Acta Herpetologica 15(2): 111-118, 2020 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/a_h-7564 Stomach histology of Crocodylus siamensis and Gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function Ryuji Takasaki1,2,*, Yoshitsugu Kobayashi3 1 Department of Natural History and Planetary Sciences, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido, 060-0810, Japan 2 Faculty of Biosphere-Geosphere Science, Okayama University of Science, Ridai-cho, Kita-ku, Okayama, Japan 3 Hokkaido University Museum, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido, 060-0810, Japan *Corresponding author. E-mail: rtakasaki@big.ous.ac.jp Submitted on: 2020, 1st December; revised on: 2020, 14th August; accepted on: 2020, 1st October Editor: Emilio Sperone Abstract. Two groups of extant Archosauria, Crocodylia and Neornithes, have two-chambered stomachs and store gastroliths inside their “gizzards”. Morphological similarities of the “gizzards” lead some previous studies to assume that the presence of this structure, organ “gizzard” is synapomorphic to Archosauria. However, the homology of archosaur “gizzards” had never been tested. This study provides general histological descriptions of stomachs of two crocodylian taxa, Crocodylus siamensis and Gavialis gangeticus, to determine the homology of crocodylian and neor- nithine “gizzards”. Our study demonstrates that both Crocodylus siamensis and Gavialis gangeticus have longer, more complex glands in the fundic stomach (crocodylian “gizzard”) than in the pyloric stomach. Additionally, we found that compound glands are present in the fundic stomach of Crocodylus siamensis. Therefore, crocodylian stomach histomorphological structures are concordant with those of other non-avian reptiles, despite the unique gross mor- phology. The pyloric regions of non-avian reptile stomachs are known to be homologous with the pyloric regions of mammalian stomachs as well as neornithine ventriculus (neornithine gizzard). Therefore, crocodylian and neornithine “gizzards” are morphologically analogous but not homologous. The presence of PAS-positive layer in the pyloric stom- ach of Gavialis gangeticus, which resembles the koilin layer of neornithine ventriculus, further supports this inter- pretation. At the same time, however, the similarity in gastroliths mass/body mass ratio and the correlations between gastroliths occurrence and diet types suggest that crocodylian gastroliths might have contributed to the digestion of ingesta, even though crocodylian and neornithine “gizzards” are not homologous. Keywords. Histology, gizzard, gastroliths, Crocodylia, stomach. INTRODUCTION Crocodylians present the most complex stomach known in existing members of non-avian reptiles (here- after referred to as reptiles) (Owen, 1866; Richardson et al., 2002). Crocodylian stomachs are composed of two distinct units: fundic and pyloric chambers. Neornithes (a least inclusive clade of living birds), the closest living relatives of crocodylians, also have two-chambered stom- achs: a glandular stomach (proventriculus) and a muscu- lar stomach (ventriculus or gizzard) (Ziswiler and Farner, 1972; Denbow, 2015). While the proventriculus excretes the mucus, pepsin, and hydrochloric acid necessary for chemical digestion, the ventriculus performs mechanical digestion of ingesta. Some neornithines, mostly herbi- vores, consume stones and store them inside gizzards as gastroliths (geo-gastroliths, Wings, 2007) to aid gastric mechanical digestion (Fritz, 1937; Hetland et al., 2003; Jin et al., 2014). Several crocodylians are also known to con- tain gastroliths inside their fundic stomachs (e.g., Corbet, 112 Ryuji Takasaki, Yoshitsugu Kobayashi 1960; Cott, 1961). Since both crocodylians and neornith- ines have two-chambered stomachs and store gastroliths inside them, some studies refer crocodylian fundic stom- ach as a “gizzard” (Reese, 1915; Grigg and Gans, 1993). Based on the phylogenetic bracket of the two-cham- bered stomachs, together with the generality of gastro- liths among archosaurs including non-avian dinosaurs, (e.g., Kobayashi et al., 1999; Cerda, 2008; Lee et al., 2014), neornithine style muscular “gizzard” had previous- ly been considered as a plesiomorphic feature of Archo- sauria (Varricchio, 2001; Fritz et al., 2011). However, the homology of avian and crocodylian “gizzards” is con- sidered ambiguous (Schwenk and Rubega, 2005). While some studies considered that crocodylian “gizzards” are homologous with neornithine gizzards (Varricchio, 2001; Fritz et al., 2011), some studies refute the homology (Jones, 1861; Huang et al., 2016). Additionally, the func- tions of crocodylian gastroliths are still under debates (e.g., food processing, hydrostatic function, accidental intake; Cott, 1961; Davenport et al., 1990; Taylor, 1993; Wings, 2007; Uriona et al., 2019). Previous studies on crocodylian stomach microstruc- tures were based only on Alligator mississippiensis (Eisler, 1889; Reese, 1915; Staley, 1925). This lack of knowledge of crocodylian stomach structures cannot allow deter- mining the plesiomorphic status of archosaur “gizzard”. Our study provides the first histomorphological informa- tion of the stomachs of Crocodylus siamensis and Gavialis gangeticus to test the homology of crocodylian and neor- nithine “gizzards”. Besides, this study conducts analyses that provide new implications of the digestive function of crocodylian gastroliths. Neornithine gastrolith mass is known to be correlated with a body mass (Wings and Sander, 2007), and the relationship is utilized as a proxy for the digestive use of dinosaur gastroliths (Wings and Sander, 2007; Cerda, 2008; Lee et al., 2014). Furthermore, avian dietary habits are strongly related to the occurrence frequencies of gastroliths (Best and Gionfriddo, 1991; Gionfriddo and Best, 1996; Gionfriddo and Best, 1999). Our study tests if crocodylian gastroliths have the same relationship as observed in neornithines to assess the digestive function of crocodylian gastroliths. The clarifi- cations of archosaur “gizzard” homology and the croco- dylian gastroliths functions are expected to contribute to better understandings of crocodylian physiology and the evolutionary history of the archosaur digestive system. MATERIAL AND METHOD Corpora of four juvenile individuals of captive Crocodylus siamensis which were dead during winter are provided from a local farmer Koike Wani Sohonpo Co. Ltd. in Shizuoka Pre- fecture of Japan, and four stomachs of captive post-mortem Gavialis gangeticus are provided from Atagawa Tropical & Alli- gator Garden in Shizuoka Prefecture of Japan (Fig. 1). All the specimens were stored frozen before sampling. Small segments were sampled from the greater curvature wall, ventral wall, and pyloric wall of the stomach. The segments are fixed in 10% formalin neutral buffer solution, then dehydrated in ascend- ing grades of ethyl alcohol, cleared with xylene, and embedded in paraffin. Sections were cut at 3μm in thickness and stained with Haematoxylin-Eosin (HE), Periodic Acid Schiff (PAS), and Alcian-Blue (AB) pH 2.5 for general histological observations. To avoid confusion due to different terminologies used in pre- vious studies, this study uses the term “gizzard” for a stomach chamber that may possess gastroliths. Terms fundic stomach and pyloric stomach are used for first and second chambers of the crocodylian stomach, respectively. Terms proventriculus and ventriculus are used for first and second chambers of the avian stomach, respectively. Crocodylian body and gastroliths weights are compiled from previous studies (Corbet, 1960; Cott, 1961; Kennedy and Brockman, 1965; Brazaitis, 1969; Pauwels et al., 2007). Stom- ach contents of crocodylians are gathered from previous studies (Corbet, 1960; Cott, 1961; Tucker et al., 1996; Platt et al., 2006; Wallace and Leslie, 2008; Platt et al., 2013). Body mass and gas- troliths mass are log10 transformed and occurrence frequencies of gastroliths and different food types are arcsine transformed before statistical analyses. Statistical analyses are conducted using the software JMP version 14.3. RESULTS The stomach walls of all of the observed specimens are composed of 4 layers: mucosa, submucosa, muscula- Fig. 1. Stomachs of Crocodylus siamensis (A) and Gavialis gangeti- cus (B). Scales: 5cm for A, 10cm for B. 113Stomach histology of Crocodylus siamensis and Gavialis gangeticus ris externa, and serosa layers from inner to outer layers (Fig. 2). The greater curvature wall is the thickest among the observed regions (Fig. 1B, 2A, 2B). Submucosa com- prises nearly half of the stomach wall in thickness in Crocodylus siamensis (~800μm), while the muscularis externa occupies more than half of the wall in thick- ness in Gavialis gangeticus (~2000μm). The gastric folds, supported by thick submucosa, are shorter than wide in both Crocodylus siamensis and Gavialis gangeticus. The mucosa (~200μm in Crocodylus siamensis and ~300μm in Gavialis gangeticus) is thinner than submucosa and has long fundic glands in both taxa. The fundic glands are tubular and branched (Fig. 2C, 2D) although postmor- tem damage obscures the details. The fundic glands are Fig. 2. Histological structures of Crocodylus siamensis (A, C, E, G, I, K) and Gavialis gangeticus (B, D, F, H, J, L). A-D, greater curvature wall; E-H, ventral wall; I-L, pyloric wall. Abbreviations: cg, compound gland; fg, fundic gland; kl, possible koilin layer; m, mucosa; me, mus- cularis externa; sm, submucosa; pg, pyloric gland. Scales: 1000μm for A, B, E, F, I, and J; 250μm for C, D, G, H, K, and L. 114 Ryuji Takasaki, Yoshitsugu Kobayashi mainly composed of dark oxynticopeptic cells, as previ- ously reported in the stomachs of Alligator mississippien- sis (Eisler, 1889; Staley, 1925). There are no morphologi- cally distinct mucous neck cells reported in most snakes (Jacobson, 2007). The ventral wall is slightly thinner than the greater curvature wall (Fig. 2E, 2F). The submucosa of the ven- tral wall is thin, and the muscularis externa comprises the largest proportion of the ventral wall. The gastric folds are well-developed in Crocodylus siamensis, but it is absent in Gavialis gangeticus. The mucosa is proportion- ally thinner than it is in the greater curvature wall, result- ing in shorter fundic glands than in the greater curvature wall in both taxa. The fundic gland structures are gener- ally the same as those in the greater curvature wall. How- ever, gastric glands in the ventral wall are markedly larger than in the other stomach walls and form a lobule-like compound gland in Crocodylus siamensis (Fig. 2G). These gastric glands are separated from each other with thick connective tissue. The lobule-like compound glands could not be observed in Gavialis gangeticus, partly because available stomachs are not well-preserved compared to Crocodylus siamensis. The pyloric walls of the two crocodylian taxa are largely different from the greater curvature and the ven- tral walls in their extremely thick muscularis externa, which represents up to 80% of the stomach wall thick- ness (Fig. 2I, 2J). On the other hand, the submucosa is reduced, unlike what was observed in the fundic stom- ach. Muscularis mucosa is also much thicker than in the other two regions. Pyloric glands are simple tubular glands and are significantly short compared to the fun- dic glands (Fig. 2K). Unfortunately, details of the pyloric glands are not available due to the impact of  postmortem damage, especially in the stomachs of Gavialis gangeticus. The internal surface of the pyloric wall is locally covered by a PAS-positive layer in Gavialis gangeticus (Fig. 2L). Body mass and gastroliths mass of Crocodylia (Table 1) demonstrates that the average proportion of gastroliths mass relative to body mass is 0.66%. The value is slightly higher than that in neornithines (0.55%), but the differ- ence is not statistically significant (Student’s t-test, P = 0.50). Regression analysis demonstrates the correlation of crocodylian body mass and gastroliths mass (Fig. 3; r2 = 0.84, P < 0.001) as in neornithines (Wings and Sander, 2007). Neither the slope nor the intercept of the regres- sion line differs from those of neornithines (P = 0.43 and 0.73, respectively), indicating that the relationship between gastroliths mass and body mass of crocodylians are statistically indistinctive from that of neornithines. Regression analyses on occurrence frequencies of gastro- liths and different food types (Table 2) demonstrate that occurrence frequency of gastroliths are positively corre- lated with those of vertebrates and negatively correlated with those of most invertebrates (Table 3). The correla- tions are statistically significant (P < 0.05) in Insecta, Pisces, Amphibia, and Mammalia. DISCUSSION Histological evaluations of Crocodylus siamensis and Gavialis gangeticus stomachs demonstrate that general stomach morphology is similar to each other. Both taxa have long, tubular branched fundic glands and short, simple pyloric glands (Fig. 2). The result is concordant with the stomach microstructure of Alligator mississippi- ensis as reported in Staley (1925), indicating that mem- bers of Crocodylia share generally the same fundic and pyloric gland structures. The long, complex fundic glands and short, simple pyloric glands are also in agreement with general features of reptilian stomachs (Luppa, 1977; Jacobson, 2007). Furthermore, the lobule-like compound fundic glands that are present in the ventral wall of Crocodylus siamensis (Fig. 2G) are also reported in fun- dic stomachs of other reptiles including Caretta caretta (Oppel, 1896), Chamaeleon afticanus (Hamdi et al., 2014), Laudakia stellio (Koca and Gurcu, 2011), Ophisops elegans (Çakici and Akat, 2013), and Varanus niloticus (Ahmed et al., 2009). Therefore, the present observations dem- onstrate that general histomorphological structures of crocodylian stomach glands are concordant with those Fig. 3. Relationships of body mass to gastroliths mass in croco- dylians (red diamond) and crown birds (blue circle). The red solid line represents the regression line for crocodylians and the blue dashed line represents the regression line for crown birds. 115Stomach histology of Crocodylus siamensis and Gavialis gangeticus of other reptiles, despite the unique gross morphology of crocodylian stomach among Reptilia. The general histomorphological features of reptilian fundic and pyloric glands resemble neornithine gastric glands of proventriculus and ventriculus, respectively. Neornithine proventriculus contains highly branched compound glands that compose lobules, and the ventric- ulus contains simple tubular glands covered by the PAS- positive koilin layer (Ziswiler and Farner, 1972). Through stomach muscle structure comparisons, Pernkopf (1929) suggested that the reptilian pyloric stomach is homolo- gous to the pyloric region of the mammalian stomach, which is homologous to neornithine ventriculus (Smith et al., 2000). The present results suggest that the croco- dylian pyloric stomach is homologous with neornithine ventriculus (neornithine gizzard), whereas the croco- dylian fundic stomach (crocodylian “gizzard”) is homolo- gous with neornithine proventriculus. Since crocodylian and neornithine “gizzards” are not homologous, the pre- vious assumption that “gizzard” is synapomorphic to Archosauria (Varricchio, 2001; Fritz et al., 2011) is dis- missed. Although crocodylian and neornithine “gizzards” are not homologous, the absence of statistical difference in the body mass-gastroliths mass relationship between the two groups suggests the digestive function of crocodylian gastroliths based on previous interpretations (Wings and Sander, 2007). The relationships between gastroliths Table 1. Mean total gastroliths mass and body mass of crocodylians compiled. Species Mean total gastrolith mass[g] Mean body mass[g] Sample size Relative weight of gastroliths [%] Reference (gastroliths) Reference (body mass) Crocodile acutus 174.00 32206 2 0.54% Brazaitis (1969) Brazaitis (1969) Alligator mississippiensis 22.00 7800 1 0.28% Kennedy and Brockman (1965) Kennedy and Brockman (1965) Osteolaemus t. tetraspis (Rabi oil fields) 5.54 3241 14 0.17% Pauwels et al. (2007) Pauwels et al. (2007) Osteolaemus t. tetraspis (Loango National Park) 4.33 8193 8 0.05% Pauwels et al. (2007) Pauwels et al. (2007) Crocodilus niloticus 0.5-1.0m 2.04 1524 101 0.13% Cott (1961) Cott (1961) Crocodilus niloticus 1.0-1.5m 11.70 4518 102 0.26% Cott (1961) Cott (1961) Crocodilus niloticus 1.5-2.0m 88.87 16540 76 0.54% Cott (1961) Cott (1961) Crocodilus niloticus 2.0-2.5m 312.50 40900 73 0.76% Cott (1961) Cott (1961) Crocodilus niloticus 2.5-3.0m 700.30 79390 69 0.88% Cott (1961) Cott (1961) Crocodilus niloticus 3.0-3.5m 1321.20 131900 52 1.00% Cott (1961) Cott (1961) Crocodilus niloticus 3.5-4.0m 1906.20 206500 16 0.92% Cott (1961) Cott (1961) Crocodilus niloticus 4.0-4.5m 2940.40 298700 5 0.98% Cott (1961) Cott (1961) Crocodilus niloticus 4.5-5.0m 3356.00 325500 3 1.03% Cott (1961) Cott (1961) Crocodilus niloticus 0.3-0.5m 4.80 146 2 3.29% Corbet (1960) Corbet (1960) Crocodilus niloticus 0.5-1.0m 2.87 1524 23 0.19% Corbet (1960) Corbet (1960) Crocodilus niloticus 1.0-1.5m 19.79 4518 18 0.44% Corbet (1960) Corbet (1960) Crocodilus niloticus 1.5-2.0m 66.80 16540 2 0.40% Corbet (1960) Corbet (1960) Crocodilus niloticus 3.5-4.0m 206.50 206500 1 0.10% Corbet (1960) Corbet (1960) R ef er en ce C or be t ( 19 60 ) C or be t ( 19 60 ) C ot t ( 19 61 ) C ot t ( 19 61 ) C ot t ( 19 61 ) C ot t ( 19 61 ) C ot t ( 19 61 ) C ot t ( 19 61 ) C ot t ( 19 61 ) C ot t ( 19 61 ) C ot t ( 19 61 ) C ot t ( 19 61 ) W al la ce a nd L es lie ( 20 08 ) W al la ce a nd L es lie ( 20 08 ) W al la ce a nd L es lie ( 20 08 ) Pl at t e t a l. (2 00 6) Pl at t e t a l. (2 00 6) Pl at t e t a l. (2 00 6) Pl at t e t a l. (2 00 6) Pl at t e t a l. (2 00 6) Pl at t e t a l. (2 01 3) Pl at t e t a l. (2 01 3) Pl at t e t a l. (2 01 3) Pl at t e t a l. (2 01 3) Pl at t e t a l. (2 01 3) Tu ck er e t a l. (1 99 6) Tu ck er e t a l. (1 99 6) Tu ck er e t a l. (1 99 6) Tu ck er e t a l. (1 99 6) Tu ck er e t a l. (1 99 6) Tu ck er e t a l. (1 99 6) Tu ck er e t a l. (1 99 6) Tu ck er e t a l. (1 99 6) O cc ur re nc e Fr eq ue nc ie s (% ) M am m al ia 6. 90 0. 00 0. 00 4. 93 10 .6 4 10 .8 1 9. 40 12 .4 0 21 .2 4 26 .5 3 47 .8 3 58 .3 3 3. 60 4. 20 10 .0 0 0. 00 0. 00 9. 92 3. 17 2. 08 0. 00 0. 00 3. 57 0. 00 0. 00 0. 00 2. 00 2. 00 0. 00 3. 00 0. 00 6. 00 10 .0 0 A ve s 0. 00 18 .1 8 0. 00 3. 52 2. 13 9. 91 5. 98 10 .8 5 9. 73 12 .2 4 13 .0 4 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 1. 65 4. 76 10 .4 2 0. 00 0. 00 3. 57 12 .5 0 11 .1 1 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 2. 00 R ep til ia 0. 00 0. 00 0. 00 2. 82 2. 84 7. 21 5. 98 9. 30 14 .1 6 20 .4 1 34 .7 8 41 .6 7 0. 00 4. 20 0. 00 0. 00 0. 85 6. 61 3. 17 2. 08 0. 00 6. 25 0. 00 12 .5 0 0. 00 0. 00 0. 00 0. 00 0. 00 3. 00 4. 00 8. 00 2. 00 A m ph ib ia 24 .1 4 9. 09 25 .0 0 11 .9 7 12 .7 7 0. 90 0. 85 0. 00 0. 00 0. 00 0. 00 0. 00 7. 10 4. 20 0. 00 0. 00 0. 85 5. 79 3. 17 0. 00 0. 00 0. 00 3. 57 0. 00 0. 00 3. 00 7. 00 16 .0 0 19 .0 0 15 .0 0 75 .0 0 56 .0 0 52 .0 0 Pi sc es 17 .2 4 45 .4 5 0. 00 9. 86 16 .3 1 37 .8 4 45 .3 0 43 .4 1 42 .4 8 44 .9 0 13 .0 4 33 .3 3 10 .7 0 12 .5 0 80 .0 0 16 .9 0 5. 98 25 .6 2 31 .7 5 31 .2 5 5. 26 12 .5 0 25 .0 0 0. 00 11 .1 1 10 .0 0 31 .0 0 34 .0 0 21 .0 0 13 .0 0 8. 00 3. 00 0. 00 M ol lu sc a 13 .7 9 9. 09 0. 00 7. 04 17 .7 3 22 .5 2 26 .5 0 17 .8 3 20 .3 5 12 .2 4 8. 70 0. 00 0. 00 0. 00 0. 00 2. 82 5. 13 20 .6 6 41 .2 7 70 .8 3 0. 00 6. 25 3. 57 0. 00 5. 56 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 C ru st ac ea 10 .3 4 18 .1 8 0. 00 23 .9 4 28 .3 7 10 .8 1 7. 69 3. 10 5. 31 2. 04 4. 35 0. 00 0. 00 8. 30 0. 00 0. 00 7. 69 18 .1 8 14 .2 9 20 .8 3 31 .5 8 68 .7 5 89 .2 9 87 .5 0 94 .4 4 0. 00 5. 00 10 .0 0 14 .0 0 5. 00 8. 00 6. 00 10 .0 0 A ra ne id a 13 .7 9 4. 55 16 .6 7 13 .3 8 2. 13 0. 90 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 57 .1 0 41 .7 0 10 .0 0 29 .5 8 26 .5 0 6. 61 1. 59 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 48 .0 0 45 .0 0 31 .0 0 40 .0 0 41 .0 0 8. 00 11 .0 0 12 .0 0 In se ct a 96 .5 5 77 .2 7 91 .6 7 82 .3 9 58 .1 6 26 .1 3 10 .2 6 2. 33 0. 88 0. 00 0. 00 0. 00 57 .1 0 45 .8 0 20 .0 0 84 .5 1 91 .4 5 68 .6 0 34 .9 2 12 .5 0 63 .1 6 75 .0 0 14 .2 9 18 .7 5 0. 00 66 .0 0 66 .0 0 59 .0 0 42 .0 0 33 .0 0 29 .0 0 11 .0 0 12 .0 0 G as tr ol ith s 76 .6 7 92 .0 0 0. 00 50 .0 0 67 .2 4 82 .2 9 89 .6 9 10 0. 00 10 0. 00 10 0. 00 10 0. 00 10 0. 00 3. 60 20 .8 0 50 .0 0 11 .2 7 5. 98 18 .1 8 17 .4 6 14 .5 8 0. 00 0. 00 28 .5 7 31 .2 5 16 .6 7 59 .0 0 89 .0 0 82 .0 0 91 .0 0 87 .0 0 96 .0 0 94 .0 0 91 .0 0 Sa m pl e # 30 25 12 14 2 14 1 11 1 11 7 12 9 11 3 49 23 12 15 1 82 53 71 11 7 12 1 63 48 19 16 28 16 18 29 62 49 43 39 24 36 42 SV L (c m ) - - - - - - - - - - - - 17 .0 -3 8. 9 39 -6 6. 3 66 .4 -1 15 .8 - - - - - <1 5 15 .1 -4 0 40 .1 -6 5 65 /1 -9 0 >9 0 10 -1 9. 9 20 -2 9. 9 30 -3 9. 9 40 -4 9. 9 50 -5 9. 9 60 -6 9. 9 70 -7 9. 9 >8 0 B od y Le ng th (c m ) <1 00 10 0- 19 9 30 -5 0 50 -1 00 10 0- 15 0 15 0- 20 0 20 0- 25 0 25 0- 30 0 30 0- 35 0 35 0- 40 0 40 0- 45 0 45 0- 50 0 - - - <3 0 30 .1 -5 0 50 .1 -1 00 10 0. 1- 15 0 >1 50 - - - - - - - - - - - - - Sp ec ie s C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s ni lo tic us C ro co dy lu s m or el et ii C ro co dy lu s m or el et ii C ro co dy lu s m or el et ii C ro co dy lu s m or el et ii C ro co dy lu s m or el et ii C ro co dy lu s ac ut us C ro co dy lu s ac ut us C ro co dy lu s ac ut us C ro co dy lu s ac ut us C ro co dy lu s ac ut us C ro co dy lu s jo hn so ni C ro co dy lu s jo hn so ni C ro co dy lu s jo hn so ni C ro co dy lu s jo hn so ni C ro co dy lu s jo hn so ni C ro co dy lu s jo hn so ni C ro co dy lu s jo hn so ni C ro co dy lu s jo hn so ni Ta bl e 2. S to m ac h co nt en ts o f c ro co dy lia ns c om pi le d. 117Stomach histology of Crocodylus siamensis and Gavialis gangeticus occurrence frequency with dietary types (Table 3) further support their digestive function. The positive correlations with vertebrate diets, although supported statistically only in mammals, may suggest that gastroliths are possibly beneficial for digesting bones. Although the gastroliths might have not served as “teeth” to strongly grind inges- ta as they do in herbivorous birds (Moore, 1998; Moore, 1999), they might have benefited digestion through inges- ta mixing and facilitating stomach juice excretion (Wings, 2007). These functions do not contradict with other possible gastroliths functions such as buoyancy control (Taylor, 1993). Therefore, the results of this study sug- gest a possibility that although crocodylian “gizzard” is not homologous with that of neornithines, their “gizzard” efficiently utilized gastroliths for digestion. ACKNOWLEDGEMENTS We deeply appreciate Dr. Yasuhiro Kon and Dr. Osa- mu Ichii for providing the facility and instructing the his- tological methods. We acknowledge Dr. Masaya Iijima, Junki Yoshida, and Tomonori Tanaka for their comments on earlier versions of the manuscript. REFERENCES Ahmed, Y., El-Hafez, A., Zayed, A. (2009): Histological and histochemical studies on the esophagus, stomach and small intestines of Varanus niloticus. J. Vet. Anat. 2: 35-48. Best, L.B., Gionfriddo, J.P. (1991): Characterization of grit use by cornfield birds. Wilson Bull. 103: 68-82. Brazaitis, P. (1969): The occurrence and ingestion of gas- troliths in two captive crocodilians. Herpetologica 25: 63-64. Çakici, Ö., Akat, E. (2013): Some histomorphological and histochemical characteristics of the digestive tract of the snake-eyed lizard, Ophisops elegans Menetries, 1832. North-West J. Zool. 9. Cerda, I.A. (2008): Gastroliths in an ornithopod dino- saur. Acta Palaeontol. Pol. 53: 351-355. Corbet, P.S. (1960): The food of a sample of crocodiles (Crocodilus niloticus L.) from Lake Victoria. P. Zool. Soc. Lond. 133: 561-572. Cott, B.H. (1961): Scientific results of an inquiry into the ecology and economic status of the Nile Crocodile (Crocodilus niloticus) in Uganda and Northern Rho- desia. Trans. Zool. Soc. London 29: 211-356. Davenport, J., Grove, D.J., Cannon, J., Ellis, T.R., Stables, R. (1990): Food capture, appetite, digestion rate and efficiency in hatchling and juvenile Crocodylus poro- sus. J. Zool. 220: 569-592. Denbow, D.M. (2015): Gastrointestinal Anatomy and Physiology. In: Anatomy and Physiology of Domestic Animals, 2nd Edition, p. 337-366. Akers, R.M., Den- bow, D.M., Eds., New Jersey, Wiley-Blackwell. Eisler, P. (1889): Zur kenntniss der histologie des alliga- tormagens. Archiv für Mikroskopische Anatomie 34: 1-10. Fritz, J., Kienzle, E., Hummel, J., Wings, O., Streich, W.J., Clauss, M. (2011): Gizzard vs. teeth, it’s a tie: food- processing efficiency in herbivorous birds and mam- mals and implications for dinosaur feeding strategies. Paleobiology 37: 577-586. Fritz, J.C. (1937): The effect of feeding grit on digestibility in the domestic fowl. Poult. Sci. 16: 75-79. Gionfriddo, J.P., Best, L.B. (1996): Grit-use patterns in North American birds: the influence of diet, body size, and gender. The Wilson Bulletin 1996: 685-696. Gionfriddo, J.P., Best, L.B. (1999): Grit use by birds: a review. In: Curr Ornithol, p. 89-148. Nolan Jr., V., Ketterson, E.D., Thompson, C.F., Eds., New York, Klu- wer Academic/Plenum Publishers. Grigg, G., Gans, C. (1993): Morphology and physiol- ogy of the Crocodylia. In: Fauna of Australia Vol 2A Amphibia and Reptilia, p. 326-336. Glasby, C.G., Ross, G.J.B., Beesley, P.L., Eds., Australian Govern- ment Publishing Service. Hamdi, H., El-Ghareeb, A.-W., Zaher, M., Essa, A., Lah- sik, S. (2014): Anatomical, histological and histo- chemical adaptations of the reptilian alimentary canal to their food habits: II-Chamaeleon africanus. World Appl. Sci. J. 30: 1306-1316. Hetland, H., Svihus, B., Krogdahl, A. (2003): Effects of oat hulls and wood shavings on digestion in broilers and layers fed diets based on whole or ground wheat. Br. Poult. Sci. 44: 275-82. Table 3. Results of regression analyses between the occurrences of different food types and gastroliths. Group Coefficient p-value R2 Correlation Insecta -0.659 0.002 0.280 Negative Araneida -0.222 0.496 0.015 Negative Crustacea -0.478 0.065 0.106 Negative Mollusca 0.000 0.999 0.000 - Pisces 0.762 0.041 0.106 Positive Amphibia 0.407 0.228 0.047 Positive Reptilia 1.342 0.004 0.237 Positive Aves 0.937 0.110 0.080 Positive Mammalia 1.398 0.000 0.352 Positive 118 Ryuji Takasaki, Yoshitsugu Kobayashi Huang, J., Wang, X., Hu, Y., Liu, J., Peteya, J.A., Clarke, J.A. (2016): A new ornithurine from the Early Creta- ceous of China sheds light on the evolution of early ecological and cranial diversity in birds. PeerJ 4: e1765. Jacobson, E.R. (2007): Infectious Diseases and Pathology of Reptiles: Color Atlas and Text. CRC press, Boca Raton, Florida. Jin, L., Gao, Y.-Y., Ye, H., Wang, W.-C., Lin, Z.-P., Yang, H.-Y., Huang, S.-B., Yang, L. (2014): Effects of dietary fiber and grit on performance, gastrointestinal tract development, lipometabolism, and grit retention of goslings. J. Integr. Agr. 13: 2731-2740. Jones, T.R. (1861): General Outline of the Organization of the Animal Kingdom: and Manual of Comparative Anatomy. John Van Voorst, London. Kennedy, J., Brockman, H. (1965): Stomach stone in the American Alligator, Alligator mississippiensis Daudin. Brit. J. Herpetol. 3: 201-203. Kobayashi, Y., Lu, J.-C., Dong, Z.-M., Barsbold, R., Azu- ma, Y., Tomida, Y. (1999): Herbivorous diet in an ornithomimid dinosaur. Nature 402: 480-481. Koca, Y.B., Gurcu, B. (2011): Morphological and histo- chemical investigations of esophagogastric tract of a lizard, Laudakia stellio (Agamidae, Linnaeus 1758). Acta Biol. Hung. 62: 376-87. Lee, Y.N., Barsbold, R., Currie, P.J., Kobayashi, Y., Lee, H.J., Godefroit, P., Escuillie, F., Chinzorig, T. (2014): Resolving the long-standing enigmas of a giant orni- thomimosaur Deinocheirus mirificus. Nature 515: 257- 60. Luppa, H. (1977): Histology of the digestive tract. In: Biol Reptil, p. 225-313. Gans, C., Ed., New York, Academic Press. Moore, S.J. (1998): Use of an artificial gizzard to inves- tigate the effect of grit on the breakdown of grass. J. Zool. 246: 119-124. Moore, S.J. (1999): Food breakdown in an avian herbi- vore: who needs teeth? Aust. J. Zool. 47: 625. Oppel, A. (1896): Lehrbuch der vergleichenden mik- roskopischen Anatomie der Wirbeltiere. Gustav Fis- cher, Jena. Owen, R. (1866): Anatomy of Vertebrates. Volume 1: Fishes and Reptiles. Longmans, Green, and Co. Pauwels, O.S., Barr, B., Sanchez, M.L., Burger, M. (2007): Diet records for the dwarf crocodile (Osteolaemus tet- raspis tetraspis) in Rabi Oil Fields and Loango Nation- al Park, Southwestern Gabon. Hamadryad 31: 258- 264. Pernkopf, E. (1929): Beiträge zur vergleichenden Anato- mie des Vertebratenmagens. Z. Anat. Entwicklung. 91: 329-390. Platt, S.G., Rainwater, T.R., Finger, A.G., Thorbjarnarson, J.B., Anderson, T.A., McMurry, S.T. (2006): Food hab- its, ontogenetic dietary partitioning and observations of foraging behaviour of Morelet’s crocodile (Crocodylus moreletii) in northern Belize. Herpetol. J. 16: 281-290. Platt, S.G., Thorbjarnarson, J.B., Rainwater, T.R., Martin, D.R. (2013): Diet of the american crocodile (Crocody- lus acutus) in marine environments of coastal Belize. J. Herpetol. 47: 1-10. Reese, A.M. (1915): The Alligator and its Allies. GP Put- nam’s Sons, New York. Richardson, K.C., Webb, G., Manolis, S.C. (2002): Croc- odiles: Inside Out: A Guide to the Crocodilians and Their Functional Morphology. Surrey Beatty & Sons, Ltd., Sydney. Schwenk, K., Rubega, M. (2005): Diversity of vertebrate feeding systems. In: Physiological and Ecological Adaptations to Feeding in Vertebrates, p. 1-41. Starck, J.M., Wang, T., Eds., Enfield, New Hampshire, Science Publishers. Smith, D.M., Grasty, R.C., Theodosiou, N.A., Tabin, C.J., Nascone-Yoder, N.M. (2000): Evolutionary relation- ships between the amphibian, avian, and mammalian stomachs. Evol. Dev. 2: 348-359. Staley, F.H. (1925): A study of the gastric glands of Alliga- tor mississippiensis. J. Morphol. 40: 169-189. Taylor, M.A. (1993): Stomach stones for feeding or buoy- ancy? The occurrence and function of gastroliths in marine tetrapods. Philos. T. R. Soc. B. 341: 163-175. Tucker, A.D., Limpus, C.J., McCallum, H.I., McDonald, K.R. (1996): Ontogenetic dietary partitioning by Croc- odylus johnstoni during the dry season. Copeia 1996: 978. Uriona, T.J., Lyon, M., Farmer, C.G. (2019): Lithophagy prolongs voluntary dives in american alligators (Alliga- tor mississippiensis). Int. Org. Biol. 1. Varricchio, D.J. (2001): Gut contents from a Cretaceous tyrannosaurid: implications for theropod dinosaur digestive tracts. J. Paleontol. 75: 401-406. Wallace, K.M., Leslie, A.J. (2008): Diet of the nile croco- dile (Crocodylus niloticus) in the Okavango Delta, Botswana. J. Herpetol. 42: 361-368. Wings, O. (2007): A review of gastrolith function with implications for fossil vertebrates and a revised clas- sification. Acta Palaeontol. Pol. 52. Wings, O., Sander, P.M. (2007): No gastric mill in sauro- pod dinosaurs: new evidence from analysis of gastro- lith mass and function in ostriches. P. R. Soc. B 274: 635-40. Ziswiler, V., Farner, D.S. (1972): Digestion and the diges- tive system. Avian Biol. 2: 343-430. Acta Herpetologica Vol. 15, n. 2 - December 2020 Firenze University Press Estimating abundance and habitat suitability in a micro-endemic snake: the Walser viper Gentile Francesco Ficetola1,2,*, Mauro Fanelli3, Lorenzo Garizio3, Mattia Falaschi1, Simone Tenan4, Samuele Ghielmi5, Lorenzo Laddaga6, Michele Menegon7,8, Massimo Delfino3,9. Potential effects of climate change on the distribution of invasive bullfrogs Lithobates catesbeianus in China Li Qing Peng1, Min Tang1, Jia Hong Liao1, Hai Fen Qing1, Zhen Kun Zhao1, David A. Pike2, Wei Chen1,* A bibliometric-mapping approach to identifying patterns and trends in amphibian decline research Claudio Angelini1,*, Jon Bielby2, Corrado Costa3 Food composition of a breeding population the endemic Anatolia newt, Neurergus strauchii (Steindachner, 1887) (Caudata: Salamandridae), from Bingöl, Eastern Turkey Kerim Çiçek1,*, Mustafa Koyun2, Ahmet Mermer1, Cemal Varol Tok3 Stomach histology of Crocodylus siamensis and Gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function Ryuji Takasaki1,2,*, Yoshitsugu Kobayashi3 Does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? A comparison between natural-habitat and agrosystem frogs Sonia Zambrano-Fernández1, Francisco Javier Zamora-Camacho2,3,*, Pedro Aragón2,4 Confirming Lessona’s brown frogs distribution sketch: Rana temporaria is present on Turin Hills (Piedmont, NW Italy) Davide Marino1, Angelica Crottini2, Franco Andreone3,* Phylogenetic relationships of the Italian populations of Horseshoe Whip Snake Hemorrhois hippocrepis (Serpentes, Colubridae) Francesco Paolo Faraone1, Raffaella Melfi2, Matteo Riccardo Di Nicola3, Gabriele Giacalone4, Mario Lo Valvo5* First karyological analysis of the endemic Malagasy phantom gecko Matoatoa brevipes (Squamata: Gekkonidae) Marcello Mezzasalma1,2,*, Fabio M. Guarino3, Simon P. Loader1, Gaetano Odierna3, Jeffrey W. Streicher1, Natalie Cooper1 Notes on sexual dimorphism, diet and reproduction of the false coral snake Oxyrhopus rhombifer Duméril, Bibron & Duméril, 1854 (Dipsadidae: Pseudoboini) from coastal plains of Subtropical Brazil Fernando M. Quintela1,*, Felipe Caseiro¹, Daniel Loebmann¹