New acoustic and molecular data shed light on the poorly known Amazonian frog Adenomera simonstuarti (Leptodactylidae): implications for distribution and conservation Author Carvalho, Thiago R. A1286A8F-C01A-421B-8135-A333D321AC95 Laboratório de Herpetologia, Departamento de Biodiversidade e Centro de Aquicultura, Universidade Estadual Paulista (UNESP), Rio Claro, SP, Brazil. thiago_decarvalho@yahoo.com.br Author Moraes, Leandro J. C. L. C10837F4-B3EF-401F-963B-A1EC285636E7 Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, AM, Brazil. leandro.jclm@gmail.com Author Angulo, Ariadne 812819A1-41EF-4CB5-A23E-BC19CC0ED7B9 IUCN SSC Amphibian Specialist Group, Toronto, Canada. aangulo@amphibians.org Author Werneck, Fernanda P. F4964B53-22D3-4B7B-A774-674C5F207A98 Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, AM, Brazil. fewerneck@gmail.com Author Icochea, Javier 59BD9040-E4A1-4B1D-8CC2-C4F1CA29D635 IUCN SSC Amphibian Specialist Group, Toronto, Canada. javiericochea@gmail.com Author Lima, Albertina P. 5E06A4D8-79DC-42EB-86C2-CC6042314ED9 Coordenação de Biodiversidade, Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, AM, Brazil. lima@inpa.gov.br text European Journal of Taxonomy 2020 2020-07-08 682 1 18 journal article 10.5852/ejt.2020.682 eae2652b-e985-466c-acbb-c5dbf7964914 2118-9773 3939336 B37BF24C-3A88-4DB6-A32E-B7EA35F6D9F7 Adenomera simonstuarti ( Angulo & Icochea, 2010 ) Material examined Holotype PERU • Cusco , La Convención , Echarate , Río Camisea ; MUSM 18218 . Paratypes PERU • 3 specs ; same collection data as for holotype; MUSM 18220 , 18221 , 18229 . Other material BRAZIL • 1 spec. , Acre , Tarauacá ; INPA-H 40967 • 5 specs ; Amazonas , Juruá ; INPA-H 5337 , 39792 , 39796 , 39813 , 39814 . Comparative material Adenomera andreae (M̹ller, 1923) BRAZIL • 3 specs ; Amapá , Serra do Navio ; AAG-UFU 5994 , 6006 , 6007 • 2 specs ; same collection data as for preceding; CFBH 43259 , 43265 • 11 specs ; Amazonas , Manaus ; INPA-H 34045 , 34048 , 34073 , 34074 , 34076 , 34081 , 34082 , 34084 to 34086 , 34090 • 5 specs ; same collection data as for preceding; ZUEC 3937 , 3969 , 3973 , 3974 , 7799 • 2 specs ; Pará , Belém ; AAG-UFU 2797 , 2798 • 7 specs ; Nova Timboteua ; AAG-UFU 2788 to 2794 . Adenomera chicomendesi Carvalho, Angulo, Kokubum, Barrera, Souza, Haddad & Giaretta, 2019 BRAZIL • holotype ; Acre , Rio Branco , Parque Zoobotânico ; CFBH 43562 • 3 specs , paratypes ; same collection data as for holotype; AAG-UFU 5862 to 5864 • 1 spec. , paratype ; same collection data as for holotype; CFBH 43563 • 4 specs , paratypes ; same collection data as for holotype; ZUEC 24528 to 245231 . PERU • 7 specs , paratypes ; Madre de Dios , Reserva Nacional de Tambopata ; MUSM 39462 , 30463 , 39467 , 39468 , 39472 to 39474 . Adenomera heyeri Boistel, Massary & Angulo, 2006 BRAZIL • 3 specs ; Pará , Oriximiná , ESEC-Grão-Pará; MPEG 30099 to 30101 . Adenomera hylaedactyla (Cope, 1868) BRAZIL • 5 specs ; Acre , Cruzeiro do Sul ; AAG-UFU 5907 to 5911 • 3 specs ; Feijó ; AAG-UFU 5895 to 5897 • 8 specs ; Amazonas , Manaus ; INPA-H 22410 to 22413 , 26606 to 26609 • 8 specs ; São Gabriel da Cachoeira ; AAG-UFU 3859 to 3866 • 4 specs ; Roraima , Cantá ; AAG-UFU 5540 to 5443 . Adenomera lutzi Heyer, 1975 GUYANA • 6 specs ; Potaro-Siparuni ; MZUSP 150799 to 150804 . Adenomera phonotriccus Carvalho, Giaretta, Angulo, Haddad & Peloso, 2019 BRAZIL • holotype ; Pará , Palestina do Pará ; MPEG 41155 • 2 specs , paratypes ; same collection data as for holotype; CFBH 43130 , 43131 • 1 spec. , paratype ; same collection data as for holotype; MPEG 41156 . Adenomera sp. ( A. andreae clade) PERU • 1 spec. ; Cusco , La Convención , Echarate , Río Camisea ; MUSM 18219 . Phylogenetic relationships and genetic diversity Both BI and ML phylogenetic reconstructions ( Fig. 1 ) yielded similar results with regard to relationships in the Adenomera andreae clade and the monophyly of A. simonstuarti . All three new sequences from southwestern Brazilian Amazonia were recovered nested within A. simonstuarti ( Fig. 1 ). The ABGD delimitation analysis recovered eight genetic lineages within A. simonstuarti ( Fig. 2 ) with noticeable geographic structure ( Figs 1–2 ). Mean genetic distances in COI among the lineages of A. simonstuarti ( Table 1 ) range from 3.2¯7.6% (uncorrected) and from 3.3¯8.0% (corrected), whereas within-lineage genetic distances reach a maximum value of 1.7% (uncorrected and corrected). Our genetic voucher INPA-H 40967 ( Fig. 3 ) is the only specimen of Adenomera simonstuarti with associated acoustic data. Morphological and color features of the specimen fully agree with those presented in the original description of A. simonstuarti ( Angulo & Icochea 2010 ) . This voucher specimen from the upper Juruá River constitutes the lineage 3 together with other specimens from the upper Amazon basin in southwestern Amazonia ( Figs 1–2 ). The lineage 3 is regarded hereinafter as conspecific with the nominal species. The other two new COI sequences (lower Juruá River) fell within the lineage 2. These two vouchers also have the recognized morphotype of A. simonstuarti ( Fig. 4 ), but acoustic data for this lineage remain unknown. Mean genetic distances between the COI lineages 2 and 3 are noticeable, ranging from 5.0% (uncorrected) to 5.3% (corrected). Advertisement call and acoustic diagnosis The call of Adenomera simonstuarti ( Fig. 5 ) is redescribed below based on combined values of calls recorded from southwestern Amazonia: the type locality (Camisea, Cusco , Peru ) and the upper Juruá River (Tarauacá, Acre , Brazil ) ( Table 2 ). The call consists of a multi-note signal given at a low repetition rate (<10 per minute), lasting 0.8–6.5 (3.4 ± 1.9) s. Calls are formed by 4–30 (15.6 ± 9.9) notes. Call notes are given at a rate of 4–5 (4.2 ± 0.6) per second. Notes last 57–79 (66.6 ± 2.6) ms, and the rise time is at 13–73 (35.8 ± 3.2) % of note duration. Notes are formed by 2–3 (2.6 ± 0.5) partly fused pulses with duration varying from 10–53 (26.4 ± 6.4) ms. Notes have the dominant frequency coinciding almost always with the fundamental harmonic ( 1873–2046 Hz, 1959.9 ± 2.3), but coinciding with the second harmonic (3596–4156 Hz, 3962.1 ± 254.8) in four notes given by the male from Brazil . The frequency modulation is upward, rising from 43–301 (194.0 ± 12.3) Hz. Fig. 1. Bayesian phylogenetic tree of Adenomera Steindachner, 1867 inferred from a concatenated dataset of four genes (two mitochondrial + two nuclear), showing the eight major clades delimited by colors. The emphasis on the diversification within the A. simonstuarti species complex shows a deep genetic divergence, with eight distinct lineages (see Fig. 2 for geographic distribution). Symbols above branches indicate posterior probabilities of Bayesian inference (BI) and those below branches indicate bootstraps of maximum likelihood inference (ML). Low support values (BI <80 and ML <70) were omitted. Branch scale is indicated in number of substitution per site. Table 1. Uncorrected (lower diagonal) and corrected (Jukes-Cantor model; upper diagonal) pairwise genetic distances of a fragment from COI mtDNA gene among the lineages of the Adenomera simonstuarti species complex (named as sim1–8). Within-lineage distances (uncorrected/ corrected), when applicable, were highlighted in bold across the central diagonal (upper left to lower right). The lineage 3 corresponds to the nominal species. Values are presented as means (in %).

sim1	sim2	sim3	sim4	sim5	sim6	sim7	sim8
sim1	1.7 / 1.7	3.3	5.2	6.1	4.8	5.9	4.3	6.9
sim2	3.2	0.2/ 0.2	5.3	7.0	5.4	5.8	5.4	7.2
sim3	4.9	5.0	0.7/ 0.7	5.3	4.7	5.1	5.2	7.3
sim4	5.8	6.7	5.1	0.0/ 0.0	3.8	7.3	5.6	8.0
sim5	4.6	5.2	4.5	3.7	–	6.1	3.9	7.1
sim6	5.7	5.6	5.0	7.0	5.9	–	6.0	7.5
sim7	4.2	5.2	5.0	5.3	3.8	5.8	–	6.7
sim8	6.6	6.9	7.0	7.6	6.8	7.1	6.3	–

Table 2. Advertisement call traits of Adenomera simonstuarti ( Angulo & Icochea, 2010 ) from southwestern Amazonia of Peru (type locality) and Brazil. One male was recorded from each locality. N = number of quantified notes and pulses, respectively. Values are presented as range (mean ± SD).

Acoustic traits	Camisea (Cusco, Peru) N = 33 / 98	Tarauacá (Acre, Brazil) N = 24 / 55
Call duration (s)	0.8–5.1 (2.1 ± 1.8)	1.5–6.5 (4.8 ± 1.7)
Notes per call	4–20 (8.6 ± 6.7)	8–30 (22.6 ± 7.6)
Note duration (ms)	57–71 (64.7 ± 3.4)	62–79 (68.5 ± 4.5)
Note rate per second	3.7–4.1 (3.8 ± 0.1)	4.5–4.9 (4.6 ± 0.1)
Note rise time (%)	13–73 (33.5 ± 14.0)	20–60 (38.1 ± 16.2)
Pulses per note	2–3 (3.0 ± 0.2)	2–3 (2.3 ± 0.5)
Pulse duration (ms)	10–31 (21.8 ± 2.3)	10–53 (30.9 ± 5.8)
Dominant frequency (Hz)	1873–2003 (1958.2 ± 20.1)	1873–2046 (1961.7 ± 56.7)
Frequency modulation (Hz)	43–301 (185.3 ± 55.4)	86–301 (202.8 ± 58.9)

The advertisement call of Adenomera simonstuarti ( Fig. 5 ) recorded from the type locality (Camisea, Peru ; Angulo & Icochea 2010 ) and from Brazil are given as multi-note calls. The multi-note call of A. simonstuarti represents a useful diagnostic character of the species by being unique among members of the A. andreae clade. The only other described species of Adenomera with multi-note call is the allopatric A. cotuba Carvalho & Giaretta, 2013 , distributed in the Cerrado savannas and dry forests of north central Brazil ( Carvalho & Giaretta 2013b ). Additionally, the following morphological and color features, when combined with acoustic data, can help distinguish nominal A. simonstuarti from the seven Amazonian congeners [ A. andreae , A. chicomendesi , A. coca ( Angulo & Reichle, 2008 ) , A. heyeri , A. hylaedactyla , A. lutzi and A. phonotriccus ; see Boistel et al. 2006 ; Kok et al. 2007 ; Angulo & Reichle 2008 ; Carvalho et al. 2019b , 2019c , 2019d ]: (1) a nearly solid, dark-colored stripe along the underside of the forearm; (2) absence of dorsolateral stripe; (3) toe tips fully expanded into discs; (4) absence of antebrachial tubercle on underside of forearm; and (5) multi-note advertisement call. Fig. 2. Geographic distribution of the Adenomera simonstuarti species complex in northwestern South America; genetic lineage 3 corresponds to the nominal species. Phylogenetic relationships among the eight lineages are shown on the upper right. Black solid-filled symbols represent the localities reported in the original description (square = type locality at Camisea, Peru; circle = Pando, Bolivia). Blackdotted symbols indicate newly collected specimens from the Juruá River in the Brazilian Amazonia. Habitat and natural history The call voucher of Adenomera simonstuarti from the upper Juruá River (INPA-H 40967; Fig. 3 ), corresponding to the lineage 3, was collected from an open bamboo forest, approximately 2 km from BR-364 road. This individual and other two were heard calling from an old clearing surrounded by decomposing fallen logs. The three individuals called hidden underneath dense leaf litter, and only one of them (the call voucher) were found while surveying the area. Adenomera simonstuarti and A. andreae were found syntopically in this area. The four specimens from the lower Juruá River (INPA-H 39792, 39796 and 39813–14; Fig. 4 ), corresponding to lineage 2, were collected in a non-flooded lowland forest ( terra firme forest) with dense understory layer. Three specimens (INPA-H 39792 and 39813–14) were found in a forest affected by anthropogenic activities (i.e., logging), located close to Comunidade Cumaru village. This could indicate a certain degree of tolerance of the lower Juruá population to habitat disturbance, given that human occupation and activities in this region have begun during the late 1980s (ICMBio 2009). The specimen INPA-H 39796 ( Fig. 4 ) was collected from a preserved forest, distant from that village. Specimens in the lower Juruá River were sympatric with A. andreae and A. hylaedactyla . Adenomera simonstuarti and A. andreae were found syntopically inside the forest, whereas A. hylaedactyla was only found along riverbanks. Fig. 3. Preserved male of nominal Adenomera simonstuarti ( Angulo & Icochea, 2010 ) (= genetic lineage 3): call voucher INPA-H 40967 (SVL = 23.4 mm) from the upper Juruá River, in Tarauacá, Brazilian state of Acre. This specimen corresponds to a call voucher (see Fig. 5). A−B . Body in dorsal and ventral views, not to scale. C−D . Detail of the ventral surface of right foot and hand, respectively. Note the nearly solid, dark-colored stripe along the underside of the forearm. Photographs by J. Magnusson. Scale bar = 5 mm. Distribution patterns Adenomera simonstuarti (= lineage 3) is distributed in the upper Amazon Basin of southwestern Brazilian and Peruvian Amazonia, and two locations in the eastern slopes of the Andes in south central Peru . Populations linked to the other seven lineages are in most cases allopatric among each other. Some lineages are widely distributed, such as lineage 1, from lowland and montane forests in the upper Amazon Basin of Peru and Brazil . Other lineages, such as lineage 2, may be narrowly distributed on the east bank of the lower Juruá River. Other distribution patterns include: lineage 4 in lowland Amazonia of northeastern Ecuador and extreme northern Peru ; lineage 5 in Venezuelan Andes montane forests; lineage 6 in the Marañón-Ucayali interfluve; lineage 7 in the upper Amazon River; and lineage 8 in the upper Negro River. Based on the geographic patterns of each of the lineages, we could expect that some of them may have distributions associated with interfluve regions, such as lineages 4, 6 and 7 ( Fig. 2 ). Another interesting pattern is that the nominal species (lineage 3) and other lineages (e.g., lineage 8) are distributed in the upper Amazon Basin, while some others are distributed in the middle-lower portions of major southern tributaries of the Amazon River (e.g., lineage 2; Fig. 2 ).