod abundance surveys from nearby sites of similar elevation, rainfall, and forest composition to those in this study suggest that amphipods are the most commonly available prey item in the leaf litter in Hawaii (Van Kleeck et al. unpublished), 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

7 Hawaiian species (one indigenous and nine endemic; Nashida 2002). Although the endemic dermapteran species are not common and were not observed in the frog’s diet, the frog’s ongoing range expansion (Figure 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 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 ants (Table 3).

Diet did not vary with frog body size, developmental stage, or with sex suggesting that frogs of both sexes and all life stages are potential predators to native arthropod species.

Differences between predatory patterns observed in frogs from Japan (Hirai and Matsui 2000) vs.

Hawaii (Table 3) 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. 2015). It is possible that differences in predatory patterns observed between native and introduced ranges of this frog reflect changes due to adaptation of prey preference, though further studies will be needed to confirm this possibility.

Our understanding of the environmental impacts and threats posed by invasive herpetofauna in Hawaii is currently inadequate considering the number of established predatory amphibian and reptile species in the islands (26). There is both a dearth and a need for additional

8 studies of native and endemic invertebrate diversity, ecosystem services, and changes in areas where new predatory species are becoming established. Data regarding declines in native biodiversity are necessary, but frequently address neither causes nor indirect, broad, and more subtle ecosystem impacts of loss of invertebrate diversity in terms of ecosystem processes such as pollination, leaf litter breakdown, and nutrient cycling. For example, a recent study identified a measurable decrease in microbial diversity in the phyllosphere of tree snail host plants where a small native tree snail has been extirpated versus plants with the snail (O’Rorke et al. 2017).

Preventing further introductions is, and needs to continue to be a primary objective of island ecosystem protection programs, because based on studies such as this, subtle ecosystem damage is occurring undetected, at scales that are frequently not addressed. Additional scientific assessments of the ecology of established invasive taxa, however inconspicuous they may be, are warranted to improve our ability to prevent, manage and ultimately restore diverse island ecosystems.

Acknowledgments.—For help with collection of specimens we thank the members of the

Hawaiian Tree Snail Conservation Lab at the University of Hawaii and 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 Dr. Jesse Eiben University of Hawaii, Hilo, for assistance in arthropod taxonomic identification. We also thank the National Parks Service and Molly Hagemann at the Bishop

Museum for access to museum collections. Field collections were conducted under Hawaii

DLNR Permit #EX 12-22. Finally, we acknowledge the efforts of Lily and Roxy Holland and

Bella Hann-Van Kleeck for inspiration and field assistance.

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13 Table 1. Summary of amphibians in the Hawaiian Islands introduced intentionally for biological control, including date of release and current status. Abbreviations for current status are: “Rel.” = released but not established and “Est.” = established. Note also that the focus of this study, the wrinkled frog Glandirana rugosa, as well as Bufo americanus are the earliest documented amphibian releases in Hawaii, although B. americanus did not become established, leaving G. rugosa as the longest standing amphibian introduction.

Year Current Family Genus/species Reference introduced status Ranidae Glandiana rugosa 1896 Est. Bryan, 1932 Lithobates clamitans 1935 Rel. Tinker, 1938 Lithobates pipiens ? Rel. Bryan, 1932; Tinker, 1938 Lithobates catesbeianus 1899-1902 Est. Bryan, 1932 Rana nigromaculata ? 1925 Rel. Tinker, 1938 Dendrobatidae Dendrobates aurauts 1932 Est. Tinker, 1938 Bufonidae Rhinella marina 1931 Est. Tinker, 1938 Bufo bufo ? 1933 Rel. Tinker, 1938 Bufo boreas ? 1857 Rel. Bryan, 1932 Bufo americanus 1892-93 Rel. Bryan, 1932

14 Table 2. Summary of stomach contents by family from 52 individuals of Glandirana rugosa from Oahu and Maui. Known Hawaiian endemic lineages are in bold according to Solem (1990), Cowie (2001), and Nashida (2002). Astrisks are placed by families with a lager number of endemic species compared to introduced species.

# Prey % Relative % Relative Order/ Taxa Family items abundance volume ARTHROPOD A Acari 3 0.67 0.00

Amphipoda* 99 22.15 10.92 Araneae Dysderidae 2 0.45 0.04 Pholicidae 1 0.22 0.02 Salticidae 3 0.67 0.85 Thomisidae 2 0.45 0.15 Unknown 12 2.68 0.93 Chilopoda 44 9.84 2.65 Coleoptera Carabidae * 13 2.91 0.43 Coccinellidae 2 0.45 0.03 Hydrophyllidae 1 0.22 0.08 Molytinae 2 0.45 0.11 Pythidae 1 0.22 0.47 Scolytinae 12 2.68 0.40 Staphalinidae * 3 0.67 0.44 Tenebrionoidea 2 0.45 0.07 Unknown Larvae 3 0.67 0.21 Dermaptera Chelisochidae * 25 5.59 59.42 Diplopoda 4 0.89 0.06 Dolichopodidae Diptera * 9 2.01 0.03 Ephrydidae * 11 2.46 0.04 Phoridae 3 0.67 0.01 Sciaridae * 1 0.22 0.00 Tipulidae * 34 7.61 0.72 Unknown 4 0.89 0.01 Hemiptera Cydnidae 37 8.28 9.22 Largidae 1 0.22 0.02 Miridae * 15 3.36 0.07 Nabidae * 3 0.67 0.27 Unknown 3 0.67 0.01

Hymenoptera Apidae 1 0.22 1.45 Chalcidoideae 2 0.45 0.00 Formicidae 49 10.96 1.06 Isopoda 3 0.67 0.26 Cosmopterygii 1 0.22 0.03 15 Lepidoptera Unknown larvae 4 0.89 1.55 Neuroptera Hemerobiidae * 1 0.22 0.24 Orthoptera Gryllidae 7 1.57 0.67 Psocoptera Psocidae * 1 0.22 0.10 Achatinellidae 12 2.68 2.96 Helicarionidae 2 0.45 1.48 Subulinidae 5 1.12 0.58 Oxychilidae 2 0.45 0.49 ANNELIDA

2 0.45 Oligochaeta 1.43 Total 447 100 100

16 Table 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 dominantly exploited prey items by number and volume from native and introduced ranges are in bold.

Hawaii Japan % Order/Prey # Prey % Relative % Relative % Relative Relative taxa items abundance abundance volume volume ARTHROPODA Acari 3 0.67 0.00 0.07 0.03 Amphipoda 99 22.15 10.92 0.17 0.13 Araneae 20 4.47 2.00 2.07 2.00 Chilopoda 44 9.84 2.65 0.03 0.10 Coleoptera 39 8.72 2.24 12.23 28.23 Dermaptera 25 5.59 59.42 0.07 0.37 Diplopoda 4 0.89 0.06 1.97 4.37 Diptera 62 13.87 0.82 13.60 6.10 Hemiptera 59 13.20 9.59 3.07 4.67 Hymenoptera 52 11.63 2.51 59.07 12.00 Isopoda 3 0.67 0.26 0.87 4.60 Lepidoptera 5 1.12 1.58 1.90 21.27 Neuroptera 1 0.22 0.24 1.00 0.97 Oligochaeta 2 0.45 1.43 0.50 7.73 Orthoptera 7 1.57 0.67 0.33 1.30 Psocoptera 1 0.22 0.10 0.00 0.00 MOLLUSCA Gastropoda 21 4.70 5.52 1.37 1.43 Total 447 100 100 98 95 Figure 1. Map of collection localities in this study. a) Hawaiian Islands and inset photo of

Glandirana rugosa, b) Kauai, c) Oahu and d) Maui. Closed circles represent 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