A New Species of Tiger Pleco Panaqolus (Siluriformes: Loricariidae) from the Xingu Basin, Brazil

Panaqolus tankei is described from the Xingu River, Brazil. The new species is diagnosed from P. albomaculatus, P. dentex, P. nix, P. nocturnus, and P. koko by its color pattern consisting of dark and light diagonal bars on the body and bands on the fins (vs. body and fins without bars or bands); from P. albivermis, P. maccus, and P. purusiensis by the width of the dark bars being more or less the same of the light bars (vs. dark bars at least two or three times wider than light bars) and from P. changae by the absence of vermiculation on the head (vs. vermiculation present on head). The new species differs from P. gnomus by the orientation of the bars from posterodorsal to anteroventral direction (vs. anterodorsal to posteroventral direction), and from P. claustellifer by the orientation of the bands in the dorsal fin that are not parallel to the margin (vs. parallel to the margin). The barcoding region (COI) was sequenced for the new species, sequences were deposited in GenBank and were compared with congeners from other drainages. With regard to the current construction of a hydroelectric power plant (a so-called mega dam) in the Xingu River, herewith we increase knowledge of the river Xingu’s ichthyofauna and, thus improve the assessment of the impacts of that construction on the river.


Introduction
After some debate [1,2,3,4,5,6], Panaqolus Isbrücker & Schraml (2001) is now a well-accepted genus easily separated from Panaque, the former genus of the Panaqolus-species  (Schaefer & Stewart, 1993), and P. purusiensis (Lamonte, 1935). The diversity of the genus is, however, nowhere near fully described. There are at least 10 undescribed species known in the aquarium trade from the main drainages of the Amazon, including Río Ucayali, Rio Negro, Rio Madeira, Rio Tapajós, Rio Curuá-Una, Rio Paru and Rio Xingu, amongst others. These species are still awaiting formal description, mainly due to a lack of specimens deposited in scientific collections.
The Rio Xingu, the fourth largest drainage on the Amazon, has an astonishing diversity of loricariids with at least 60 species from more than 25 genera occurring in its Lower and Middle courses, of which 18 species are endemic and 15 are still undescribed (LMS, pers. obs.; [7]).
At the time of writing, a huge hydroelectric power plant is under construction in the middle reaches of the Rio Xingu, with yet unknown consequences for the often highly specialized local fish fauna. Thus, the aim of the present study is to contribute to the effort of improving our knowledge of these endemic fishes by describing a new species of Panaqolus from the Lower Xingu River.

Morphological analysis
Counts, measurements and terminology follow Lujan et al. [8]. All measurements were made to the nearest 0.1 mm with the use of digital calipers. The following abbreviations were used: SL, standard length; HL, head length; and c&s, cleared and stained. For clearing and double staining we followed the protocol from Tayler & van Dyke [9]. Institutional abbreviations are as listed in Sabaj Pérez [10]. Specimens were collected with LMS' IBAMA permit #31089-1 which is valid in the whole Brazilian territory. Collected fish were first euthanized using clove oil and then fixed in formalin, following the guidelines of the Brazilian Society of Ichthyology [11]. No endangered species were collected. Only fixed museum material was used for the present work, therefore, according to the Brazilian legislation, no approval by an ethics committee was necessary.
The type specimens were publicly deposited in the permanent collections of the following institutions: INPA, Instituto Nacional de Pesquisas da Amazônia, Manaus/AM, Brazil; USNM, Smithsonian Institution National Museum of Natural History, Washington, D.C., United States of America (USA); MNRJ, Museu Nacional da Universidade Federal do Rio de Janeiro, Rio de Janeiro/RJ, Brazil; MPEG, Museu Paraense Emílio Goeldi, Belém/PA, Brazil; ANSP, Academy of Natural Sciences of Drexel University, Philadelphia, USA; MZUSP, Museu de Zoologia da Universidade de São Paulo, São Paulo/SP, Brazil.

DNA sequencing, editing, and alignment
Total DNA was extracted from ethanol-preserved fin samples using DNeasy Tissue Kit (Qiagen), following manufacturer's instructions. Partial sequences of Cytochrome c Oxidase subunit 1 [12] were amplified and sequenced using the protocol and the primers from Roxo et al. [13]. The DNA sequences were edited and aligned using BioEdit 7.0.9 [14]. Nucleotide variation was examined using MEGA 6.0 [15].

Nomenclatural Acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:F416B09C-018D-441F-88F5-9284A1330EF9. The electronic edition of this work was published in a journal with an ISSN and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Diagnosis
Panaqolus tankei can be differentiated from all congeners except P. albivermis, P. changae, P. claustellifer, P. gnomus, P. maccus and P. purusiensis by the presence of dark bars on the trunk and bands on the fins alternating with light interspacing (vs. body and fins without bars or bands). It differs from P. albivermis, P. maccus, and P. purusiensis by the width of the dark bars, which is more or less the same width of the light bars (vs. dark bars at least two or three times wider than light bars) and from P. changae by the absence of vermiculation on the head (vs. vermiculation present on head). The new species differs from P. claustellifer by by the number of dorsal-fin bands (2-4 vs. 2) and their orientation being not parallel to the margin of the fin (vs. parallel to the margin), and from P. gnomus by the orientation of the bars on the body from posterodorsal to anteroventral direction (vs. anterodorsal to posteroventral direction). Panaqolus

Description
Proportional measurements in Table 1. Medium to small-sized loricariid with standard length of measured specimens up to 83.0 mm SL. Dorsal profile of head and snout strongly convex from snout tip to posterior tip of supraoccipital, straight and posteroventrally slanted between dorsal-fin origin and adipose-fin origin, gently concave through caudal peduncle to posterior tip of last procurrent caudal fin ray. Dorsal orbit margin slightly raised, forming a small ridge, narrowing anteriorly, from anterior orbit margin to area lateral to nares. Dorsal surface of trunk transversely flattened from dorsal-fin origin to adipose-fin base. Ventral profile of head and body flat from oral disk to anal-fin origin. Caudal peduncle oval in cross-section and moderately deep (10-12% SL).
Greatest body depth at dorsal-fin origin. Pectoral-fin origin just posterior to orbit, pelvic-fin origin at vertical through origin of second dorsal-fin ray, anal-fin origin shortly posterior after vertical through origin of last dorsal-fin ray. Adipose fin with well-ossified leading spine bearing odontodes.
Dorsal fin II,7, pectoral fin I,6, pelvic fin I,5, anal fin I,3-4 (mode 4), caudal fin I,14-15,I (mode 14 branched rays). Spinelet triangular, dorsal-fin spine lock functional, posterior fin margin straight, margin of last two rays rounded. Dorsal fin origin closer to vertical line passing through pelvic-fin origin than to vertical line passing through pectoral fin origin; not reaching adipose fin when adpressed. Adipose fin triangular; adipose-fin spine slanted posteroventrally, tip straight to curved ventrally, pointed; posterior margin of adipose-fin membrane concave to nearly vertical. Pectoral-fin spine robust, posterior fin margin straight, when adpressed reaching 1/3 of pelvic fin. Pelvic-fin spine robust, posterior margin slightly rounded, when adpressed reaching mid-length of anal-fin spine. Caudal fin forked with short filamentous extensions on rays.
Head and body entirely plated except for small naked area along dorsal-fin base, snout without naked area near tip. Abdomen of adults mainly naked, only triangle formed by the anus and the two pelvic-fin origins covered by small plates. Abdomen of juveniles not plated. Area dorsal to pelvic-fin base below ventral margin of inframedian plate row usually naked. Supraoccipital bordered posteriorly by 2 scutes on each side. Body with pronounced lateral ridge extending from cleithrum to posterior margin of fifth or sixth plate of the inframedian plate row; ridge gradually decreasing in prominence posteriorly. Trunk without elevated ridges. 6-7 (mode 7) scutes on dorsal-fin base, 5-6 (mode 6) scutes between dorsal and adipose fin, 5-7 (mode 6) scutes between adipose and caudal fin, 2 scutes on anal-fin base, 9-12 (mode 10) scutes between anal and caudal fin, 22-25 (mode 24) scutes on lateral line. 28 vertebrae including 5 in the Weberian apparatus and 1 the ural centra.
Head and body covered by odontodes of uniform size and distribution. Enlarged odontodes on anterodorsal border of pectoral-fin spine. Cheek odontodes hypertrophied with tips recurved distally, longest odontode extending to posterior cleithrum margin. Interorbital space straight or slightly convex. Eye moderately large, dorsolaterally placed; orbit diameter 15.8-25.2% HL. Iris diverticulum present. Nares small and ovoid, slightly longer than wide.
Oral disk more or less circular, distal margin of upper lip well separated from maxillary barbel base (Fig 3a). Maxillary barbels of moderate length (7-17% HL), tip of barbel sometimes bior trifurcated. Lips moderately rugose, small patch of elongate fleshy papillae medial to each tooth row. Border of lips smooth. Teeth spoon-shaped with small lateral cusp. Premaxillary teeth 4-7 per ramus (mode 6), mandibular teeth 4-9 per ramus (mode 7). In contrast to Panaque [8], no correlation of SL and number of teeth was found. Premaxillae tooth rows angled at approximately 80°, dentary tooth rows at approximately 60° (Fig 3).  (Figs 4 and 5a). In larger individuals, the first three dark bands on the body in phase of splitting, forming a D (Fig 5b), the second and third band sometimes completely split, resulting in two separate thin bands. Fourth and fifth bands sometimes in form of an inverted Y shape (Fig 5c and 5d). Head with dark lines going from supraoccipital to anterior margin of snout. 3-4 (mode 3) lines under the eye, often being split at the ventral margin of the head, forming an inverted Y shape. One dark line between eye and nare and 2 between the nares. These lines may be partially split, forming an inverted Y, an elongated 0 or a sequence of 0s (see Fig 6). Dark lines on head usually thicker than light interspaces. Belly uniformly yellowish white or with light brown dots or vermiculations. Oral disk yellowish white. Live coloration is shown in Figs 4 and 7.

Sexual dimorphism
Mature males strongly display elongated odontodes on the caudal peduncle, whereas females and juveniles only have short odontodes.

Distribution and habitat
The species is only known from the lower Xingu River, downstream Belo Monte Waterfalls (Fig 8). Panaqolus tankei are usually found on fallen trees and sunken wood alongside the riverbank, in depths varying from 1 to 10 m (Figs 9 and 10).

Etymology
A patronym in honor of Andreas Tanke, a German aquarist very dedicated to the genus Panaqolus, studying its behavior, reproduction, and differences between known forms, keeping these fishes in the aquarium, visiting their habitats, and publishing his findings (e.g. [17]). He probably was the first to reproduce Panaqolus tankei in captivity [18]. For his (successful) efforts to improve communications between aquarists and scientists to join their forces in an era of less and less money for research and an ever accelerating destruction of natural habitats.

Molecular Analyses
Six hundred sixty-seven (667) basepairs of the Cytochrome c Oxidase subunit 1 (COI), the so called barcode region [12], have been amplified for 12 specimens (localities, collection numbers and GenBank accession numbers in Table 2). Seven of them are P. tankei; the other five specimens belong to a putative new species from Rio Tocantins (from locations close to the cities Cametá and Portel). As only few specimens from that probable new species are available in scientific collections, analyses on its taxonomic status have not been possible. However, differences in the coloration compared to described species indicate that they might represent a new species. GenBank sequences from P. changae (EU359435.1) and P. albivermis (EU359436.1), the only congeners of which sequences were available, were used for comparison. The   New Panaqolus from Xingu River intraspecific variability was 0% to 1.1% for P. tankei, and 0.2% to 0.5% for the putative new species from the Rio Tocantins. Interspecific variability was 0.3% to 1.7% for P. tankei-Panaqolus sp. Tocantins, 0.8 to 1.8% for P. tankei-P. changae, and 4.1% to 4.6% for P. tankei-P. albivermis. Further distances were 0.8% to 1.1% for Panaqolus sp. Tocantins -P. changae, 4.1% to 4.5% for Panaqolus sp. Tocantins -P. albivermis, and 4.0% for P. changae-P. albivermis. The intraspecific variation in P. tankei was higher than the distances between P. tankei and the putative new species. Similar results were found in the loricariid genus Rineloricaria [19]. Identification via DNA barcodes is based on the observation that intraspecific genetic divergence is usually lower than interspecific divergence, leading to the so called 'barcoding gap' [20]. At first, DNA barcoding studies suggested that a 2% threshold typically represented a cut-off between species [12], but later it was showed that the divergence between species can fall well below this value, depending on the taxa of interest [21,22]. To be able to characterize species with very low interspecific variability, a character bases approach was successfully tested [23,24]. This approach is comparable to the traditional morphology-based methods where a species diagnosis would be based on a binary signal-the presence or absence of a diagnostic character (in this case a DNA character), therefore bypassing the uncertainty found in an analogue measurement of sequence similarity. These diagnostic sites of interest are referred to as nucleotide diagnostics (ND) [24]. Such a ND was found for P. tankei in position 351 (T vs. A in all other analyzed species). Another ND was found for Panaqolus sp. Tocantins on position 198 (C vs. T in all other analyzed species).
An exhaustive comparison of the barcode region of all described species of Panaqolus was not possible due to difficulties in getting tissue samples. Many species occur in remote regions and are rarely caught by scientists.

Discussion
Together with P. changae, P. claustellifer and P. purusiensis, P. tankei is part of a group of very similar species that differ mainly in details of their coloration (e.g. wormlines on the head vs. straight lines; number and width of the dark bars on the body). The forms most akin to P. tankei are a putative new species distributed in the Rio Negro basin [25] and P. claustellifer. Unfortunately, no fixed material of the former is available for comparison. Preliminary results from an analysis of DNA sequences from five genes show these (putative) species to be clearly distinct (Lujan (Royal Ontario Museum), personal communication). Furthermore, adult P. tankei have 3 to 4 dark bands in the dorsal fin which are orientated non-parallel to the margin of the fin, whereas P. claustellifer has only two bands and these are parallel to the margin of the fin [26]. Barcode sequences [12] from seven P. tankei and five specimens of Panaqolus sp. from rio Tocantins were very similar, with very low interspecific variation. Similar color patterns have been found for a broad variety of loricariids from the Rios Xingu and Tapajós [27]. Low genetic distances between congeners have already been reported for other members of the subfamily Hypostominae [4,19,28,29]. This pattern can also be found in other diverse groups like piranhas (Serrasalmus & Pygocentrus) [30]. This supports the theory of recent radiation of the Neotropical fish fauna [28,30]. New Panaqolus from Xingu River Nucleotide diagnostics suggest that all specimens from the Rio Tocantins represent a single lineage, distinct from P. tankei. One ND could be found for each P. tankei and the new species from Rio Tocantins. Also of interest is the greater distance of all broad banded species from P. albivermis, a species with thin bands and/or dots, showing that the coloration is a strong character for differentiation in this genus. Costa-Silva et al. [19] proposed that recognition as a formal new species is justified even if only morphological traits or high molecular divergence distinguish a population from others.
From the region of Porto de Moz, near mouth of the Xingu, a population of Panaqolus is known which has slightly more and finer lines on the snout (A. Tanke (Neustadt am Rübenberge), personal communication; Fig 7) than P. tankei from the middle reaches of the Xingu river. As no material for comparison has been available, we cannot state here if it is a variant of P. tankei or a distinct species. Other similar populations which potentially represent distinct and undescribed species have been reported from the rivers Tocantins [31], Jari, Curuá-Una, Tapajós [32], Anapu [33], and Rio do Pará [34]. A molecular analysis is in preparation to give us further insights into the relationships between the species and populations of the genus.
Many species of Panaqolus are common in the aquarium trade and P. tankei has been introduced to fishkeeping hobbyists as L398 [16]. Although being an endemic species from Xingu, the geographic distribution of P. tankei does not directly overlap the impacted area of the recently implemented Belo Monte Dam. The habit of living and feeding on sunken wood in lowland riverbanks will likely remain unharmed in most parts of its distribution. However, hobbyist observations in the aquarium show that spawning of these fish appears to depend on seasonal changes in the water quality and temperature [18] and these water parameters should be constantly monitored to guarantee the natural cycles downstream the impacted area.