70 (3): 357 – 366 © Senckenberg Gesellschaft für Naturforschung, 2020. 2020 Molecular phylogenetic relationships and generic placement of Dryaderces inframaculata Boulenger, 1882 (Anura: Hylidae) Diego A. Ortiz 1, 4, *, Leandro J.C.L. Moraes 2, 3, *, Dante Pavan 3 & Fernanda P. Werneck 2 1 College of Science and Engineering, James Cook University, Townsville, Australia — 2 Coordenação de Biodiversidade, Programa de Coleções Científicas Biológicas, Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, AM, Brazil — 3 Ecosfera Consultoria e Pes- quisa em Meio Ambiente Ltda., São Paulo, SP, Brazil — 4 Corresponding author; email: [email protected] — * These authors con- tributed equally to this work Submitted January 4, 2020. Accepted July 9, 2020. Published online at www.senckenberg.de/vertebrate-zoology on July 31, 2020. Published in print Q3/2020. Editor in charge: Raffael Ernst Abstract Dryaderces inframaculata Boulenger, 1882, is a rare species known only from a few specimens and localities in the southeastern Amazonia rainforest. It was originally described in the genus Hyla, after ~ 130 years transferred to Osteocephalus, and more recently to Dryaderces. These taxonomic changes were based solely on the similarity of morphological characters. Herein, we investigate the phylogenetic re­ lationships and generic placement of D. inframaculata using molecular data from a collected specimen from the middle Tapajós River region, state of Pará, Brazil. Two mitochondrial DNA fragments (16S and COI) were assessed among representative species in the sub­ family Lophiohylinae (Anura: Hylidae) to reconstruct phylogenetic trees under Bayesian and Maximum Likelihood criteria. Our results corroborate the monophyly of Dryaderces and the generic placement of D. inframaculata with high support. Dryaderces inframaculata is sister to an undescribed taxon, Dryaderces pearsoni Ca1 Jungfer et al., 2013, and both are sister to Dryaderces pearsoni Gaige, 1929. These fndings are relevant for further research on the systematics and biogeography of the genus. Key words Amazonia, integrative taxonomy, Lophiohylinae, mtDNA, phylogeny, rare species, Santarém tree frog, Tapajós River. Introduction Tree frogs of the Amazonian genera Dryaderces Jung­ cies boundaries, described new taxa, and transferred taxa fer et al., 2013, Osteocephalus Steindachner, 1862, and among genera and among species groups within Osteo­ Tepuihyla Ayarzagüena, Señaris & Gorzula, 1993 (Lo­ cephalus (MORAVEC et al., 2009; JUNGFER, 2010; RON et phio hylinae) are among the most speciose Neotropical al., 2010, 2012, 2016; JUNGFER et al., 2013, 2016; FER­ hylids. These genera are often taxonomically problematic RÃO et al., 2019). Such increasing knowledge has helped as they contain high levels of cryptic diversity, cases of to clarify the diversity and evolutionary history of these synonymy and resurrection of names, old descriptions genera; however, some taxa remain absent from molecu­ of species, and type series with low representation of the lar phylogenies. species’ variation and distribution (TRUEB & DUELLMAN, In a vast systematic revision using molecular data, 1971; DUELLMAN & MENDELSON, 1995; JUNGFER, 2010; JUNGFER et al. (2013) noted some distinct and early diver­ JUNGFER et al., 2013; FERRÃO et al., 2019). Using integra­ gent lineages in the genus Osteocephalus, and allocated tive approaches, recent studies have re­evaluated the spe­ them in a new genus, Dryaderces. Currently, Dryaderces ISSN 1864-5755 | eISSN 2625-8498 | DOI: 10.26049/VZ70-3-2020-08 357 Ortiz, D. A. et al.: Molecular phylogenetic position of Dryaderces inframaculata Fig. 1. Map of south­central Amazonia with a hydrographic and elevational background showing the known localities of Dryaderces. Data compiled from literature records (JUNGFER et al., 2013; MAFFEI et al., 2018; HOOGMOED, 2019) and material examined at INPA­H collection (Appendix). The black­dotted red symbol represents the locality of the newly collected D. inframaculata specimen (INPA­H 41311 with molecular data). Brazilian state abbreviations are: AM (Amazonas), MT (Mato Grosso), PA (Pará), and RO (Rondônia). The type locality of D. inframaculata is Santarém, PA (northern­most symbol). is considered the sister taxon of Osteocephalus (DUELL­ (as Hyla inframaculata). JUNGFER (2010) reviewed the MAN et al., 2016; BLOTTO et al., 2020); and contains two holotype, reported new morphological information, described species (D. pearsoni Gaige, 1929, and D. in­ and transferred it to Osteocephalus (as Osteocephalus framaculata Boulenger, 1882), and an undescribed spe­ inframaculatus), within the Osteocephalus buckleyi cies labelled as D. pearsoni Ca1 (JUNGFER et al., 2013). species group. JUNGFER et al. (2013), in the absence of The Santarém tree frog (Dryaderces inframaculata) DNA sequences of O. inframaculatus, kept the generic is known from primary fooded and non-fooded forests placement suggested by JUNGFER (2010). HOOGMOED of southeastern Amazonia, with confrmed localities in (2013) studied the morphology of the holotype and new the Brazilian states of Pará, Mato Grosso, and Rondônia, specimens, re­diagnosed the taxon, and transferred it to south of the Amazon and east of the Madeira rivers (Fig. Dryaderces based on the morphological similarity with 1; HOOGMOED, 2013; PINTO et al., 2017a; MAFFEI et al., D. pearsoni, the only other described species in this ge­ 2018). Despite its apparent wide geographic distribution, nus. Therefore, the generic placement of D. inframacu­ the species has been rarely collected and for more than a lata has been so far exclusively based on morphological century it was only known from the holotype (JUNGFER, data, and its phylogenetic relationships remain specula­ 2010). After HOOGMOED (2013), new information was re­ tive (BLOttO et al., 2020). ported on its morphological variation and taxonomy, dis­ Based on a newly collected D. inframaculata speci­ tribution range, natural history, and reproduction (PINTO men from the middle Tapajós River region, we present et al., 2017a, b; MAFFEI et al., 2018; HOOGMOED, 2019). here the frst molecular-based study addressing its ge­ Since its formal description in 1882, the absence neric placement and phylogenetic relationships, through of new specimens and data led FAIVOVICH et al. (2005) comparison of mitochondrial DNA sequences among re­ to consider the species incertae sedis within Hylidae lated taxa in Lophiohylinae, Hylidae. 358 VERTEBRATE ZOOLOGY — 70 (3) 2020 Materials and Methods CHmL4, CHE et al., 2012), 0.15 µL of Taq polymerase (1U), and 7.45 μL of ddH2O. Reaction conditions fol­ lowed standard cycling protocols (PALUMBI et al., 1991), Study area and specimen examination with an annealing temperature of 48 ºC and 50 ºC for 16S and COI, respectively. PCR products were purifed with We collected an adult female D. inframaculata (SVL = ExoSAP­IT (USB Corporation) and sequenced using Big 67.9 mm), found perched on a palm leaf in a non-food­ Dye Terminator kit (Applied Biosystems) in an automat­ ed (terra frme) primary forest, on the east bank of the ed sequencer ABI 3130 XL (Applied Biosystems). Con­ middle Tapajós River, municipality of Itaituba, state of sensus sequences were assembled in GENEIOUS 8 (KEARSE Pará, Brazil (4°45′ S, 56°36′ W, 132 m above sea level) et al., 2012) and deposited in GenBank (Table 1). [Fig. 1; see Moraes et al. (2016) for further details on locality]. This locality is close (ca. 97 km southwest) to a recently included locality from the known distribution Phylogenetic analyses range of the species (Itaituba sensu MAFFEI et al., 2018). The new specimen (Figs. 2, 3) was identifed based on We combined our newly generated sequences of D. in­ external morphology according to the known species’ framaculata with available GenBank material of closely variation (JUNGFER 2010; HOOGMOED, 2013; MAFFEI et al., related taxa (Table 1), according to recent molecular sys­ 2018), which we compared to other available specimens tematic reviews of Lophiohylinae (RON et al., 2012, 2016; of Dryaderces deposited at the Amphibians and Rep­ JUNGFER et al., 2013; DUELLMAN et al., 2016). We includ­ tiles Collection of the Instituto Nacional de Pesquisas ed all available homologous sequences of Dryaderces, all da Amazônia (INPA­H), including additional specimens described species with sequences of Osteocephalus and of D. inframaculata collected close to the type locality Tepuihyla (closest related genera), and one species per (Fig. 1; Appendix). The specimen with molecular data genus from other genera within Lophiohylinae (FROST, was deposited in this same collection under the voucher 2020; Table 1). More recently published, molecular number: INPA-H 41311; feld series: DT3584. studies propose the transference of species from Argen­ Main diagnostic characters used for the species iden­ teohyla, Aparasphenodon (most of), and Corythomantis tifcation included: medium body size; snout truncate in (part) to Nyctimantis (BLOTTO et al., 2020), as well as the dorsal view, and rounded, slightly protruding in lateral inclusion of new species in Osteocephalus (FERRÃO et al., view; granular skin over most of body, except on hidden 2019; CHASILUISA et al., 2020). We could not account for surfaces of hind limbs (smooth); outer edge of forearms these modifcations in our analyses; however, they were and tarsus with a row of tubercles; brown dorsum with not relevant for Dryaderces. The root of Lophiohylinae a V­shaped mark extending from upper eyelid to shoul­ was defned using as outgroups: