Zoosyst. Evol. 100 (1) 2024, 233-253 | DOI 10.3897/zse.100.110133 Gee BERLIN A new species of terrestrial foam-nesting frog of the Adenomera simonstuarti complex (Anura, Leptodactylidae) from white-sand forests of central Amazonia, Brazil Bryan da Cunha Martins', Alexander Tamanini Ménico*, Cianir Mendonca!, Silionama P. Dantas’, Jesus R. D. Souza!, James Hanken®, Albertina Pimentel Lima’, Miquéias Ferrao!?*° Programa de Pos-graduagao em Zoologia, Instituto de Ciéncias Bioldgicas, Universidade Federal do Amazonas, Manaus, Amazonas, Brazil Instituto Nacional de Pesquisas da Amazonia, Manaus, Amazonas, Brazil Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, USA Programa de Poés-graduagdo em Biodiversidade Animal, Instituto de Ciéncias Biolégicas, Universidade Federal de Goids, Goidnia, Goids, Brazil oF WN FP Centro Nacional de Pesquisa e Conservagdao de Répteis e Anfibios, Instituto Chico Mendes de Conservagdo da Biodiversidade, Goidnia, Goids, Brazil https://zoobank. org/1F8E43A D-2A 15-4244-AA77-F914D3BFAF2F Corresponding author: Miquéias Ferrao (uranoscodon@gmail.com) Academic editor: Pedro Taucce # Received 27 July 2023 Accepted 14 February 2024 @ Published 7 March 2024 Abstract By using integrative taxonomy, we describe a new species of terrestrial foam-nesting frog of the genus Adenomera from white-sand forests of the Rio Negro Sustainable Development Reserve, Central Amazonia, Brazil. Within the A. andreae clade, the new species belongs to the A. simonstuarti complex where it is sister to the lineage from the lower Jurua River. The new species is assigned to the genus Adenomera by having adult SVL smaller than 34.1 mm, by its lack of fringing and webbing between toes and by the absence of spines on the thumb of adult males. It differs from other Adenomera by the following combination of characters: antebrachial tubercle absent; toe tips flattened or slightly flattened, with visible expansions; nearly solid, dark-coloured stripe on underside of forearm; single-note advertisement call; notes formed by 11—21 incomplete pulses; call duration varying between 100 and 199 ms; fundamental frequency 1,765—2,239 Hz; dominant frequency 3,448—4,349 Hz; and endotrophic tadpoles with spiracle present and labial teeth absent. Over the last decade, we have inventoried many permanent sampling modules in ombrophilous forests in the Manaus Region and in the Purus-Madeira interfluve, but the new species was found only in the white-sand forest from West Ne- gro-Solimées Interfluve. Adenomera sp. nov. may be endemic to, or at least a specialist in, this environment. Key Words campina, campinarana, integrative taxonomy, tadpoles, West Negro-Solim6es Interfluve Introduction al. 2006; Pyron and Wiens 2011; de Sa et al. 2014). The genus was originally described by Steindachner (1867) to accommodate a single species, A. marmorata. Later, Lutz (1930) synonymised the genus with Parvulus, a subgenus Leptodactylid frogs of the genus Adenomera Stein- dachner, 1867 comprise 30 described species distributed throughout South America east of the Andes (Frost 2024). The taxonomic history of this genus 1s very complex and, over the last 50 years, numerous systematic studies have reviewed its taxonomic validity, phylogenetic po- sition and species diversity (Heyer 1973, 1974; Frost et of Leptodactylus Fitzinger, 1826, but Parker (1932) soon gave priority to the name Adenomera and elevated it as a subgenus of Leptodactylus. Four decades later, Adeno- mera was resurrected by Heyer (1974) to accommodate taxa of the Leptodactylus marmoratus species group. Copyright Martins, B. da C. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 234 Martins, B. da C. et al.: New Adenomera from white-sand forests of Brazilian Amazonia To avoid paraphyly of Leptodactylus rendered by Van- zolinius Heyer, 1974, Frost et al. (2006), supported by evidence from Heyer (1998) and Kokubum and Giaret- ta (2005), declared Adenomera a synonym of Lithodytes Fitzinger, 1843 and the latter taxon a synonym (subge- nus) of Leptodactylus. Based on molecular data, Pyron and Wiens (2011) recovered Adenomera as sister to Lithodytes and this clade as sister to Leptodactylus. The authors also removed the two former taxa from the synon- ymy with Leptodactylus. The sister relationship between Adenomera and Lithodytes was corroborated by de Sa et al. (2014) through a total evidence analysis, which recov- ered the clade comprising the two genera as sister to the one grouping Hydrolaetare and Leptodactylus. Fouquet et al. (2014) performed a comprehensive phylogenetic analysis and recovered eight major clades within Ade- nomera: A. lutzi clade, A. heyeri clade, Adenomera sp. I clade, A. andreae clade, A. marmorata clade, A. thomei clade, A. martinezi clade and A. hylaedactyla clade. How- ever, Carvalho et al. (2021) recovered Adenomera sp. I as A. juikitam and, based on acoustic, morphologic and genetic data, concluded that A. juikitam instead belongs to the A. heyeri clade. The genus Adenomera displays a high prevalence of morphologically cryptic species (e.g. Angulo and Icochea (2010); Carvalho and Giaretta (2013a); Carvalho et al. (2020a); Zaracho et al. (2023)). Some species also show high levels of intraspecific polymorphism (e.g. Cassini et al. (2020)) and congeneric sympatry and syntopy are com- mon; up to three species may occur 1n the same region (e.g. Carvalho et al. (2021)). These factors make species delim- itation in Adenomera challenging. Nevertheless, 15 of the 30 currently recognised species were described in the last 10 years (Frost 2024) and several candidate species still await formal description (Fouquet et al. 2014). The mas- sive advance in the taxonomy of Adenomera has been made possible by the use of integrative taxonomy (Carvalho et al. 2019a, 2019c). In particular, despite morphological cryp- sis, advertisement calls are markedly divergent amongst Species and represent a powerful source of reliable diag- nostic characters (Angulo and Icochea 2010; Carvalho and Giaretta 2013a, b; Carvalho et al. 2019c, 2021). The Adenomera andreae clade comprises four described species—A. andreae (Miller, 1923); A. chicomendesi Carvalho, Angulo, Kokubum, Barrera, Souza, Haddad & Giaretta, 2019; A. guarayo Carvalho, Angulo, Barrera, Aguilar-Puntriano & Haddad, 2020; and A. simonstuarti (Angulo & Icochea, 2010)}—and two candidate species, Adenomera sp. D and Adenomera sp. T (Fouquet et al. 2014). While the A. andreae clade is restricted to Amazonia, none of the nominal species has a restricted geographic distribution. Adenomera andre- ae shows the widest range, being distributed throughout Amazonia (Carvalho et al. 2019c), while A. chicomendesi and A. guarayo are widely distributed in south-western Amazonia (Carvalho et al. 2019a, 2020a). Adenomera simonstuarti 1s distributed in western and south-western Amazonia (Carvalho et al. 2020b). zse.pensoft.net Adenomera simonstuarti was described from Peruvian Amazonia, based on morphological and acoustic data of four males and two females (Angulo and Icochea 2010). Subsequently, Fouquet et al. (2014) reported that the species was more widespread than previously thought, also occurring in Venezuela, Ecuador and Brazil (States of Acre and Amazonas). They also suggested the exis- tence of more than one species hidden under the name A. simonstuarti (Fouquet et al. 2014, appendix S2a). Recently, Carvalho et al. (2020b) sequenced additional specimens from Brazil referred to as A. simonstuarti and their delimitation analysis recovered eight lineages within this name (hereafter, the A. simonstuarti complex). They also re-described the species’ advertisement call, based on recordings from the type locality in Peru and an addi- tional locality in the upper Jurua River Basin (Acre, Bra- zil). Based on molecular, morphological and bioacoustic data, Carvalho et al. (2020b) recognised their lineage 3 as A. simonstuarti sensu stricto. They also identified the oth- er lineages as putative new species, pending confirmation with additional data (e.g. acoustic and morphologic data). Poorly sampled environments in Amazonia usually harbour undocumented biodiversity of anurans (Ferrao et al. 2016; Vacher et al. 2020). Physiognomies comprising the white-sand ecosystems (hereafter, WSE) exemplify such environments (Adeney et al. 2016). The WSE oc- cupies an area of 5% of the Amazonia and comprises two main physiognomies in Brazil: campina—open environ- ments characterised as patches of grasslands or scrublands (canopy < 7 m) on a matrix of exposed sandy soil; and campinarana—closed-canopy, forested environments characterised by thin-trunked trees of low stature (canopy < 20 m) (Anderson 1981; Ferreira 2009; Adeney et al. 2016). Despite the increasing interest in WSE organisms (Capurucho et al. 2013; Fine and Baraloto 2016; Vicenti- ni 2016; Lamarre et al. 2016; Borges et al. 2016; Fraga et al. 2018; Gonella et al. 2020), studies of anurans from WSE are rare. The few such studies recently published show that WSE represents a source of poorly known and new species of anurans, many of which appear to be spe- cialists in or endemic to these environments (Carvalho et al. 2019a:; Ferrdo et al. 2019, 2022; Monico et al. 2023). In the present study, we sampled an unreported lineage of the Adenomera simonstuarti complex from the white- sand forests of Central Amazonia and, by using integra- tive taxonomy, describe it as a new species. Methods Sampling Fieldwork was conducted between 2019 and 2023 in three long-term ecological research sites (RAPELD) in the Rio Negro Sustainable Development Reserve (here- after, RDS Rio Negro), Municipality of Iranduba, State of Amazonas, Brazil (Fig. 1). Modules are located near km 18 (3°06'33.6"S, 60°40'29.0"W; 73 m above sea level Zoosyst. Evol. 100 (1) 2024, 233-253 0.0 -10.0 -20.0 © A. simonstuarti 2 ee A. simonstuarti 6 © A. sp. nov. paratype locality © A. simonstuarti 1 @ A. simonstuarti 5 Sx A. sp. nov. type locality Novo Airao lranduba 230 9°C- 1 8°C- Manaus |, jo % A. simonstuarti SS ® A. simonstuarti 4 VA. simonstuarti 7 A A. simonstuarti 8 [] A. sp. nov. call record Figure 1. Geographic distribution of the Adenomera simonstuarti species complex (left) and a detailed view of the geographic distribution of the new species in central Amazonia, Amazonas, Brazil (right). Green area: Rio Negro Sustainable Development Reserve. Numbers: permanent sampling modules at (1) km 18, (2) km 26 and (3) km 50 along the AM-352 highway; (4) Vale da Bengéo Community, Ramal do 25, Manacapuru. South American countries: ARG, Argentina; BOL, Bolivia; CHL, Chile; COL, Colombia; ECU, Ecuador; PAR, Paraguay; PER, Peru; VEN, Venezuela. [hereafter [a.s.1.]), km 26 (3°03'31.0"S, 60°45'42.0"W; 73 ma.s.l.) and km 50 (2°50'10.0"S, 60°50'20.0"W; 19 m a.s.l.) of the AM-352 highway. Adults were euthanised with 2% aqueous benzocaine topical solution, fixed in 10% neutral-buffered formalin and preserved in 70% ethanol. Before fixation, tissue samples of each speci- men were collected and stored in 100% ethanol. Tadpoles were collected from two foam nests in the calling site of two uncollected males near the sampling module at km 18. They were euthanised as described above, fixed and preserved in 5% neutral-buffered formalin. Adults were deposited in the herpetological collections of the Instituto Nacional de Pesquisas da Amazonia (INPA-H; Manaus, Brazil), Museu Paraense Emilio Goeldi (MPEG; Belém, Brazil) and Museu de Zoologia da Universidade de Campinas (ZUEC-AMP; Campinas, Brazil); tadpoles were deposited at INPA-H. Advertisement calls of six males of the new species (IN- PA-H 44867 [holotype], MPEG 44649, INPA-H 44868— 69, MPEG 44652 and INPA-H 44877) were recorded with a Sennheiser K6/ME66 unidirectional microphone (Senn- heiser, Germany) coupled to a Marantz PMD660 digital recorder (Kanagawa, Japan) and with a Sony PCM-D50 digital recorder with built-in microphone. Recordings were stored in wav files with a sampling rate of 44.1 kHz and sample size of 16 bits. The microphone was positioned 50-100 cm from the calling male. Air temperature during all recordings was 25 °C. Recordings were deposited in the Neotropical Jacques Vielliard sound repository of the University of Campinas (FNJV; Campinas, Brazil) under accession numbers FNJV 59561-66. To facilitate interspecific comparisons, 16 specimens and the advertisement calls of six males of Adenomera simonstuarti sensu stricto were collected and recorded, respectively, at Unidade de Gestéo Ambiental Acuraua, Municipality of Tarauaca, State of Acre, Brazil. A speci- men from this locality (INPA-H 40967) was included in the phylogenetic inference of Carvalho et al. (2020b) and nests with samples of A. simonstuarti sensu stricto from Peru and male advertisement calls of the Acre population match with those in the original description by Angulo and Icochea (2010). All males of the Acre population were found in the field by their vocalisation, ensuring that we collected the target species. zse.pensoft.net 236 Martins, B. da C. et al.: New Adenomera from white-sand forests of Brazilian Amazonia Morphology The description of external morphology of adults of the new species is based on 21 males and five females. Sex was determined through direct assessment of sexual characters: the presence of vocal slits, vocal sac and a fleshy ridge on the snout tip in males and absence in females. Maturity was determined, based on breeding behaviour in males (calling activity) and examination of secondary sexual characters in females (mature oocytes visible through the belly skin). The following 16 morphometric measurements (Watters et al. 2016) were taken to the nearest 0.1 mm using digital calipers and an ocular micrometer coupled to a stereomi- croscope: snout-vent length (SVL), head length (HL), head width (HW), snout length (SL), eye-nostril distance (EN), eye diameter (ED), interorbital distance (IOD) internari- al distance (IND), tympanum diameter (TD), upper arm length (UAL), hand length (HAL), forearm length (FLL), thigh length (THL), tibia length (TL), foot length (FL) and tarsus length (TSL). Toe tip development (character states) follows Heyer (1973). Snout shape follows Heyer et al. (1990). Terminology for other morphological charac- ters follows Carvalho et al. (2020a). We follow the colour catalogue of Kohler (2012): colour names are italicised; cc, colour code. Repeated colours do not repeat codes. See Suppl. material 1: table S1 for morphometric raw data. The larval developmental stage was determined ac- cording to Gosner (1960). The following morphometric measurements were taken with a micrometer coupled to a stereomicroscope from 10 tadpoles at stages 35 (n = 7) and 41 (n = 3): total length (TL), body length (BL), tail length (TAL), maximum tail height (MTH), tail muscle height (TMH), tail muscle width (TMW), inter-nostril distance (IND) and interorbital distance (IOD) (Altig and McDiarmid 1999); body height (BH), body width at spiracle level (BW), eye-nostril distance (END), eye diameter (ED) and oral-disc width (ODW) (Lavilla and Scrocchi 1986); body width at eye level (HW) (Lima et al. 2015); and vent-tube length (VTL) (Lins et al. 2018). Morphological description is based on seven tadpoles at stage 35. Terminology and diagnostic characters follow Altig and McDiarmid (1999) and Schulze et al. (2015). Vocalisation Description of the advertisement call and the following acoustic parameters follow Carvalho et al. (2019a) and Kohler et al. (2017): call duration (CD), notes per call (NpC), note duration (ND), note repetition rate (NrR) note rise time (NrT), pulses per note (PpN), pulse duration (PD; measured for the first, central and last pulses of each note), pulse repetition rate (PrR), dominant frequency (DF), fun- damental frequency (FF) and frequency modulation (FM). See Suppl. material 1: table S2 for bioacoustic raw data. Calls were analysed with Raven 1.5.1 (Bioacous- tics Research Program 2014) configured as follows: Hamming window (size = 20 ms), filter bandwidth 65 Hz, overlap 90%, hop size 2 ms and Discrete Fourier zse.pensoft.net Transform 1,024 samples. The dominant frequency and rise time were measured with the peak frequency and peak time relative functions. Figures were produced in R platform (R Core Team 2021) with the packages seewave 2.1.0 (Sueur et al. 2008) and tuneR 1.3.2 (Ligges et al. 2017). Seewave was set as follows: Hamming window, Fast Fourier Transform 256 points, overlap 90%. Molecular phylogenetics Genomic DNA was extracted from tissues of four spec- imens of the new species using a Wizard genomic DNA Purification Kit (Promega Corp., Madison, WI, USA) according to the manufacturer’s protocol. Fragments of cytochrome c oxidase subunit I (COI) were ampli- fied through polymerase chain reaction (PCR) using the primers CHmL4 (5’-TYTCWACWAAYCAYAAAGAY- ATCGG-3’) and CHmR4 (5’-ACYTCRGGRTGRC- CRAARAATCA-3’) (Che et al. 2012). Reaction condi- tions were: 60 s at 94 °C followed by 35 cycles of 94 °C (20 s), 50 °C (50 s) and 72 °C (90 s) and final extension of 10 min at 72 °C. The final volume of the PCR reaction was 15 wl and contained 0.6 wl of 50 mM MgCl, 1.2 ul of 10 mM dNTPs (2.5 mM each dNTP), 1.5 pl of tampon 10x (75 mM Tris HCI, 50 mM KCl, 20 mM (NH,),SO,), 0.5 wl of each primer (10 uM), 9.55 wl of ddH,O, 0.15 wl of 1 U Taq DNA Polymerase and 1 ul of DNA (30-50 ng/ul). The PCR products were purified using Exonuclease I and Thermosensitive Alkaline Phosphatase (Thermo Fish- er Scientific, Waltham, MA, USA). Subsequent sequenc- ing reactions were performed using standard protocols of the Big Dye™ Terminator Kit (Applied Biosystems, Waltham, USA). We used an automated sequencer ABI Prism 3130 (ThermoFisher Scientific, Waltham, USA) to sequence the amplicons. Sequences were edited with Geneious 5.3.4 (Kearse et al. 2012). Newly-generated se- quences are deposited in the online repository GenBank under accession numbers OQ974333-—36. To infer phylogenetic relationships, we inserted the generated sequences into a dataset containing sequences retrieved from GenBank (Suppl. material 1: table S3). Our dataset contains the genes cytochrome b (Cytb), cyto- chrome c oxidase subunit I (COI), recombination activat- ing gene 1 (RAG1) and pro-opiomelanocortin C. These sequences represent all species of the Adenomera andreae clade, including all known lineages of A. simonstuarti, as well as Adenomera sp. D and Adenomera sp. T (Fouquet et al. 2014; Carvalho et al. 2020b) and species belong- ing to the other six clades (Suppl. material 1: table S3). Lithodytes lineatus was used to root the tree. To align se- quences of each gene, we used the MAFFT online server following default parameters under the G-INS-i strategy. The final matrix was concatenated in Geneious 5.3.4 and comprises 53 terminals and 3,293 base pairs (bp) (667 for Cytb, 657 for COI, 1,422 for RAGI and 547 for POMC). We divided the dataset considering first, second and third codon positions for each protein-coding gene and we used PartitionFinder 2.1.1 (Lanfear et al. 2017) under Zoosyst. Evol. 100 (1) 2024, 233-253 the corrected Akaike Information Criterion (AICc) to infer partition schemes and evolutionary models. The best evolutionary models for partitions in the concate- nated matrix were TIM+G for Cytb 1* and COI 3" po- sitions; SYM+I+G for Cytb 2™ position; GTR+I+G for Cytb 3 position, TRNEF+I+G for COI 1* position; F81+I+G for COI 2™ position; TRN+I+G for RAGI 1* and 2™ positions; GTR+G for RAGI 3™ and POCM 1* positions; TVM+I+G for POMC 2™ position; and GTR+I for POMC 3" position. Phylogenetic relationships were reconstructed through Maximum Likelihood (ML) using IQTREE (Nguyen et al. 2015) implemented in the online server http://iqtree.cibiv.univie.ac.at/ (Trifinopoulos et al. 2016). Clade support was estimated with 10,000 ultrafast bootstrap replicates (Hoang et al. 2018) using 5,000 max- imum iterations, 3,000 replicates and a minimum correla- tion coefficient of 0.99. Lineage numbering within the A. simonstuarti species complex follows Carvalho et al. (2020b), except by A. simonstuarti 3, which is referred to as A. simonstuarti sensu stricto (SS) in the present study. Based on COI alignment, we calculated pairwise genetic distances (uncorrected p-distance and Kimura two-parameter distance; Kimura (1980)) between the new species and closely related taxa of the A. simonstu- arti species complex using MEGA 6 (Tamura et al. 2013). Mean distances are presented as in the main text, mini- mum—maximum values in the Suppl. material 1: table S4. Morphometric analysis Due to the phenotypic similarity between A. simonstuarti sensu stricto and the new species, we performed a Principal Component Analysis (PCA) associated with a Multivariate Analysis of Variance (MANOVA) to test for a statistical difference between the morphometric multidimensional spaces of each species. Analysis was performed only for males due to the low number of females collected for A. si- monstuarti sensu stricto. The same 16 morphometric mea- surements taken from the new species were also taken from 14 adult males of A. simonstuarti. To perform morphomet- ric PCA, we transformed the raw data into 15 morphomet- ric ratios: HL/SVL, HW/SVL, SL/SVL, END/SVL, IND/ SVL, ED/SVL, IOD/SVL, TD/SVL, FAL/SVL, UAL/ SVL, HAL/SVLL, TL/SVL, FL/SVL, THL/SVL and TAL/ SVL. The PCA and MANOVA were run using the functions prcomp and manova of the package stats 4.1 (R Core Team 2021); an ellipse representing the standard errors of points in the graphic representation of PCA was drawn using the function ordiellipse of the package vegan 2.5-7 (Oksanen et al. 2020) with parameter kind set as se. See Suppl. material 1: table S1 for morphometric measurements of Adenomera sp. nov. and A. simonstuarti sensu stricto. Interspecific morphological comparisons Succinct morphological comparisons of adults were made with all 30 nominal congeners but detailed morphological 23/7 and acoustic comparisons were restricted to species of the Adenomera andreae clade (A. andreae [Miller, 1923]; A. chicomendesi Carvalho, Angulo, Kokubum, Barrera, Souza, Haddad & Giaretta, 2019; A. guarayo Carvalho, Angulo, Barrera, Aguilar-Puntriano & Haddad, 2020; and A. simonstuarti) and species distributed in Amazonia (A. amicorum Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel- Oliveira & Haddad, 2021; A. aurantiaca Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira & Haddad, 2021; A. glauciae Carvalho, Simédes, Gagliardi-Urrutia, Rojas-Runjaic, Haddad and Castrovejo-Fisher, 2020; A. gridipappi Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira & Haddad, 2021; A. heyeri Boistel, Massary & Angulo, 2006; A. hylaedactyla (Cope, 1868); A. inopinata Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira & Haddad, 2021; A. kayapo Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira & Haddad, 2021; A. /utzi Heyer, 1975; A. martinezi (Bokermann, 1956); A. phonotriccus Carvalho, Giaretta, Angulo, Haddad & Peloso, 2019; and A. tapajonica Carvalho, Moraes, Lima, Fouquet, Peloso, Pavan, Drummond, Rodrigues, Giaretta, Gordo, Neckel-Oliveira & Haddad, 2021). Larval comparisons were made with all nominal species for which tadpoles are described (A. andreae, A. guarani, A. hylaedactyla, A. marmorata, A. saci and A. thomei), except for A. bokermanni because the tadpole described for it might correspond to another species (Carvalho and Giaretta 2013b). Comparisons were made based on published data (i.e. taxonomic descriptions, re-descriptions and revisions), except the one for Adenomera simonstuarti, which is based on direct analysis of specimens (Appendices 1, 2). Results Phylogenetic relationships and genetic distances Individuals of Adenomera sp. nov. nest together as a new monophyletic lineage (bootstrap support = 100) with- in the A. simonstuarti species complex (sensu Carvalho et al. (2020b)), which nests within the A. andreae clade (Fig. 2). The new species is sister to the lineage A. simonstuarti 2 from the lower Jurua River in Brazil. Clades representing A. simonstuarti 2 and Adenomera sp. nov. are the shallowest within the species complex; the average p-distance for COI between them equals 2.9% (2.5-3.3%) (Table 1; Suppl. material 1: table S4). Peru- vian and Brazilian individuals of A. simonstuarti sensu stricto are recovered as sister to the clade comprising A. simonstuarti 1, A. simonstuarti 2 and Adenomera sp. nov. Genetic p-distance between the new species and A. simonstuarti sensu stricto averages 5.2% (4.3—5.9%). zse.pensoft.net 238 Martins, B. da C. et al.: New Adenomera from white-sand forests of Brazilian Amazonia Outgroup Adenomera lutzi Clade ——_8___ | Adenomera heyeri Clade Adenomera marmorata Clade = ——= Adenomera thomei Clade 0 Adenomera martinezi Clade ey | Adenomera hylaedactyla Clade Adenomera sp. T MHNC8385 100 Adenomera guarayo AM29 00 » Adenomera guarayo MNCN34687 'Adenomera guarayo USNM268935 100 Adenomera andreae LSU17479 bi Adenomera andreae MTR18580 Adenomera andreae PV2060 Adenomera sp. D ZSM751 Adenomera chicomendesi AA9972 afeh wired Adenomera chicomendesi MNCN4004__ Adenomera simonstuarti 8 APL13097 Adenomera simonstuarti 7 MNCN27344 Adenomera simonstuarti 6 MHNC10058 97 A V oF Adenomera simonstuarti 5 CB5696 a 98 | Adenomera simonstuarti 4 LSU12840 Oo Adenomers simonstuarti 4 MHNC6302 O 94 ,Adenomera simonstuarti SS ZSM748 % ‘e’adenomera simonstuarti SS MNCN23203 > 4 = Adenomera simonstuarti SS INPAH40967 9% Adenomera simonstuarti 1 LSU13787 oe EAdenomera simonstuarti 1 MHNC10092 ©} Adenomera simonstuarti 1 AJC2777 © 95 Adenomera simonstuarti 2 QU5337 O | Adenomera simonstuarti 2 INPAH39792 © AdeHavnera simonstuarti 2 INPAH39814 O Adenomera sp. nov. INPAH44871 *& Adenomera sp. nov. INPAH44868 * Adenomera sp. nov. INPAH44873 * Adenomera sp. nov. INPAH44867 wy 94 100 0.04 I 1 1 I I { I 1 I I I I 1 I I I I I I I I I 74 I I I I I I I I I I I I I I I I I I I I I I x9|dwoo salseds IL/eNJSUOLUIS BJOLIOUBPYY SS ee ee ee ee eee apelo saloeds aeaspue BJeLUOUapY Figure 2. Phylogenetic relationships of the Adenomera andreae species clade with a focus on the A. simonstuarti species complex. Maximum Likelihood values are inferred from sequence data for Cytb, CO], RAGI and POMC genes. Lineage numbering within A. simonstuarti species complex follows Carvalho et al. (2020b), except for A. simonstuarti sensu stricto (SS). Species names are followed by the corresponding museum voucher numbers. Symbols are as in Fig. 1. Morphometric analysis significantly different (Pillai = 0.286, df = 32, p = 0.004) and do not overlap (Fig. 3). The three morphometric ra- The first two principal components (PCs) of morphomet- _ tios that contribute most of the variation along PCI are ric PCA explained ~ 53% of data variance. Spaces occu- END/SVL, ED/SVL and DSL/SVL. See Table 2 for data pied by Adenomera sp. nov. and A. simonstuarti SS are regarding other PCA variables. zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 233-253 239 Table 1. Average pairwise genetic distances (%) between lineages of the Adenomera simonstuarti species complex and related species of the A. andreae clade. Interspecific uncorrected p-distances (lower diagonal) and Kimura 2-parameter distances (upper di- agonal) are based on a fragment of the COI gene. Intraspecific p-distances are shown along the diagonal in bold. See Supplementary file 4 for minimum and maximum values. Species 1 2 3 a 5 1 Adenomera sp. nov.(n=4) 0.3 40 30 55 £57 2 A. simonstuarti 1 (n = 3) 39) 3241 a bf. ‘6k 3 A. simonstuarti 2 (n = 3) 29. 3:6" “Oiae 355 = 0 4 A. simonstuarti SS (n = 3) bie Coe Bis 0B 5 5 A. simonstuarti 4 (n = 2) Beals 2b, E65 og bn, DOO 6 A. simonstuarti 5 (n = 1) 49 49 563 48 3:9 7 A. simonstuarti 6 (n = 1) Bie “G65 §5.64 Fize =40 8 A. simonstuarti 7 (n = 1) 5A ade 662. 20 5A 9 A. simonstuarti 8 (n = 1) bot 16477 “20. 26:9" 22 10 A. andreae (n = 3) 12:35 E75 EA = 22 29 11 A. chicomendesi (n = 2) 124 12:3 28. tS) 12.5 12 A. guarayo (n = 3) 15:2, 14.6- 14-9. 146~ +1379 13 Adenomera sp. D (n = 1) 12:4. 12:6: 225. V2 12:8 14 Adenomera sp. T (n = 1) 140° 15.7~ 15a. 15:6° 4:9 A Adenomera sp. nov. @ Adenomera simonstuarti -3 -20 = 0 1 2 c) PC 1 (35.4%) Figure 3. Morphometric Principal Component Analysis. Analyses were based on 15 morphometric ratios of 21 males of Adenomera sp. nov. and 14 males of A. simonstuarti sensu stricto. Ellipse rep- resents the standard error with 95% confidence interval. Table 2. Loadings of 15 morphometric and 9 bioacoustic ratios on the respective first principal components. Values were gener- ated from data for 21 males of Adenomera sp. nov. and 14 males of A. simonstuarti sensu stricto. Variables PC 1 PC 2 HL/SVL 0.480 0.697 HW/SVL 0.378 OEFET SL/SVL O.732 -0.325 END/SVL 0.845 -0.355 IND/SVL 0.348 0.168 ED/SVL 0.743 -0.112 IOD/SVL 0.720 0.145 TD/SVL 0.199 -0.420 FAL/SVL 0.571 -0,152 UAL/SVL -0.174 -0.046 HAL/SVL 0.097 0.009 TL/SVL 0.099 -0.118 FL/SVL -0.041 O72 THL/SVL -0.165 -0.051 TSL/SVL 0.448 -0.215 6 7 8 9 10 11 12 13 14 Sale 45.5" Fae “le 1376" 15.0) chy ALO e169 52 67 46 $7.1 #140 144 167 142 18.1 56 61 #55 75 146 144 17.1 141 17.3 50 54 53 74 136 148 16.7 140 18.0 Ad “29° Ofe Ve Tab” 14:06 T5747) 14:55 V1 NA 622. 9:9" '2:3. “la, o.l- 16:6 J13.5° [733 6.2 NA 61 69 141 144 17.6 14.7 17.6 36. qo.0!) INAD 6.7 815500147" 16:6) 3:0: “19-5 69 65 64 NA 13.0 126 165 141 17.3 12°38 126 TSF 11a e6:6- 1:3 TOM E28" 16.) 133. 126 913.0 1A 8S 4:90 “12s 10.35 “19.4 14:5. 15.2 laos 14.98 14:9" 16.6. 2:7. 1719) 8:8 12-0...12:9° “ISAC “I2oF. LI 9A S156, wNA 19 Pols tors. 16:8 lol) Aa} WG? Bute pry “NA Taxonomic account Order Anura Fischer von Waldhein, 1813 Family Leptodactylidae Werner, 1896 Subfamily Leptodactylinae Werner, 1896 Genus Adenomera Steindachner, 1867 Adenomera albarena sp. nov. https://zoobank.org/OFAB2CE9-329 1-453D-8C58-E092BD288DCA Tables 3, 4, Figs 4-7, 9B—D Chresonymy. Adenomera gr. heyeri (Lima et al. 2021). Type material. Holotype. INPA-H 44867, an adult male collected at km 26 of the AM-352 highway, Rio Negro Sustainable Development Reserve (03°05'35"S, 60°40'36" W: 76 m a.s.l.), Municipality of Iranduba, State of Amazonas, Brazil, on 11 December 2020 by M. Fer- rao, A. P. Lima and W. E. Magnusson. Paratypes. Twenty-four adults collected at the same locality as the holotype; eight males MPEG 44649, IN- PA-H 44868—73 and ZUEC-AMP 25694 collected on 11 December 2020 by M. Ferrao, A. P. Lima and W. E. Magnusson; four females INPA-H 44874—75, ZU- EC-AMP 25695 and MPEG 44650 collected on 10 De- cember 2021 by M. Ferrao, A. P. Lima and B. Martins; four males INPA-H 44876—77 and MPEG 44651-52 collected on 11 December 2021 by M. Ferrao, A. P. Lima and B. Martins; four males INPA-H 44878-—80 and ZUEC-AMP 25696 collected on 12 December 2021 by M. Ferrao, A. P. Lima and B. Martins; a male INPA-H 44881 collected on 19 January 2022 by B. Martins; a male INPA-H 44882 collected on 3 February 2022 by B. Martins; a male ZUEC-AMP 25697 and a female IN- PA-H 44883 collected on 14 May 2022 by B. Martins. One adult male INPA-H 44885, collected at km 50 of the AM-352 Highway, Rio Negro Sustainable Devel- opment Reserve (2°50'10.0"S, 60°50'20.0"W), Munic- ipality of Iranduba, State of Amazonas, Brazil, on 12 January 2023 by B. Martins. zse.pensoft.net 240 Table 3. Morphometric measurements of the type series of Adenomera albarena (Rio Negro Sustainable Development Reserve, Iranduba, Amazonas, Brazil) and A. simonstuarti sen- su stricto (Tarauaca, Acre, Brazil). Values depict average, stan- dard deviation and range. Abbreviation: SS, sensu stricto. Trait acronyms are explained in the text. * Holotype included. Adenomera albarena Adenomera simonstuarti SS Trait Holotype Males Females Males Females (n=21)* (n=5) (n = 14) (n = 2) SVL 22.9 21.9+0.5 23.740.9 249+0.7 244+2.0 (21.2-23.0) (22.1-24.3) (23.9-26.4) (23.0-25.8) HL 8.4 7.9+0.3 81404 89203 8520.5 (7.4-8.4) (7.4-8.5) (8.2-9.4) — (8.1-8.8) HW 8-5 8.1+0.3 842406 9.2+03 9.2+0.6 (7.6-8.7) (7.6-9) (8.7-9.7) (8.79.6) SL 3.8 3.54+0.2 34403 3.8402 3.4+0.5 (3.3-3.9) (2.9-3.8) (3.5-4.0) = (3.1-3.8) EN 2.0 2.0+0.1 2340.2 20+01 2.0+04 (1.9-2.2) (2.0-2.5) (2.0-2.2) = (1.8-2.3) IND 2.4 2.3+0.1 2.3401 26401 25201 (2.0-2.4) (2.1-2.5) 9 (2.5-2.7) — (2.42.5) ED 2.4 2.5+0.1 2640.2 23402 2.3+01 (2.2-2.7) (2.3-2.8) (2.1-2.6) = (2.22.4) lOD 54 54+02 56+04 582+0.2 56+0.2 (5.0-5.8) (4.9-5.9) (5.6-6.3) (5.4-5.7) TD ie 14+01 14+01 152401 15+01 (1.2-1.5) 9 (1.2-1.5) = (1.3-1.8) — (1.5-1.6) FAL A} 45+0.3 51405 50203 5140.3 (4.0-5.0) (4.7-6) (4.7-5.8) (5.1-5.6) UAL 4.4 41+04 46405 50203 5340.3 (3.1-4.8) (41-5.4) (4.5-5.5) = (5.1-5.6) HAL 4.9 4540.2 47402 52+02 51+04 (4.0-4.9) (4.5-4.9) (4.8-5.6) (4.8-5.3) TL 9.5 97+0.5 10.9+0.2 11.1406 11.4+04 (8.9-10.8) (10.5-11.1) (10.0-12.1) (11.1-11.6) FL 10.5 100+04 10.7+03 11.5+05 116+0.6 (9.5-10.7) (10.5-11.2) (10.5-12.2) (11.2-12.0) THL 9.4 92+0.3 97408 108+0.7 11.140. (8.9-10) (8.4-10.2) (9.8-11.8) (10.6-11.5) TSL 5:5 56+0.3 6140.2 62+04 62+0.5 (5.0-6.2) (5.9-6.5) (5.5-6.8) — (5.8-6.5) Martins, B. da C. et al.: New Adenomera from white-sand forests of Brazilian Amazonia Etymology. The specific epithet a/barena is formed by the combination of two Latin words: “alba” (white) and “arena” (sand). This is a reference to the white-sand forests of central Amazonia, the distinctive environment inhabited by this species. Vernacular names. White-sand terrestrial foam-nest- ing frog (English), rana terrestre de arena blanca (Span- ish) and razinha da areia branca (Portuguese). Diagnosis. The species Adenomera albarena is rec- ognised by the following combination of characters. (1) Medium size (adult male SVL = 21.2—23.0 mm, n = 21; adult female SVL 22.1—24.3, n = 5); (2) snout of males subovoid in dorsal view and acuminate in lateral view; (3) absence of antebrachial tubercle; (4) toe tips moder- ately to fully expanded (character states C, D sensu Hey- er (1973)); (5) throat in males with condensed melano- phores near the jaw and scattered melanophores on the central portion; 6) nearly solid dark-coloured stripe pres- ent on the underside of the forearm; (7) Advertisement call composed of a single pulsed note; (8) notes formed by 11-21 pulses; (9) pulses incomplete; (10) dominant frequency 3,448-4,349 Hz; (11) dominant frequency co- inciding with the second harmonic; (12) Endotrophic tad- poles; (13) with labial teeth absent; (13) spiracle present; and (14) internarial distance 44-52% of IOD. Interspecific comparisons. Adenomera albarena ditf- fers from all congeners, except A. simonstuarti by having a nearly solid dark-coloured stripe on the underside of the forearm (Heyer 1973, 1975; Kwet and Angulo 2002; Almeida and Angulo 2006; Kok et al. 2007; Kwet 2007; Angulo and Reichle 2008; Berneck et al. 2008; Kwet et al. 2009; Angulo and Icochea 2010; Carvalho and Gia- retta 2013a, 2013b; Carvalho et al. 2019a, 2019b, 2019c, 2019d, 2020a, 2020c, 2021; Cassini et al. 2020; Zaracho et al. 2023). Amongst Amazonian congeners, adult male Ade- nomera albarena have SVL 21.2—23.0 mm, which is smaller than A. glauciae (SVL 27.6—30.4; Carvalho et al. (2020b)), A. gridipappi (SVL 25.4—27.7 mm; Carvalho et Table 4. Spectral and temporal parameters of the advertisement calls of Adenomera albarena and A. simonstuarti sensu stricto. Values depict average, standard deviation and range. Symbols: *, same values of call duration because the call is composed of only one note; **, measured by Carvalho et al. (2020b) from calls at the type locality in Cusco, Peru and Tarauaca, Brazil; ***, dominant frequency correspond to the first harmonic in A. simonstuarti and to the second harmonic in the new species. Abbreviation: SS, sensu stricto. Trait acronyms are described in the main text. Call traits Adenomera albarena (n = 6) A. simonstuarti SS (n = 6) A. simonstuarti SS (n = 2) ** CD (ms) 142 + 19.0 (100-199), n = 148 4,700 + 1,400 (1,800-7,000), n = 93 800-6,500 ND (ms) 142 + 19.0 (100-199), n = 148* 64 + 10 (40-93), n = 93 57-79 NpC 1 + 0 (1-1), n= 148 22.4 + 6.5 (9-33), n = 93 4-30 NrT (%) 28.4 + 19.9 (2-73), n = 90 50 + 14 (16-76), n= 93 13-73 NrR 0.8 + 0.15 (0.6-1.2), n = 30 4.8 + 0.3 (4.3-5.4), n = 30 4.6 + 0.1 (4.5-4.9) PpN 14.8 + 1.9 (11-21), n = 148 3.4 + 0.7 (2-6), n = 93 2-3 PD (ms) 10 + 3.3 (4-23), n = 444 26.4 + 6.4 (10-53), n= 192 10-53 PrR 107 + 9.7 (94-138), n = 60 53 + 8.7 (37-78), n = 30 - FF (Hz) 1,986 + 0.1 (1,765-2,239), n = 148*** 3,987 + 0.16 (3,617-4,263), n = 93*** 3596-4156*** DF (Hz) 3,899 + 1.3 (3,448-4,349), n = 148*** 1,991 + 0.05 (1,851-2,224), n = 93*** 1,873-2,046*** FM (Hz) 273.8 + 238.1 (-173-861), n = 90 261.7 + 119.7 (-173-517), n = 93 43-301 zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 233-253 al. (2021)), A. /utzi (SVL 25.7-—33.5 mm; Kok et al. (2007)) and A. simonstuarti (SVL 23.4—26.2 mm; Angulo and Icochea (2010); Carvalho et al. (2020b); present study), but larger than A. kayapo (SVL 17.5—21.0 mm; Carvalho et al. (2021)). Adenomera albarena has a snout round- ed in dorsal view, which differs from A. martinezi (snout pointed in dorsal view; Carvalho & Giaretta (2013b)). The absence of an antebrachial tubercle distinguishes A. albarena from A. amicorum, A. aurantiaca, A. cotu- ba, A. glauciae, A. gridipappi, A. inopinata, A. kayapo, A. lutzi, A. tapajonica and A. phonotriccus (antebrachial tubercle present; Kok et al. (2007); Carvalho and Giaretta (2013b), Carvalho et al. (2019b, 2020c, 2021)). Adeno- mera albarena has toe tips moderately to fully expand- ed (states C and D, sensu Heyer (1973)), which differs from A. cotuba, A. hylaedactyla and A. martinezi (toe tips pointed to poorly expanded, states A and B, respectively; Carvalho et al. (2019c); Carvalho and Giaretta (2013a, 2013b)). Adenomera albarena differs from A. heyeri by having white ventral colouration (yellow colouration; Carvalho et al. (2021)). Although A. albarena differs morphologically from A. andreae, A. chicomendesi and A. guarayo only by having a nearly solid dark-coloured stripe on the underside of the forearm (absence in A. an- dreae, A. chicomendesi and A. guarayo; Carvalho et al. (2019a, 2019b, 2019c)), it also differs acoustically from these species (see below). The advertisement call of Adenomera albarena 1s composed of incomplete pulses, which differs from A. aurantiaca, A. guarayo, A. inopinata and A. phono- triccus (complete pulses; Carvalho et al. (2019b, 2020a, 2021)). Amongst those with incomplete pulses, calls of A. albarena sp. nov. differ from A. amicorum, A. glauci- ae, A. gridipappi and A. simonstuarti sensu stricto by hav- ing a single note (multi-note calls; Angulo and Icochea (2010); Carvalho et al. (2020b, 2021)). Adenomera al- barena has calls composed of 11—21 pulses, which differ from A. amicorum (4-10 pulses; Carvalho et al. (2021)), A. andreae (3-10 pulses; Carvalho et al. (2019c)), A. chicomendesi (22-35 pulses; Carvalho et al. (2019a)), A. gridipappi (2-4 pulses; Carvalho et al. (2021)), A. hey- eri (4-12 pulses; Carvalho et al. (2021)), A. hylaedactyla (4-10 pulses; Carvalho et al. (2019c)), A. tapajonica (3-5 pulses; Carvalho et al. (2021)), A. glauciae (unpulsed; Carvalho et al. (2020b)) and A. simonstuarti sensu stric- to (2-6 pulses; this study). Adenomera albarena has a dominant frequency of 3,448-4,349 Hz, which differs from A. kayapo (4,570-4,990 Hz; Carvalho et al. (2021)) and A. simonstuarti sensu stricto (1,851—2,224 Hz; this study). The dominant frequency of Adenomera albarena Sp. nov. is placed in the second harmonic, which differs from A. simonstuarti sensu stricto (dominant frequency in the fundamental harmonic). Lack of labial teeth distinguishes tadpoles of Adeno- mera albarena from exotrophic tadpoles of A. guarani, A. saci and A. thomei (present in all mentioned species; De la Riva (1995); Almeida and Angulo (2006); Carvalho 241 and Giaretta (2013a)). Endotrophic tadpoles of A. albare- na differ from those of A. hylaedactyla and A. andreae by the presence of a spiracle (absent in all mentioned spe- cies; Menin et al. (2009); Menin and Rodrigues (2013)); from A. marmorata by an internarial distance 44-52% of IOD (IND/IOD = 74%; Heyer et al. (1990)). Description of the holotype. Adult male (Figs 4A, B, C, 5A, C). Dorsal skin glandular, warty on flank. Dor- solateral fold indistinct. Sacral region, dorsal surface of tibia and posterior surface of tarsus with white-tipped tubercles. Vertebral stripe in sacral region. Throat, bel- ly and ventral surface of limbs smooth. Pair of lumbar glands. Posterior surface of thigh with a pair of paracloa- cal glands. Snout subovoid in dorsal view and acuminate in profile. Nostril closer to the snout tip than to the eye and orientated dorsolaterally; fleshy ridge on the snout tip. Eye nostril distance 83% of eye diameter, eye diam- eter equals internarial distance. Head wider than long. Internarial distance > 25% of head width. Canthus ros- tralis defined; loreal region slightly concave. Triangular interorbital blotch. Tympanum distinct, nearly 60% of eye diameter; black-coloured supratympanic fold well developed, extending from posterior corner of eye to base of arm. Postcommissural gland ovoid. Subgular vo- cal sac; vocal slits present. Vomerine teeth in two straight rows posterior to choanae and arranged in transverse se- ries parallel to choanae. Tongue lanceolate (sensu Du- ellman (1970)) and free behind. Relative finger lengths IV