The complete mitochondrial genome and description of a new cryptic Brazilian species of Metopiellus Raffray (Coleoptera: Staphylinidae: Pselaphinae)

Field collection and sequencing

A total of 12 specimens of Metopiellus crypticus Asenjo sp. nov. were collected in forest areas from the Serra dos Carajás in Pará, Brazil, in which the individuals were more abundant, and only two specimens from the canga, a savanna-like environment from the region (Figs. 1B–1C), according to the sampling permit 49.994, granted by ICMBio/MMA. Various collection methods were used in all sites (hand collection, litter sampling and hay-bait traps and soil sampling by flotation), but specimens were found only in litter-associated methods (hay-bait traps and soil sampling by flotation). Therefore, it is likely that the edaphic environment is characterized as a preferred habitat for populations of Metopiellus crypticus Asenjo sp. nov. The specimens were immediately fixed in 99% ethanol within a 2 mL centrifuge tube and transported to the laboratory.

Total genomic DNA was extracted from three specimens from the type population with the DNeasy Blood & Tissue kit (Qiagen), following the manufacturer’s protocol for insect samples, being deposited at the DNA bank of the Instituto Tecnológico Vale (ITV) under the accession numbers ITV10661, ITV21026 and ITV21027. Paired-end libraries were constructed from ∼50 ng of genomic DNA using the QXT SureSelect kit (Agilent Technologies, Santa Clara, CA, USA), with which the DNA samples were subjected to an enzymatic random fragmentation and simultaneously bound to adapters, following the manufacturer’s instructions. Then, the samples were purified and subjected to an amplification reaction using primers complementary to the adapters. Afterwards, the libraries were quantified using a Qubit 3.0 (Invitrogen, Waltham, MA, USA) fluorimeter and checked for fragment sizes in a 2100 Bioanalyzer (Agilent Technologies). Finally, the libraries were diluted in a solution of 0.1% Tris–HCl and Tween and pooled to be sequenced in an Illumina NextSeq 500 with the high-output v2 kit (300 cycles, 2 × 150 bp). Resulting raw sequencing reads with base quality <Phred 20 and length <70 bp were trimmed with AdapterRemoval v.2 (Schubert, Lindgreen & Orlando, 2016), resulting in 21,140,835 (ITV10661), 48,275,325 (ITV21026) and 49,851,478 (ITV21027) high quality pairs of reads, which were used to assemble the mitochondrial genomes using NovoPlasty 3.6 (Dierckxsens, Mardulyn & Smits, 2017). A bash script containing the commands for the cleaning and assembling steps is available as Data S1. Finally, the annotations were performed with MITOS2 (Bernt et al., 2013), with subsequent minor manual corrections using Geneious Prime 2021 (Biomatters), by comparing the annotated mitogenomes with previously available data for Pselaphinae species.

Morphological study

Specimens. The apical segments were cleared in a double boiler using 10% KOH during three minutes. Dissections were made under a Leica S8APO (16×–128×) stereo-microscope. Pictures were obtained using the AxioCam 506 color camera connected to an Axio ZoomV16 (ZEISS) stereo microscope and Photoshop CC 2021 was used for image processing, with final plates being assembled in Adobe Illustrator CC 2021. Morphological character terminology, including foveation and its abbreviation followed Chandler (2001). All measurements were made using the Leica S8APO (16×–128×) stereo-microscope, and the width/length ratios were acquired using the widest and longest parts of the respective structures, being presented in millimeters, based on the holotype. We also performed additional measurements using the paratypes, comparing with the data obtained for the holotype (Data S2).

Measurements symbols:

BL body length (from margin of prolongation of head to tergite IX posterior margin)

BW body width (maximum width of elytra)

EL elytral length (maximum)

EW elytral width (maximum)

HL head length (from anterior margin of prolongation of head to head disc posterior margin)

HW head width (maximum)

NW neck width (minimum)

PL pronotum length (maximum)

PW pronotum width (maximum)

In the type label data, quotation marks (“ ”) separate different labels and a slash (/) separates different lines within a label. Text within square brackets [] is explanatory and is not included in the original labels.

Depositories. The specimens examined in this revision are deposited in the following collections (curators in parenthesis):

CEMT - Setor de Entomologia da Coleção Zoológica da Universidade Federal de Mato Grosso, Departamento de Biologia e Zoologia, Cuiabá, Mato Grosso, Brazil (Fernando Vaz-de-Mello).

ISLA - Coleção de Invertebrados Subterrâneos de Lavras, Setor de Zoologia, Departamento de Biologia, Universidade Federal de Lavras, Lavras, Minas Gerais, Brazil (Rodrigo Lopes Ferreira).

ITV - Coleção de DNA do Instituto Tecnológico Vale, Belém, Pará, Brazil (Santelmo Vasconcelos).

MPEG - Museu Paraense Emilio Goeldi, Belém, Pará, Brazil (Orlando Tobias Silveira).

Nomenclatural acts

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively 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 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:85F589F2-AECD-4D52-B545-6363C4CE8E18

The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Description

Metopiellus crypticus Asenjo, new species

(Figs. 1, 2, 3 and 4)

urn:lsid:zoobank.org:act:EE11C828-CCDD-4FDC-B090-A3CC80A1A2E1

Type material (seven males, two females). Holotype: BRAZIL, male, labeled “BRAZIL: Pará, /Curionópolis, Serra/Leste, 22M, 650137mE, /9339970mN, WGS84, [5°58′10.59″S, 49°38′36.86″W]/25.iv[April].2017, BioEspeleo leg”.; “Hay-bait trap, /Transect: T2, /Quadrant: E, Parcel: d”; “HOLOTYPE ♂ [red label]/Metopiellus/crypticus sp. nov./Desig. Asenjo et al. 2023” (CEMT-00120424). Paratype: (six males, two females), labeled: “BRAZIL: Pará, /Curionópolis, Serra/Leste, 22M, 652136mE, /9339073mN, WGS84, [5°8′39.63″S, 49°37′31.79″W]/26.iv[April].2017, BioEspeleo leg”.; “Soil sampling, /Transect: T3, /Quadrant: C, Parcel:-” (one male, ISLA-103823). “BRAZIL: Pará, /Curionópolis, Serra/Leste, 22M, 650360mE, /9339477mN, WGS84, [5°58′26.62″S, 49°38′29.57″W]/25.iv[April].2017, BioEspeleo leg”.; “Soil sampling, /Transect: T1, /Quadrant: D, Parcel:-” (one female, ISLA-103824). “BRAZIL: Pará, /Curionópolis, Serra/Leste, 22M, 652013mE, /9339211mN, WGS84, [5°58′35.15″S, 49°37′35.80″W]/26.iv[April].2017, BioEspeleo leg”.; “Soil sampling, /Transect: T4, /Quadrant: C, Parcel:-” (one male, MPEG-01051329). “BRAZIL: Pará, /Curionópolis, Serra/Leste, 22M, 650095mE, /9339732mN, WGS84, [5°58′18.34″S, 49°38′38.21″W]/25.iv[April].2017, BioEspeleo leg”.; “Hay-bait trap, /Transect: T2, /Quadrant: A, Parcel: d” (1 male, MPEG-01051330). “BRAZIL: Pará, /Curionópolis, Serra/Leste, 22M, 650095mE, /9339732mN, WGS84, [5°58′18.34″S, 49°38′38.21″W]/25.iv[April].2017, BioEspeleo leg”.; “Hay-bait trap, /Transect: T2, /Quadrant: A, Parcel: c” (two male, CEMT-00120425 and CEMT-00120426, and one female, CEMT-00120427). “BRAZIL: Pará, /Curionópolis, Serra/Leste, 22M, 650070mE, /9339845mN, WGS84, [5°58′14.67″S, 49°38′39.03″W]/25.iv[April].2017, BioEspeleo leg”.; “Hay-bait trap, /Transect: T2, /Quadrant: C, Parcel: b” (1 male, MPEG-01051331). All paratypes with label “PARATYPE [yellow label]/Metopiellus/crypticus sp. nov./Desig. Asenjo et al. 2023”.

Additional specimens. BRAZIL: Pará, Curionópolis, Serra Leste, 22M, 650137mE, 9339970mN, WGS84, [5°58′10.59″S, 49°38′36.86″W], 25.iv[April].2017, BioEspeleo leg., HBT-T2 E(B) (1 male, ITV10661). BRAZIL: Pará, Curionópolis, Serra Leste, 22M, 650070mE, 9339845mN, WGS84, [5°58′14.67″S, 4938′39.03″W], 25.iv[April].2017, BioEspeleo leg., HBT-T2 C(A), (1 female, ITV21026). BRAZIL: Pará, Curionópolis, Serra Leste, 22M, 650070mE, 9339845mN, WGS84, [5°58′14.67″S, 4938′39.03″W], 25.iv[April].2017, BioEspeleo leg., HBT-T2 C(A), (1 female, ITV21027).

Diagnosis. Among the members of Metopiellus, the new species Metopiellus crypticus Asenjo sp. nov. is very similar to M. painensis because both have a similar habitus (Figs. 2A–2B; Fig. 1 in Asenjo, Ferreira & Zampaulo Rde, 2017) and eyes nearly absent (Figs. 2A–2C; Fig. 3 in Asenjo, Ferreira & Zampaulo Rde, 2017), but the new species differs by having the antennomere 7 rounded (Fig. 2E) (rectangular in Metopiellus painensis [Fig. 5 in Asenjo, Ferreira & Zampaulo Rde, 2017]). Furthermore, Metopiellus crypticus Asenjo sp. nov. further differs by the paramere asymmetric elongate with apex bifurcated (Figs. 2H–2J) (paramere asymmetric not bifurcated in M. painensis [Figs. 10–12 in Asenjo, Ferreira & Zampaulo Rde, 2017]). Also, Metopiellus crypticus sp. nov. differs by the median lobe curved and edge with long line of small teeth (Figs. 2H–2J) (median lobe almost right with the apex curved without small teeth in M. painensis [Figs. 10–12 in Asenjo, Ferreira & Zampaulo Rde, 2017]).

Holotype male, BL: 2.68. Body, mouthparts, antennae and tarsi light brown (Figs. 2A–2B).

Head: pyriform (Figs. 2A and 2C), length (HL: 0.44) similar to width (HW: 0.44), anterior region narrower and ending in an emarginated, antennal tubercle. Posterior margin of head narrowing, with posterolateral angles rounded. Neck almost half of width (NW: 0.19) of head. Head with two vertexal foveae [VF] (Figs. 2A and 2C), foveae connected by a transverse sulcus near posterior margin. Vertex longitudinally impressed, with weak sulcus running from anterior margin of antennal tubercle to neck. Ventral surface of head without gular sulcus and posterior region with two gular foveae [GF] connected by curved sulcus. Eyes (Figs. 2A–2C) composed of some ommatidia situated at middle of head length in lateral view. Antennae (Fig. 2E) almost 3/4 body length, scape almost half antenna length, last three antennomeres gradually broadening. Scape length 0.92 mm, width 0.09 mm, pedicel shorter than scape (0.39: 0.08), antennomere 3 (0.06: 0.06), antennomere 4 (0.05: 0.06), antennomere 5 (0.07: 0.06), antennomere 6 (0.06: 0.06), antennomere 7 (0.06: 0.08), antennomere 8 (0.03: 0.06), antennomere 9 (0.06: 0.1), antennomere 10 (0.06: 0.12), antennomere 11 (0.15: 0.14); all antennomeres covered by long microsetae.

Thorax: pronotum (Figs. 2A, 2C) slightly wider than long (PL: 0.45; PW: 0.52) widest at anterior half. Pronotum convex with weak median longitudinal sulcus, each side with lateral sulcus, with transversal antebasal sulcus. Pronotum with basal and anterior margins weakly emarginated; with median antebasal fovea (MAF) and lateral antebasal fovea (LAF). Prosternum with lateral procoxal fovea (LPCF). Mesoventrite with median mesocoxal fovea (MMNF), lateral mesosternal foveae (LMNF) lateral mesocoxal foveae (LMCF), and with lateral metasternal foveae (LMTF). Metaventrite with median metasternal fovea (MMTF) and one flat median triangle area before metacoxal cavities.

Elytra: subquadrate (EL: 0.74; EW: 0.80), sides gradually broadening apically (Fig. 2A). Posterior margins slightly concave, discal stria (DS) and sutural stria (SS) present. Elytron with two basal elytral foveae (BEF) at anterior margin, one at side of base of the elytral sutural stria, second on the base of discal stria. Apico-lateral margin of elytra with a small notch. Flight wings absent.

Legs: Legs long and slender (Figs. 2A–2B). Femora thickened in apical half. Tibiae curved and similar in length to femora, all tibiae thickened at apex. Protibiae carinate and open at base, lacking microsetae on the concave mesial face (Fig. 2D). Tarsi 3-segmented (Fig. 2D), first tarsomeres short, last 2 tarsomeres longer, tarsomere 2 longer than segment 3; all tarsi with single claw and minute accessory seta. Procoxae conical and prominent, mesocoxae rounded and prominent, metacoxae transverse, region articulated with trochanter conical in shape. Procoxae with small, apically pointed prosternal process, mesocoxae weakly separated, metacoxae contiguous.

Abdomen: strongly margined (Fig. 2A), with five visible tergites (morphological tergites IV-VIII), tergite III reduced to translucent plate beneath elytra, tergite IV with basolateral fovea (BLF), tergite VIII with apex straight. Tergites IV-VII bordered by distinct paratergites, paratergite in abdominal segment IV with one small tooth in the middle. Sternite III with transverse depressed plate completely bare and beneath metacoxae, transverse plate with longitudinally projecting carina at middle. Sternite IV with baselateral fovea (BLF). Tergum IX divided into two plates; right plate (Fig. 2K) larger and more sclerotized than left (Fig. 2J). Sternite VIII (Fig. 2F) with apex deeply emarginate.

Aedeagus (Figs. 2H–2J): asymmetric with paramere partially fused to form elongate plate with apex forked, the median lobe slightly bulbous at base, elongate and narrow, stronger curved laterally at apex, on edge with line of small teeth.

Female. Similar to male, except apex of tergite VIII convex (Fig. 2G).

Distribution. Only known from Serra Leste, Curionópolis, Pará, Brazil (Figs. 1A–1C).

Etymology. The specific epithet “crypticus” is a noun in apposition.

Mitogenome sequence and phylogenetic placement

All three assembled mitogenomes (GenBank accession numbers MZ576843 (ITV10661), MZ576844 (ITV21026) and MZ576845 (ITV21027)) presented the standard structure sequence and gene content for Metazoa, consisting of 13 PCG, 22 tRNA genes and two rRNA genes (Fig. 3). The three mitogenome assemblies ranged in size from 14,353 to 14,984 bp, with similar GC contents between 16.2% and 16.5%, and 98.3% identical sites (1.7% differences). We observed differences in nucleotide composition among the three mitogenomes of Metopiellus crypticus Asenjo sp. nov., with indel events mostly occurring in the rRNA genes (three in each locus). Also, rrnL presented 33 site substitutions as indicated by the mismatches in the alignment, one of the highest proportions of polymorphic sites within the analyzed mitogenomes (2.68% of the 1232 bp), behind only of NAD6 (3.73% of the 456 bp), excluding the tRNAs, which are considerably shorter with 63 bp on average (Table 1).

Most genes were encoded in the L-strand, including nine PCGs (ATP6, APT8, COB, COX1, COX2, COX3, NAD2, NAD3 and NAD6) and 14 tRNA genes (trnA, trnD, trnE, trnG, trnI, trnK, trnL2, trnM, trnN, trnR, trnS1, trnS2, trnT and trnW). Also, ATT was the most frequent start codon, being observed in seven genes, followed by ATA in four genes, and ATG in three (Table 1). On the other hand, almost all genes presented the TAA stop codon, except for NAD3 with TAG, and the three COX genes with an incomplete stop codon (Table 1).

Previously published mitogenome sequences of Staphylinidae species from 11 subfamilies, plus one species of Hydrophilidae (Cercyon borealis) and one of Histeridae (Euspilotus scissus) to be used as outgroups, were obtained from GenBank, totaling 61 accessions. Sequences of the 13 protein coding genes (PCG) were aligned with MAFFT v7.45 (Katoh et al., 2002; Data S3) and maximum likelihood (ML) phylogenetic trees were obtained using RAxML v8 (Stamatakis, 2014), implemented in raxmlGUI v2 (Edler et al., 2021) using the model GTR+PROTGAMMA and the rapid bootstrapping option with 1,000 replicates (Data S4).

In the phylogenetic analysis, most of the subfamilies were recovered as monophyletic and well supported, except for Tachyporinae and Paederinae, which were polyphyletic and paraphyletic, respectively, and Staphylininae, presenting a low bootstrap support (BS = 55) (Fig. 4). Within Pselaphinae, the relationships among the sampled species were mostly unsupported (BS <70). The three specimens of Metopiellus crypticus sp. nov. grouped with maximum statistical support (BS = 100) in the longer branch within the subfamily, being recovered as sister to Batrisodes sp., although with low statistical support (BS = 42) (Fig. 4).