Sampling

Field surveys were conducted at two sites: (1) Xuan Nha Nature Reserve (NR), Son La Province, north-western Vietnam (21°20'020"N 103°34'302"E, elevation: 1260 m a.s.l.) from 1 to 5 May 2021 and (2) Huu Lien NR, Lang Son Province, north-eastern Vietnam (20°37'242"N, 104°41'369"E, elevation: 610 m a.s.l.) from 3 to 14 May 2023.

Frogs were captured by hand at ponds (approximately 30.0–50.0 m in length and 20.0–40.0 m in width) between 20:30 h and 24:00 h following the guidelines approved by the American Society of Ichthyologists and Herpetologists for animal care (Beaupre et al. 2004). Frogs were found on the ground or on tree branches at the edges of ponds in the secondary forest. The surrounding habitat was second forest with medium and small wood and shrubs. Air temperature was 25-32°C and relative humidity was 70-85%. The stomach-flushing technique was used to obtain stomach contents without sacrificing frogs (Griffiths 1986, Leclerc and Courtois 1993, Solé et al. 2005). Prey items were preserved in 70% ethanol. Frogs were subsequently released at the collecting site after measurements of snout-vent length (SVL) and mouth width (MW) with a digital caliper to the nearest 0.01 mm and measured weight (BM) using electronic scales to the nearest 0.1 g.

Species identification

For taxonomic identification, two individuals were collected for voucher specimens. Field surveys were permitted by the Directorates of Xuan Nha and Huu Lien NRs (permit No. 126/KBTTNXN issued on 27 April 2021 and permit No. 126/KBTTNHL issued on 3 May 2023). After having been photographed in life, animals were anaesthetised and euthanised in a closed vessel with a piece of cotton wool containing ethyl acetate (Simmons 2002), fixed in 85% ethanol and subsequently stored in 70% ethanol. Measurements were taken with a digital calliper to the nearest 0.1 mm. Abbreviations are as follows: a.s.l.: above sea level; terminology of morphological characters followed Yu et al. (2019): SVL (snout-vent length): from tip of snout to vent; HL (head length): from posterior corner of mandible to tip of snout; MW (mouth width): at the angle of jaws; IND (internarial distance); DNE: distance from anterior corner of eye to posterior edge of nostril; ED (eye length); TD (maximum tympanum diameter); DTE: distance between anterior margin of tympanum and posterior corner of eye; SL: distance from anterior corner of eye to tip of snout; IOD (minimum distance between upper eyelids); UEW (maximum width of upper eyelid); FHL (hand length): from elbow to tip of third finger; THL (thigh length): from vent to knee; TL (tibia length); FL (foot length): from proximal end of inner metatarsal tubercle to tip of fourth toe; TFL (combined length of foot and tarsus): from base of inner metatarsal tubercle to tip of fourth toe.

Stomach content analysis

Prey items were identified under a microscope (Olympus SZ 700), based on identification keys (i.e. Naumann et al. (1991), Johnson and Triplehorn (2005), Brusca et al. (2016), Thai (2022)). The maximum length (L) and width (W) of each prey item were measured to the nearest 0.1 mm using either a caliper or a calibrated ocular micrometer fitted to a microscope. Body parts of the same individual were assembled before taking measurements, otherwise prey with incomplete bodies were measured separately. The volume (V) of prey item was calculated using the formula for a prolate spheroid (π = 3.14; Pham et al. (2022)): V = (4π/3)×(L/2)×(W/2) (mm³). To evaluate the relative importance of each prey category, we calculated the following three indices: %F, the frequency of occurrence (the percentage of stomachs containing a specific prey category amongst stomachs containing prey categories); %N, the relative number (the percentage of a specific prey category amongst the number of the bulk of prey categories); and %V, the relative volume (the percentage of the volume of a specific prey category amongst the volume of the bulk of prey categories (Nakamura and Tominaga 2021).

The index of relative importance (IRI) was used to determine the importance of each food category. This index provides a more informed estimation of prey item consumption than any of the three components alone by using the following formula (Caldart et al. 2012): IRI = (%F + %N + %V)/3, where F is the frequency of prey occurrence in stomachs and N is the total number of prey items concerning all prey items.

We used the reciprocal Simpson’s heterogeneity index, 1-D, to calculate dietary heterogeneity: D = ∑[n_i(n_i – 1)]/[N(N – 1)], where n_i is the number of prey items in the i^th taxon category and N is the total number of prey items (Pham et al. 2024).

To estimate prey evenness, we used Shannon’s index of evenness. Evenness is calculated from the equation: J’ = H’/H_max = H’/ln S. The maximum diversity (H_max) that could occur is that which would be found in a situation in which all taxa had equal abundance (H’ = H_max = ln S), S is the total number of prey taxa and H’ is the Shannon-Weiner index of taxon diversity. The value of H’ is calculated from the equation: H′ = –∑(Pi×lnPi),

where Pi is the ratio of food items in the taxon to the total number of food items in the sample (Magurran 2004, Muñoz-Pedreros and Merino 2014).

We used linear regression to examine the relationship between mouth width (MW), snout-vent length (SVL), measured weight (BM) and prey size, as well as prey volume (Nakamura and Tominaga 2021).

Statistic analyses were performed using software package SPSS 20.0 (SPSS Inc., Chicago, Illinois, USA) and with the significance level set to P < 0.05 for all analyses. Data were presented as mean ± standard deviation (SD) unless otherwise noted. We used Kendall’s_tau b statistics to examine the number of prey items, prey volume from frogs of different sexes. We used one-way analysis of variance (ANOVA) to examine the size of prey items and the SVL, MW and BM. Symbols: r is the correlation coefficient; the F₁-value is an analysis of variance (ANOVA) test between two groups; the p-value represents the probability of obtaining a different result.

Zhangixalus pachyproctus is the sister taxon of Z. smaragdinus (Frost 2024). Yu et al. (2019) excluded records of Z. smaragdinus from Yunnan, Vietnam and Thailand and subsequently described the taxon from these localities as the new species Z. pachyproctus, based on morphological and molecular evidence. Specifically, Z. pachyproctus can be clearly distinguished from Z. smaragdinus by having the protruding posterior end of the body in males that forms an arc-shaped swelling (versus vent not protruding in Z. smaragdinus); interspace between vomerine ridges narrower than that in Z. smaragdinus; large grey reticular mottles below the white stripe on flank (versus fine in Z. smaragdinus) and more sloped snout in profile (Yu et al. 2019) (Figs 3, 4).

Most tropical-subtropical frogs have often been reported feeding on spiders, cockroaches, beetles, grasshoppers and ants (e.g. Biavati et al. (2004), Quiroga et al. (2009), Brito et al. (2013), Pham and Nguyen (2018), Pham et al. (2024)). The diet of Zhangixalus pachyproctus was primarily composed of invertebrates, with cockroaches, beetles, crickets, grasshoppers, ants and termites, which is nearly similar to the diet of many frogs in Vietnam (Pham and Nguyen 2018, Pham et al. 2023, Pham et al. 2024). In the population of Polypedates megacephalus from north-western Vietnam studied by Pham et al. (2023), the diet was dominated by arthropods (Orthoptera, Isopoda, Araneae, Coleoptera, Opiliones and Hymenoptera) and the most important prey of Z. pachyproctus (Orthoptera, Coleoptera, Blattidae and Mantodea) were absent or had little importance for Z. pachyproctus. Despite differences in diet between Z. pachyproctus and P. megacephalus, Orthoptera and Coleoptera represented the most important prey categories for the two species. On the other hand, Pham et al. (2022) reported the dietary composition of Zhangixalus feae (Boulenger 1893), another representative of the genus Zhangixalus from northern Vietnam. Both Z. pachyproctus and Z. feae inhabit similar environment conditions, viz. on the ground or tree branches near ponds or streams in evergreen forest with hardwood, bamboo and shrub (Pham et al. 2017, Pham et al. 2022). The prey categories of Z. pachyproctus were more diverse than those in Z. feae (12 vs. 10) (Pham et al. 2022). Interestingly, the prey categories of Z. pachyproctus are similar to those of Z. feae, with Blattodea, Coleoptera, Hemiptera, Lepidoptera, Orthoptera and Gastropoda (Pham et al. 2022). Nonetheless, Oligochaeta, Scolopendromorpha, Hymenoptera, Isoptera, Mantodea and Thysanura were found exclusively in the diet of Z. pachyproctus, whereas Araneae, Dermaptera, Phasmatodea and Aves occurred only in the diet of Z. feae (Pham et al. 2022). The differences in the diet between Z. pachyproctus and Z. feae may reflect differences in foraging activity and microhabitat use. In this study, we only collected food from one female and only one prey category (Gastropoda) was found in the female's stomach. Further comprehensive studies on the diet of both sexes of Z. pachyproctus are needed in the future.