Hyla appendiculata Boulenger, 1882

Caminer, Marcel A. & Ron, Santiago R., 2020, Systematics of the Boana semilineata species group (Anura: Hylidae), with a description of two new species from Amazonian Ecuador, Zoological Journal of the Linnean Society 190, pp. 149-180 : 162-166

Hyla appendiculata Boulenger, 1882 .

Lectotype ( Fig. 4): BMNH 1947.2 .13.1 (designated by Parker, 1933), an adult female from ‘Canelos’, Ecuador. Common names : Proposed standard English name: Canelos treefrog. Proposed standard Spanish name: rana arbórea de Canelos

Diagnosis ( Figs 5, 7B): Boana appendiculata is characterized by: (1) mean SVL 45 mm in males (range 38.5–53.1; N = 96), 63.8 mm in females (range 50.2–72.6; N = 40; Table 5); (2) thighs long (TL/SVL 0.45–0.56); (3) dorsal skin finely granular; (4) dorsum coloration dull tan, brown, dark brown or cream usually with irregular dark brown markings frequently including an X- shaped mark in scapular region; (5) flanks and hidden surfaces of thighs creamy or pale grey with black vertical stripes (either single or paired); (6) areolate skin on the posterior surface of throat, belly and ventral surfaces of thighs; (7) venter vary from creamy white to yellowish white or orange, with or without dark brown spots; (8) in life, webbing yellow, orange or brown; (9) palpebrum reticulated; (10) nuptial excrescences present in breeding males; (11) projecting prepollices absent in both sexes; (12) tympanum large with distinct tympanic ring; (13) small, triangular calcar on heel, often coloured

;

0.4

)

2.27 0.4) 2.7 0.5 1.6) 0.4 2.8) 0.4) 0.5 3.7) length ± – ± – ± – ± – ± 2.4 ± –

CL 1.5 (0.7 2.1 (1.1 1.1 0.9 (2.3 (1.9 1.7 1 (– 2.5 1.6 (=

Tibia

TL

;

Analysis FL 16.3 1.8 ± (–) 19.8 13 24 ± 2.1 28 –) (18.4 ± 16.4 0.9 – 14.9 (

)

18.6 ± 2.8 25.2 21.2 (28.8 –

)

1.9 ± 19.2 23.6 –

)

15.8 (± 2.3 31 (27.8

)

– 34.4 diameter Component ± 2 – 26.2) 2.2 ±) – 37.7 0.8 ± 23.6 –) ± 4.4 40.2) – 1.9 ± 30.9 –) 2.8 ± – 47.9) Tympanum = TD Principal FEL 22.8 2.1 19.1 () 25.8 2.7 33.9 26.9 (37.8) 1.2 21.9 (23.5) 20.4 34.5 31.1 38.2 () 27.3 23.8) 29.8 (42.4 2.9 () 47.8 36.9; diameter for ± – ± – ± – 4.1 – ± 2 – ± – Eye = used TL 21.8 18.1 (32.9 26.9 (20.7 (18.5 33 ± 29.3 (26.8 (22.8 41.9 (36.3; ED ventrimaculata TD ± 3.9 0.4 3) – 4.8 (0.5 ± 5.1 (4– 5.8) 0.3 3.3 ± – (2.8) 3.9 0.3 4.6 ±) – (4.3 5.1 ± 0.5 4.8 5.6 (3.7) – 6.4 ± 0.8 7.7 – (5.1) width Head = HW; mm. . B length in and nigra ± 0.5) 5.6 – 0.5 5.9) 0.5 ± 5.5) – 0.5 ± – 5.8) 0.6 ± 6.3 –) ± 0.8) 8.4 – = Head are B

,

. ED 4.6 (3.7 5 ± (– 4 4.1 3.4 (5.3 (4.6 5.3 (3.9 6.7 (5.3 measurements

appendiculata HW ± 16.6 1.5) – 18.4 11.9 (1.5 ± 22.7 – 19.1 25 () 1.2 ± 14.5 10.2 – () 16.4 2.8 ± 23.5 27.9) (– 21 ± 18.8 1.6 16.2) 22.2 (– ± 2.3 29.9 () 26.6 34.9 – – = Snout = M HL; length vent. All males; of Boana 1.5 ±) 14.3 1.9 ± 18.6 –) ± 1.3 –) 13.9 3.1 ±) 19.5 – ± 1.9) 17.3 – ± 2.6) 22.9 – SVL: females morphometric HL 3.5 ± 11.5 51.9 8 –) (– ± 4.8 15.6 – 72.6 12.1) (± 10.8 2.0) 46.3 – 8.8 (7.9 ± 15.2 () 11.2 – 76.7 4.6 ± 12.9 (9.8 –) 60.7 ± 5.4 18.9 14.6 () 90.5 – Abbreviations are = F length;. Calcar = for SVL 44.3 38.5 (64.4 53.5 (42.2 38.5 (64.8 56.4 (52.7 44.5 (81.6 72.9 (parentheses in length; CL statistics M F M F M F range Foot = Descriptive appendiculata appendiculata ventrimaculata ventrimaculata with FL Table.

5

Species. B (= 35 N) B. = () 30 N. B nigra ) (= N 26 nigra B. N) 5 (=. B) 20 = (N B. =) (17 N Mean FEL is given ± SD length; = Femur with a cream spot; (14) advertisement call consists of two types of calls: Type I with one note and Type II with four to six notes; (15) pulses per note varying from 31 to 37 (Type I) and 18 to 22 (Type II); and (16) call dominant frequency varying from 1076–1645 Hz (Type I) and 818–947 Hz (Type II).

Comparisons with other species: Boana appendiculata is most similar to B. semilineata , B. diabolica , B. geographica and B. hutchinsi . It differs from the last three species by having yellow to orange or brown webbing in life (webbing black in B. hutchinsi , red in B. diabolica and B. geographica ; Fouquet et al., 2016). Although webbing between fingers III–IV and toes IV–V is grey dorsally in B. geographica , colour between the other fingers differs as indicated above. Bright webbing colours fade to cream in preservative and become indistinguishable among species. Boana appendiculata can be further distinguished by the presence of black vertical stripes on the flanks (speckled pattern in B. diabolica , B. hutchinsi and B. semilineata ; Fouquet et al., 2016). Boana appendiculata differs from B. ventrimaculata sp. nov. in having black vertical stripes on the flanks (a speckled pattern or dark blotches in B. ventrimaculata sp. nov.) and by its smaller size (differences are significant in males: Student’s t = 7.16, d.f. = 31.9, P <0.001). Boana appendiculata is similar to B. nigra sp. nov. but it differs in having a combination of yellow or orange ventral coloration in life (cream to pale orange in B. nigra sp. nov.) and black vertical stripes on the flanks and hidden surfaces of thighs (uniform black in males of B. nigra sp. nov.; Fig. 7A, 10). However, small adult specimens of B. appendiculata (<42 mm) may present a similar colour pattern to B. nigra sp. nov. (cream ventral coloration with uniform black flanks). Both species also differ in environmental envelope ( Fig. 3D; scores along PC I and PC II are significant different: PC I, t = 8.51, P <0.001; PC II, t = 2.55, P = 0.034). Boana appendiculata occurs in warmer localities than B. nigra sp. nov. as shown by annual mean temperature (bio01): B. appendiculata mean = 24.28 ˚C (SD = 1.53, N = 59) vs. B. nigra mean = 19.48 ˚C (SD = 1.78, N = 8). Despite high genetic distances (4.2–5.2% for gene 16S; Table 6), both species are sympatric in at least one locality in Pastaza Province. This suggests the existence of non-geographic reproductive barriers and, therefore, lineage independence.

Boana appendiculata is easily distinguished from B. boans , B. icamiaba , B. pombali and B. wavrini by its prepollex not modified into a projecting spine and by the presence of pigmented nuptial excrescences. Boana appendiculata further differs from B. boans in having well-defined vertical stripes on the flanks and hidden surfaces of thighs (diffuse vertical bars and barely distinguishable in B. boans ), and webbing extends to about the middle of the outer finger (fully webbed hands in B. boans ).

Variation in preservative: Morphometric data for adult specimens are summarized in Table 5, while variation in dorsal and ventral coloration of preserved specimens is shown in Figure 5. The most noticeable variation occurs in the dorsal coloration and in the shape of the calcars. Calcar size varies from inconspicuous (e.g. QCAZ 44174) to prominent (e.g. QCAZ 43850). Sexual dimorphism was observed in SVL, with 38.5–51.9 mm (44.3 ± 3.5 mm, N = 35) in males and 53.5–72.6 mm (64.4 ± 4.8 mm, N = 30) in females. Females are significantly larger than males (t = –20.13, d.f. = 52, P <0.001).

Head shape varies between subacuminate and rounded in dorsal view; in lateral view it varies between truncate and short, rounded. The head is slightly wider than the body in most of the specimens. Breeding males have keratinized nuptial excrescences on the inner surfaces of the thumbs. Webbing on the hand varies slightly among specimens: fingers I basal II (1 + –1½)—(2–3 –) III (1½–2½)—(1–2) IV. Variation of webbing between toes is I (1–2 –)—(1–2 –) II (1–1½)— (1–2 +) III (1–1 +)—(1–2 +) IV (1–2 +)—(1 –– 1 +) V. Vomerine teeth (N = 15): 10–14 (right = 11.5) and 10–15 (left = 12.1).

Background dorsal coloration varies from dull tan (e.g. QCAZ 36932), brown (e.g. QCAZ 25955), dark brown (e.g. QCAZ 64085) to cream (e.g. QCAZ 44167) with irregular faint to well-defined dark brown marks in diverse patterns. Most frequently there is an X- shaped mark on the scapular region (e.g. QCAZ 25797, 44168, 52734). A dark brown transversal band (e.g. QCAZ 36934, 52610) or sometimes two narrow transversal stripes interconnected (e.g. QCAZ 44168) are usually present on the sacral region. In some individuals in F

range Clade = N

17 = N 12 N = 25 = N 4 41 N = = 9 N N 2 = 0 with E given Clade = N

17

= N

12

N

25

= 4 N =

41

= N = N 9 0 0.035 is))

group

.

Mean

distances B. ventrimaculata Clade D N = 24 N = 19 = 32 N = N 11 48 = N (0

0.017

–

)

0.008 0.049 0.049

– 0.052

(

0.052

0.049 (0.044

–

semilineata genetic) 0.066) – 0.059) – – 0.059) Boana the intra-clade semilineata

B

. complex species = 56

N N

= 51 =

N

64 43 =

N

0.022 (0.054 0 – 0.029 (0.046 (0.044 0.032 (0.035 0.045

of shows)

members Diagonal B –

0.093 0.069) – 0.093 0.088

among.. nigra Clade = 19 14 = = 27 0.080 ((0.09) pb diagonal B N N N 0)))) 0.086) 0.077

409

(sequences

is shown above

B

appendiculata

.

A Clade =

N

30 = 35

N

(0.03 – 0) 0.017 0.046 0.052 (0.042 – (0.082 0.054 – 0.068 0.064 0.089 0.079 – (0.074 0.061 (0.068 – 0.057 0.062 – 0.069 (

DNA S

of

16

compared) – 0.057)) 0.061 –) – 0.076 0.077) 0.067) – 0.064) –

) uncorrected

p

individuals. geographica B 27 N = 0.012 – 0.004 0 (0.047 0.039 (0.057 0.059 (0.052 (0.060 – 0.07 0.064 (0.061 0.059 (0.059 (0.057 of))))))) (

distances Number). diabolica – 0) 0.005 0.04 0.052 – 0.052 – 0.071 0.069 – 0.059 0.064 0.094 – 0.079 0.091 – – 0.079 0.088 0.074 – 0.086

genetic diagonal. B

(

0.001

(

0.045 0.06 (

(

0.062

(

0.078

(

0.086

(

0.082 0.078

(

Pairwise

.

Table

6

(

below parentheses diabolica B. B geographica . appendiculata . B

Clade A B nigra . Clade B. B semilineata complex species B ventrimaculata . Clade D E Clade Clade F

there are scattered minute black flecks on the dorsal surfaces (e.g. QCAZ 25797, 36932, 39416). A dark mid-dorsal line may extend from the tip of the snout to the mid-sacrum, but in some specimens, the line is restricted to the head. Individuals may display asymmetrically distributed small to large cream or dark blotches on the dorsum (e.g. QCAZ 5076, 28227, 44160, 64085). The coloration of flanks and hidden surfaces of thighs varies from creamy to pale grey, with vertical dark stripes to black bars (e.g. QCAZ 30921, 36934, 39416); bars on the flanks, and less frequently on the thighs, are sometimes arranged in pairs. Flanks are uniform black in juveniles, some subadults and a few small adults (<42 mm). In some individuals, the bars can also be present on the hidden surfaces of the shanks (e.g. QCAZ 25797). Dark brown transversal bands are present on the dorsal surfaces of the limbs (one or two on the upper arm and forearm, and three to five on the thigh, shank and foot). The heels are usually cream, contrasting with the darker brown coloration of the dorsum (e.g. QCAZ 5076, 64085), but in some individuals, heels have the same colour as the rest of the body (e.g. QCAZ 36932, 44160).

Ventral surfaces of preserved specimens vary from creamy white (e.g. QCAZ 30921) to yellowish white (e.g. QCAZ 39416). In some individuals there are dark brown spots on the belly (e.g. QCAZ 5076). Coloration of webbing and discs varies from yellowish white to cream, brown or grey. Coloration of bones is white or green (e.g. QCAZ 64085).

Coloration in life ( Fig. 7B): Dorsal surfaces vary from brown (e.g. QCAZ 51182) to dark brown (e.g. QCAZ 23855), reddish brown (e.g. QCAZ 26267) or yellowish tan (e.g. QCAZ 52845); transversal bands on dorsal surfaces on limbs and marks on dorsum vary from light brown to dark brown, yellow or white (e.g. QCAZ 52733, 61767, 64101); in some individuals there are scattered minute black flecks on dorsal surfaces (e.g. QCAZ 36934); flanks and hidden surfaces of thighs are cream or yellowish with dark brown vertical stripes or bars (e.g. QCAZ 51179, 51182, 64085), uniform black in some small adult individuals; belly and ventral surfaces of limbs vary from cream to yellowish or orange (e.g. QCAZ 37879, 41009, 64099); throat cream white or yellowish; webbing varies from yellow to orange or brown; fingers and toes are dorsally grey or with same coloration of webbing; iris brown or bronze (e.g. QCAZ 52845, 55356); palpebrum finely reticulated with golden yellow.

In recently metamorphosed individuals, flanks, hidden surfaces of thighs and webbing are black; venter is grey; dorsal surfaces of the limbs and sides of the head are dark brown; dorsum is brown with scattered black flecks ( Fig. 8A).

Vocalization: Based on one male, not collected, from Yasuní on the road to Pompeya at km 73 (recorded by Morley Read); two males from Santa Cecilia ( KU 143144–45 ; recorded by W. E. Duellman on 10 October 1971; 20:50–21:00 h; 23 °C) and two males from Miazi-Nuevo Paraíso ( QCAZ 67113–14 ; collected by D. Paucar on 4 March 2017; 21:00 h). The call that lacks voucher specimen was assigned to Boana appendiculata based on the similarity of its structure compared to vouchered recordings of B. appendiculata . The call varies from a long groan (Type I, mean duration of 0.45 s and 31–37 pulses/note) to a series of short chuckle-like notes (Type II, mean 2.07 s and four to six notes/call) with fewer pulses per note (18–22 pulses/note). The long note ( Fig. 9A, B) has a mean dominant frequency of 973.2 Hz, rise time of 0.34 s and frequency bandwidth 1316.2 Hz. The serial of short notes ( Fig. 9C, D) has a mean dominant frequency of 1387.6 Hz, rise time of 0.44 s, frequency bandwidth of 1841.9 Hz and interval between notes of 0.16 s. Other call parameters are listed in Table 2 .

Distribution and ecology: Boana appendiculata occurs in the Amazon basin of Ecuador (Morona Santiago, Napo, Orellana, Pastaza, Sucumbíos and Zamora Chinchipe provinces), Brazil (Amazonas, Pará and Rondônia states) and Colombia ( Fig. 6). Localities with known elevation range from 14 to 1050 m above sea level. The elevation at Shell (1050 m) is the highest, while Caxiuanã (14 m) is the lowest. Specimens of B. appendiculata occur in terra firme forest, swamps, semi-flooded and flooded forests and artificial open areas. They are generally found at night in primary and secondary forest, perching on vegetation 20 to 600 cm above the ground, next to lakes, flooded areas and temporary ponds in clearings. Few individuals were found in flooded areas with pastures or forest away from water bodies. Their occurrence in secondary forests and artificial open areas suggest at least some tolerance of anthropogenic habitat disturbance.

Vegetation types for Ecuadorian localities are: (1) Amazonian evergreen foothill forest, characterized by a mixture of Amazonian and Andean vegetation with a canopy of 30 m; (2) Amazonian lowland evergreen forest, characterized by high plant alphadiversity and a canopy height of 30 m with emergent trees that reach 40 m; (3) floodplain lowland forest of white-waters, characterized by periodical flooding with white-waters from large rivers, with the vegetation reaching 35 m in height and several horizontal strata of vegetation; and (4) lowland forest of palms and black-waters characterized by a canopy height of 30 m with dense understory and dominance of the palm Mauritia flexuosa L.f. ( Sierra et al., 1999). Vegetation types at localities in Brazil include Purus-Madeira moist forests, Purus Varzea and Xingu-Tocantins-Araguaia moist forests (according to the World Wildlife Fund, 2017 classification scheme; availableathttp://www.eoearth.org/view/article/151948).

Remarks: A 16S sequence of ‘ Boana cf. geographica ’ from Valle del Guamuez, Colombia, published by Meza-Joya et al. (2019; GenBank accession number MF583739 ) is identical to the homologous sequence of specimen QCAZ 61767 of B. appendiculata from Limoncocha, Ecuador (539 bp). This similarity indicates the presence of B. appendiculata in Colombia. The Meza-Joya et al. (2019) publication became available while our manuscript was under review. Therefore, we did not include its sequence into our phylogeny.