**1. Introduction**

The eastern slopes of the Peruvian Andes are one of the regions with the greatest diversity of flora and fauna [1]. During the last few years, many species of plants and animals from the Peruvian Andes have been discovered (e.g., [2,3]). In particular, researchers have discovered many species of lizards of the family Gymnophthalmidae in poorly explored regions [4–9]. Recent phylogenetic inferences using molecular sequences have uncovered the phylogenetic relationships of many gymnophthalmid taxa [10–13]. However, there are still species whose phylogenetic position remains unclear, such as some populations currently assigned to the genus *Cercosaura* Wagler, 1830 and *Pholidobolus anomalus* Müller, 1923. Furthermore, the existence of poorly explored areas in the Peruvian Andes suggests that knowledge of the species richness of this group is still incomplete.

The genus *Cercosaura* is composed of 16 species that are widely distributed from the Andes to the Amazon [13–17]. The taxonomy of this lineage has long been confusing, but recent phylogenetic studies based on morphological and molecular data [11,15,18–20] have improved our knowledge of the composition and phylogenetic relationships of *Cercosaura*. On the basis of a morphological phylogenetic hypothesis, Doan [18] redefined the genus *Cercosaura* and synonymized it with the genera *Pantodactylus* and *Prionodactylus*. This arrangemen<sup>t</sup> was subsequently corroborated by molecular studies [19,20]. Later, Doan and Lamar [17] and Echevarría et al. [16] assigned two more gymnophthalmid taxa to the genus *Cercosaura,* increasing its richness to 14 species. Finally, Sturaro et al. [15] used integrative taxonomy to describe a new species of *Cercosaura*, and to resurrect *C. olivacea*. Despite these advances, the position of some species and populations within *Cercosaura*, such as *C. manicata boliviana*, remained unresolved.

*Pholidobolus anomalus* was described by Müller [21] from a single male specimen collected in the Department of Cusco, southeastern Peru. The holotype was deposited in the herpetological collection of the Zoologische Staatssammlung München, Germany (ZSM). Bombings during the Second World War damaged the ZSM collection, causing the loss and destruction of many type specimens, including the holotype of *Pholidobolus anomalus* [22]. Müller [21] considered the presence of a pair of small prefrontal scales in an almost rudimentary state as an outstanding character for this species. Due to this character, Müller [21] avoided assigning *P. anomalus* to the genus *Placosoma*. Instead, on the basis of similarities in pholidosis, he assigned the species to the genus *Pholidobolus* [21]. In some gymnophthalmid species, the condition of the prefrontal scales is variable at the intraspecific level [23,24]. Since its description in 1923, *Pholidobolus anomalus* has been reported once in the Department of Cusco [25]. Montanucci [25] analyzed two specimens of *P. anomalus* deposited by Thomas H. Fritts in the herpetological collection at the University of Kansas (KU 134857–58), both collected in the montane forests of Machupicchu (Cusco). On the basis of his morphological observations, Montanucci [25] concluded that *P. anomalus* was erroneously assigned to *Pholidobolus*. Generic reallocation of *P. anomalus* has since been hypothesized by several authors [25,26], but to date no taxonomic change has been proposed.

The molecular phylogenies carried out so far for the genus *Pholidobolus* included almost all species of the genus (*Pholidobolus <sup>a</sup>*ffi*nis*, *P. condor*, *P. dicrus*, *P. dolichoderes*, *P. hillisi*, *P. macbrydei*, *P. montium*, *P. paramuno*, *P. prefrontalis*, *P. samek*, *P. ulisesi*, and *P. vertebralis*), but not *P. anomalus* because biological material was unavailable [11,12,27,28]. Previous authors highlighted the need to obtain new material of this taxon to reassess its taxonomic status and to examine its phylogenetic relationships [11,12,27,29].

In this study, we investigated the phylogenetic relationships of two specimens of *P. anomalus*, two specimens of *Cercosaura manicata boliviana*, and a specimen of *Cercosaura* that we identified as a new species. We analyzed four mitochondrial genes (12S, 16S, ND4, Cytb), and one nuclear gene (c-mos) using maximum likelihood inference. As a result of these analyses, and after examination of external morphology of the material assigned to *P. anomalus*, we reassign *P. anomalus* to the genus *Cercosaura,* provide a new description, and designate a neotype. Additionally, we describe the new species of the genus *Cercosaura*, and comment on the taxonomic status of *Cercosaura manicata boliviana* from southern Peru.

## **2. Materials and Methods**

## *2.1. Biological Material and Taxon Sampling*

We analyzed specimens of *Cercosaura* and *Pholidobolus anomalus* from central and southern Peru deposited in the Centro de Ornitología y Biodiversidad, Lima (CORBIDI), Museo de Biodiversidad del Per ú, Cusco (MUBI), and Museo de Historia Natural de la Universidad Nacional de San Agustín, Arequipa (MUSA) (Table 1, Appendix A). We examined eight specimens of *P. anomalus* from Cusco, two specimens of *Cercosaura manicata boliviana* from Cusco and Puno, and two specimens of *Cercosaura pacha* sp. nov. from Oxapampa (Figure 1).

**Figure 1.** Geographic distribution of species of the genus *Cercosaura* in the Cordillera de los Andes. Orange triangles = "*Cercosaura manicata boliviana*"; light-blue circle = *C. manicata*; purple circle = *C. hypnoides*; blue circle = *C. doanae*; green square = *C. anomala*; red star = *C. pacha* sp. nov.; yellow circle = *Cercosaura* sp. Data taken from the literature [16,17,30] and museum records.


**Table 1.** Localities (all in Peru), coordinates, voucher numbers, and GenBank accession codes of newly sequenced specimens included in this study. (\* described herein)

## *2.2. DNA Extraction, Amplification, and Sequencing*

We obtained DNA sequences from five voucher specimens (Table 1). We extracted DNA from muscle tissues preserved in ethanol 96% using a commercial kit (Catalog #B47282, IBI Scientific). We obtained fragments of the nuclear oocyte maturation factor gene (c-mos), and the four mitochondrial genes: small subunit rRNA (12S), large subunit rRNA (16S), NADH dehydrogenase subunit 4 (ND4), and protein-coding cytochrome b (Cytb). We used standard primer and protocols for the polymerase chain reaction (PCR) (Table 2). We purified PCR products with Exosap-IT (Affymetrix, Santa Clara, CA, USA), and shipped purified products to MCLAB (San Francisco, CA, USA) for sequencing in both directions. We deposited new sequences in GenBank (Table 1). Additionally, we obtained 476 sequences of GenBank (https://www.ncbi.nlm.nih.gov/genbank/) of 124 terminals: 12S (124 sequences), 16S (123 sequences), ND4 (101 sequences), Cytb (19 sequences), and c-mos (109 sequences) (Table S1). We choose outgroups according to Moravec et al. [6].

**Table 2.** List of primers used in this study.


## *2.3. Phylogenetic Reconstruction and Genetic Distances*

We aligned the sequences of each fragment independently in MUSCLE [36], implemented in MEGA-X [37]. We concatenated sequences of the five fragments using Mesquite V3.61 [38].

Three phylogenetic analyses were conducted by maximum likelihood (ML) for mitochondrial genes (12S, 16S, Cytb, ND4), a nuclear gene (c-mos), and combined data (mitochondrial + nuclear). We inferred the optimal partition scheme using PartitionFinder 2.1.1 under the Bayesian information criterion (BIC) [39]. The best scheme were nine partitions (12S, 16S, c-mos-pos1 and cmos-pos2, c-mos-pos3, Cytb-pos1, Cytb-pos2, Cytb-pos3 and ND4-pos3, ND4-pos1, and ND4-pos2), and the evolution models were GTR+I+G for 12S, 16S, and ND4-pos1, TIM+G for c-mos-pos1 and c-mos-pos2,

K81UF+G for c-mos-pos3, SYM+I+G for Cytb-pos1, HKY for Cytb-pos2, TRN+G for Cytb-pos3 and ND4-pos3, and TVM+I+G for ND4-pos2. We inferred a phylogenetic tree using IQTREE 2 [40] and branch supports was estimated from 1000 pseudoreplicates using the ultrafast Bootstrap approach [41]. We uses *Alopoglossus viridiceps*, *Bachia flavescens*, *Ecpleopus gaudichaudii*, *Gymnophthalmus leucomystax*, *Rhachisaurus brachylepis* as outgroup taxa [6].

We estimated genetic distances between species (uncorrected p-distances) for 16S, which is the gene most commonly sequenced gene in gymnophthalmid lizards [6], and separately for 12S, ND4, and c-mos genes using Molecular Evolutionary Genetics Analysis (MEGA-X) [36] (Tables 3 and 4). Because the Cytb gene is poorly sampled in gymnophthalmid lizards, and available for only three species of *Cercosaura*, we omitted p-distances for this gene.


15. *Cercosaura manicata boliviana* CORBIDI 18716 0.023 0.052 0.042 0.026 0.039 0.029 0.029 0.049 0.052 0.042 0.055 0.042 0.042 0.02

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## *2.4. Designation of Neotype and Species Descriptions*

In the original description of *Pholidobolus anomalus*, Müller [21] was not very precise about the type locality, and mentioned Cusco as the collecting locality. However, the most probable place where the holotype was collected is the Historical Sanctuary of Machupicchu (HSM), in the Cordillera de Vilcabamba. After its discovery by Hiram Bingham in 1911, the HSM became very popular with naturalists. The resulting scientific collections were deposited in di fferent natural history museums [42,43].

We designed a neotype of *Pholidobolus anomalus* because the holotype was lost. This designation is covered by Article 75.3 of the International Code of Zoological Nomenclature (ICZN) [44]. We used the neotype to redescribe the species, and support the generic allocation. We followed Uzzell [45], Kizirian [46], and Doan and Cusi [24] for character definitions and measurements, and Chávez et al. [7] for description format. One of us (LM) observed morphological characters of species and took all measurements using a caliper with a precision of 0.1 mm. We referred to the literature for patterns of scalation and coloration of the following taxa: *Cercosaura anordosquama, C. ocellata, C. bassleri* and *C. olivacea* [13]; *Cercosaura argulus*, *C. eigenmanni*, and *C. oshaughnessyi* [47]; *C. hypnoides* [17]; *C. quadrilineata, C. schreibersii,* and *C. phelpsorum* [18]; *C. doanae* [16]; *C. manicata* [30]; *C. nigroventris* [48]; *C. parkeri* [49]; and *C. steyeri* [50]. We also examined specimens of *Cercosaura* deposited in the CORBIDI, MUBI, and MUSA collections (Appendix A).

This research was approved by the Institutional Animal Care and Use Committee of Southern Illinois University Carbondale (protocol #16-006). The Dirección General Forestal y de Fauna Silvestre, Ministerio de Agricultura y Riego, and Servicio Nacional Forestal y de Fauna Silvestre issued the permit authorizing this research (permits #210-2013-MINAGRI- DGFFS/DGEFFS, 064- 2013-AG-DGFFS-DGEFFS, 359-2013-MINAGRI-DGFFS- DGEFFS, 292-2014-MINAGRI-DGFFS-DGEFFS, 024–2017–SERFOR/DGGSPFFS, and 369–2019–MINAGRI-SERFOR-DGGSPFFS).

The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature, and hence the new names contained in the electronic version are e ffectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The Life Sciences Identifier (LSID) for this publication is: urn:lsid:zoobank.org:pub:FF0EC17F-965E-410D-AF0A-356B19BD4431.
