Research Article |
Corresponding author: Paula Dias Moreira ( paulaentomobio@gmail.com ) Academic editor: Steffen Pauls
© 2022 Paula Dias Moreira, Leandro Lourenço Dumas, María Paula Rozo, Gleison Robson Desidério, Daniela Maeda Takiya.
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.
Citation:
Moreira PD, Dumas LL, Rozo MP, Desidério GR, Takiya DM (2022) Integrative taxonomy supports two new species of Chimarra Stephens, 1829 from Brazil (Trichoptera: Philopotamidae). Arthropod Systematics & Phylogeny 80: 169-185. https://doi.org/10.3897/asp.80.e76559
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Chimarra Stephens, 1829 is the largest genus of the Philopotamidae with about 930 species and cosmopolitan distribution. Recent taxonomic revisions have subdivided the genus into four subgenera: Chimarra, Curgia Walker, 1860, Chimarrita Blahnik, 1997, and Otarrha Blahnik, 2002, the last three restricted to the New World. In this paper, we describe and illustrate two new species of Chimarra from Brazil, C. (Otarrha) paraodonta sp. nov. from Rio de Janeiro State and C. (Chimarrita) truncata sp. nov. from Amazonas and Pará states. Partial sequences of cytochrome oxidase subunit I (COI, DNA barcodes) were generated and integrated with morphological evidence to delimit the new species and evaluate their phylogenetic relationships within the genus. A maximum likelihood analysis of 48 COI sequences representing 19 species of Chimarra corroborated their subgeneric assignment based on morphology and highlighted their putative sister species. Both new species showed high K2P divergences when compared to their sister species: Chimarra (O.) paraodonta sp. nov. and C. (O.) odonta (17.4–21.3%) and Chimarra (C.) truncata sp. nov. and C. (C.) simpliciforma (20.0–21.3%). These distances are comparable to the range of interspecific distances calculated for the whole genus (13.6–22.7%), adding support to their description as new species. This analysis was especially important because of the high morphological similarity of C. paraodonta sp. nov. and C. odonta. Finally, analysis of the sequences of Chimarra odonta suggests that the nominal species may represent a complex of cryptic species with high intraspecific divergences (up to 18.1%), with at least two of those lineages co-occurring with C. paraodonta sp. nov. at Parque Nacional do Itatiaia.
Amazon, Atlantic Forest, caddisfly, DNA barcoding, finger-net caddisflies, Neotropics
Philopotamidae comprise approximately 1,500 species in 26 extant genera distributed worldwide, but most of the diversity is found in the tropics (
Currently, Chimarra is subdivided into four subgenera and the New World species have been the subject of relatively recent taxonomic revisions (
Recent phylogenetic analyses inferred from molecular data (
In the past few decades, molecular techniques have been used widely for species separation and identification (
In Trichoptera, studies using DNA barcodes have increased in recent decades. Initially, molecular data were used mainly to facilitate association of immatures with adults (e.g.,
Although the use of COI sequences has become common in taxonomic studies at the species level, their use is still rare for Neotropical caddisflies. The first comprehensive work using this tool in the Neotropics was made by
In this study two new species of Chimarra are described and illustrated in the subgenera Chimarrita and Otarrha through an integrative taxonomic approach based on adults from the Amazon and Atlantic Forest biomes of Brazil. In addition, DNA barcodes were generated and integrated in order to evaluate species delimitation and relationships of the new taxa within the genus Chimarra.
Specimens of the new species were collected with Malaise traps (Gressit and Gressit 1962) and Pennsylvania light traps (
The distribution map was prepared using QGIS Las Palmas 2.18.10 software (QGIS Developed Team 2016). Morphological terminology for male genitalia follows that of
Genomic DNA was extracted from legs of male adults through nondestructive methods using the DNeasy Blood & Tissue kit (Qiagen Inc., Hilden, Germany), optimizing the original protocol by incubating the tissue for lysis in Proteinase K for 48 hours and generating two separate 50µl elutions of DNA extract, instead of 100µl. Voucher specimens were deposited at DZRJ and
Species sampled for the phylogenetic analysis of Chimarra with GenBank accession numbers for COI sequences and information on specimens sequenced herein (accession numbers in bold), such as voucher specimen code at DZRJ, collection locality, and adult gender.
Species | Voucher code | Gender | Collection locality | Accession number |
---|---|---|---|---|
OUTGROUP | ||||
Chimarrhodella peruviana | Venezuela: Barinas | KX107274 | ||
Sortosa chilensis | Chile | KM225345 | ||
Philopotamus montanus | Switzerland: Grisons | MZ046700 | ||
Wormaldia planae | Costa Rica: Puntarenas | KX292642 | ||
INGROUP – Chimarra spp. | ||||
C. usitatissima | Brazil: Minas Gerais | KX106978 | ||
C. (Chimarra) forcipata | French Guiana | KM225357 | ||
C. (Chimarra) marginata | Finland: Etela-Suomen Laani | KX295044 | ||
C. (Chimarra) obscura | Canada: Ontario | KM537514 | ||
C. (Chimarrita) camella | Brazil: Rio de Janeiro | KX102673 | ||
C. (Chimarrita) camura | Brazil: Rio de Janeiro | KX104186 | ||
C. (Chimarrita) kontilos | Brazil: São Paulo | KX103574 | ||
C. (Chimarrita) simpliciforma | Guyana: Upper Demerara-Berbice | HQ967557 | ||
C. (Chimarrita) simpliciforma | ENT4288 | Male | Brazil: Pará, Tailândia, Rod. PA-150, Km 74, Agropalma | OM964809 |
C. (Chimarrita) truncata sp. nov. | ENT4292 | Male | Brazil: Amazonas, Novo Airão, Rod. AM352, km-68, Igarapé Mato Grosso | OM964817 |
C. (Chimarrita) truncata sp. nov. | ENT4293 | Male | Brazil: Amazonas, Novo Airão, Rod. AM352, km-68, Igarapé Mato Grosso | OM964816 |
C. (Chimarrita) truncata sp. nov. | ENT4294 | Male | Brazil: Amazonas, Novo Airão, Rod. AM352, km-68, Igarapé Mato Grosso | OM964815 |
C. (Chimarrita) truncata sp. nov. | ENT4295 | Male | Brazil: Amazonas, Novo Airão, Rod. AM352, km-68, Igarapé Mato Grosso | OM964814 |
C. (Chimarrita) truncata sp. nov. | ENT4296 | Male | Brazil: Amazonas, Novo Airão, Rod. AM352, km-68, Igarapé Mato Grosso | OM964813 |
C. (Chimarrita) truncata sp. nov. | ENT4297 | Male | Brazil: Amazonas, Novo Airão, Rod. AM352, km-68, Igarapé Mato Grosso | OM964811 |
C. (Chimarrita) truncata sp. nov. | ENT4298 | Male | Brazil: Amazonas, Novo Airão, Rod. AM352, km-68, Igarapé Mato Grosso | OM964812 |
C. (Chimarrita) truncata sp. nov. | ENT5580 | Male | Brazil: Pará, Belterra, BR 163, Km 85, Igarapé do Branco | OM964819 |
C. (Chimarrita) truncata sp. nov. | ENT5581 | Male | Brazil: Pará, Belterra, BR 163, Km 85, Igarapé do Branco | OM964810 |
C. (Chimarrita) truncata sp. nov. | ENT5582 | Male | Brazil: Pará, Belterra, BR 163, Km 85, Igarapé do Branco | OM964823 |
C. (Chimarrita) truncata sp. nov. | ENT5583 | Male | Brazil: Pará, Belterra, BR 163, Km 85, Igarapé do Branco | OM964820 |
C. (Chimarrita) truncata sp. nov. | ENT5584 | Male | Brazil: Pará, Belterra, BR 163, Km 85, Igarapé do Branco | OM964821 |
C. (Chimarrita) truncata sp. nov. | ENT5591 | Female | Brazil: Pará, Belterra, BR 163, Km 85, Igarapé do Branco | OM964818 |
C. (Chimarrita) truncata sp. nov. | ENT5592 | Female | Brazil: Pará, Belterra, BR 163, Km 85, Igarapé do Branco | OM964822 |
C. (Curgia) braconoides | Brazil: Al Ibateguara | KX144398 | ||
C. (Curgia) peruviana | Bolivia: Santa Cruz | KX104022 | ||
C. (Otarrha) odonta | Brazil: Rio de Janeiro, Parque Nacional da Serra dos Órgãos, Rio Beija-flor | KX104378 | ||
C. (Otarrha) odonta | Brazil: Rio de Janeiro, Parque Nacional do Itatiaia, Rio Campo Belo | KX105279 | ||
C. (Otarrha) odonta | ENT5640 | Male | Brazil: Rio de Janeiro, Maricá, Caranguejo stream | OM964825 |
C. (Otarrha) odonta | ENT5642 | Male | Brazil, Rio de Janeiro, Nova Iguaçu, Tinguá | OM964827 |
C. (Otarrha) odonta | ENT5641 | Male | Brazil: Rio de Janeiro, Petrópolis, Araras, Araras stream | OM964826 |
C. (Otarrha) odonta | ENT5635 | Male | Brazil: Rio de Janeiro, Itatiaia, Parque Nacional do Itatiaia, Córrego Maromba | OM964828 |
C. (Otarrha) odonta complex sp. | Brazil: Rio de Janeiro, Parque Nacional do Itatiaia | HQ582429 | ||
C. (Otarrha) odonta complex sp. | Brazil: Rio de Janeiro, Parque Nacional do Itatiaia | HQ582430 | ||
C. (Otarrha) odonta complex sp. | Brazil: São Paulo, Bananal | HQ582431 | ||
C. (Otarrha) odonta complex sp. | Brazil: Rio de Janeiro, Rio Macaé, Macaé de Cima | KX141696 | ||
C. (Otarrha) odonta complex sp. | Brazil: Santa Catarina, Parque Ecológica Spitzkopf, confl. Rio Ouro & Rio Caeté | KX142040 | ||
C. (Otarrha) odonta complex sp. | Brazil: Rio de Janeiro, Parque Nacional da Serra dos Órgãos, Rio Beija-flor | KX144047 | ||
C. (Otarrha) nr. odonta | Brazil: São Paulo | KX104578 | ||
C. (Otarrha) nr. odonta | Brazil: Rio de Janeiro, Parque Nacional do Itatiaia, Rio Campo Belo | KX105682 | ||
C. (Otarrha) nr. odonta | Brazil: São Paulo, 11 km SE Bananal, small stream on São Paulo Route 247 | KX106200 | ||
C. (Otarrha) parilis | Peru: Madre de Dios | KX105173 | ||
C. (Otarrha) patosa | Bolivia: La Paz | KX106455 | ||
C. (Otarrha) paraodonta sp. nov. | ENT5577 | Male | Brazil: Rio de Janeiro, Itatiaia, Parque Nacional do Itatiaia, Córrego Maromba | OM964824 |
C. (Otarrha) peruana | Peru | KM225365 | ||
C. (Otarrha) phthanorossi | Colombia: Choco | KX102748 | ||
C. (Otarrha) rossi | Costa Rica: Guanacaste | KX106996 | ||
C. (Otarrha) tachuela | Venezuela: Merida | KX103258 |
Partial mitochondrial cytochrome oxidase c subunit 1 (COI) gene was amplified by using primers C1-J-1718 (5-GAGGATTTGGAAATTGATTAGTTCC-3) (
PCR products were stained with GelRed™ (Biotium) and underwent agarose gel electrophoresis in 1.0% TBE and visualized under UV light. Amplicons were purified using ExoSAP-IT® (USB Affymetrix). DNA sequencing was performed in both forward and reverse directions using the same PCR primers by Macrogen (Seoul, South Korea). All sequences generated as part of this study were deposited in GenBank under accessions OM964809-OM964828.
Consensus sequences were constructed based on electropherogram assemblies in Geneious® v9.1.2 (Kearse et al. 2012) and used in a comparative similarity search at GenBank using the BLAST program (
In addition to sequences generated herein of the new species and two other Chimarra species, COI sequences publicly available from GenBank were included in the alignment to a total of 52 sequences (Table
Phylogenetic relationships were estimated by maximum likelihood using RAxML v.8.2.11 (
Intra- and interspecific genetic divergences were calculated using the Kimura 2-parameter model – K2P (
The maximum likelihood tree (–lnL = 5254.453966, Fig.
Both new species showed high interspecific K2P divergences when compared to its respective sister species (see Supplementary file 1: Table S1). Chimarra (Chimarrita) truncata sp. nov. and C. (Chimarrita) simpliciforma had distances ranging from 20.0−21.3% and C. (Otarrha) paraodonta sp. nov. and C. (Otarrha) odonta Blahnik ranging from 17.4−21.3%. These distances fall into the range of all Chimarra interspecific K2P distances calculated herein (13.6−22.7%), which provides support to description of the new taxa as distinct species.
It is important to note that, although we are treating C. (Otarrha) odonta (Fig.
Maximum likelihood tree (–lnL = 5254.453966) of COI sequences of Chimarra species showing the phylogenetic placement of the two new species proposed, C. (Chimarrita) truncata sp. nov. (in green) and C. (Otarrha) paraodonta sp. nov. (in blue). Chimarra (Otarrha) odonta species complex in red. Terminals sequenced herein in bold. Values above branches are bootstrap percentages (>50%).
Igarapé Mato Grosso, Novo Airão, Amazonas State, Brazil.
Chimarra (Chimarrita) truncata sp. nov. is most similar to C. (Chimarrita) xingu Blahnik, 1997 by the short, fully divided tergum X with dorsal ridges and foldlike areas on outer margin of each lobe and the simple, almost uniform in width inferior appendages. However, the new species has an apically subtruncated ventral process of segment IX, while in C. (Chimarrita) xingu this process is pointed. Furthermore, C. (Chimarrita) truncata sp. nov. can be recognized by inferior appendages with truncated apices both in lateral and ventral view (rounded in C. xingu).
Adult male: forewing length 2.6–3.0 mm (n=6; holotype = 2.6 mm). General color (in alcohol) uniformly pale brown, except dorsum of the head dark brown, antennae and palps pale brown. Head with anterior, anteromesal, posterior, and posterolateral setal warts; posterior setal warts large, triangular, meeting broadly on median portion; postocular parietal sclerite triangular, slightly extending below eye. Maxillary palps relatively short, 2nd segment longer than 3rd segment, apicomesally with stout setae. Wing venation typical for the subgenus (Fig.
Male genitalia (Fig.
The specific epithet is an allusion to the characteristic inferior appendages, which are apically truncated. Derived from the Latin, “truncata” = piece cut off, tip, end.
Holotype. BRAZIL • ♂; Amazonas State, Novo Airão, Rod. AM352, km-68, Igarapé Mato Grosso; 02°48’58”S, 60°55’18”W; 21–31.vii.2016; J.A. Rafael & F.F. Xavier leg.; Malaise trap;
Cachoeira Véu da Noiva, Parque Nacional do Itatiaia, Itatiaia, Rio de Janeiro State, Brazil.
The new species is closely similar to C. (Otarrha) odonta Blahnik, 2002 by some shared primitive characters of the subgenus, like hindwing venation pattern with Rs 4-branched and the undivided anterior head setal warts. Both species also have a simple, subtriangular, and completely divided tergum X and an inner process on each inferior appendage. However, the new species has the Otarrha synapomorphic hindwing venation with Sc+R1 fused, narrower and more uniform lobes of tergum X, and inferior appendage rhomboidal (in lateral view) and more elongated and spatulated (in ventral view). Additionally, the dorsomesal process of the inferior appendage in C. (Otarrha) paraodonta sp. nov. is thorn-like, more robust, and positioned subapically; while in C. odonta, this process is tooth-like, blunt, and positioned more apically. Furthermore, C. (Otarrha) paraodonta sp. nov. can be recognized by its differently shaped tergum IX as viewed dorsally, the more robust ventral process, and simple phallotremal sclerite.
Adult male: forewing length 5.2–5.8 mm (n=3; holotype = 5.8 mm). General color (in alcohol) uniformly golden brown, except dark brown dorsum of head. Dorsum of head with anterior, anteromesal, posterior, and posterolateral setal warts; posterolateral setal warts large; anterior setal warts each elongate and undivided; postocular parietal sclerite large, slightly extending below the eye. Maxillary palps relatively short, 2nd segment shorter than 3rd segment, apicomesally with stout setae. Wing venation typical for the subgenus (Fig.
Male genitalia (Fig.
The specific epithet is a reference to the close similarity of the new species to Chimarra (Otarrha) odonta. Derived from the Greek, “para” = beside or near.
Material examined. Holotype. BRAZIL • ♂; Rio de Janeiro State, Itatiaia, Parque Nacional do Itatiaia, Complexo da Maromba, Cachoeira Véu da Noiva; 22°25’38.6”S, 44°37’9.7”W; el. 1140 m a.s.l.; 02−19.ii.2015; D.M. Takiya & A.P.M. Santos leg.; Malaise trap; DZRJ 7828 (DNA voucher ENT5579). — Paratypes. BRAZIL • 1 ♂; Rio de Janeiro State, Itatiaia, Parque Nacional do Itatiaia, Complexo da Maromba, Cachoeira Véu da Noiva [PNI-M2A]; 22°25’36.1”S, 44°37’05.80”W; el. 1153 m a.s.l; 02.x–02.xi.2015; M.L. Monné, J.P. Botero, Â.P. Pinto, L.H. Gil-Azevedo; Malaise trap,
Currently, four subgenera are recognized in Chimarra: Chimarra, Chimarrita, Curgia, and Otarrha, all of them recently reviewed (
Chimarrita was originally divided into three species groups by
Otarrha was established in 2002 to accommodate eighteen species formerly placed either in the subgenus Chimarra or unplaced to subgenus (informally referred to as patosa Group) and thirteen new species. The subgenus is particularly well represented in the Lesser and Greater Antilles, and northern South America, with a few species recorded from Central or southern South America (
Although there is weak morphological support for most subgenera, recent species-level molecular data phylogenies, using a large subset of taxa and multiple molecular markers (
Of a total of 33 species, only three species of Otarrha are recorded from Brazil: C. diakis Flint, 1971 (Amazon), C. odonta (Atlantic Forest), and C. machadoi Camargos, 2016 (Cerrado) (
Considering the high morphological resemblance of C. paraodonta sp. nov. to C. odonta, we decided to include in the present study all C. odonta barcode sequences available (from
Our study shows that the combination of detailed morphological observation and molecular sequence data is constructive in discovering and describing new species and provides another example of the effectiveness of DNA barcodes as a tool for species delimitation. Due to efforts of the global initiative Trichoptera Barcode of Life (TBOL) that started in 2007, a comprehensive COI barcode reference library is available for about one-third of the described caddisfly species (
Caddisflies are excellent freshwater biological indicators due to their ecological diversity and intolerance of most species to pollution and disturbances (
All authors contributed to the study conceptualization and design. PDM, LLD, and GRD identified specimens and produced illustrations and maps. PDM and MPR carried out the molecular laboratory work under supervision of DMT. All authors contributed to the draft of the manuscript. All authors read and approved the final manuscript.
R. J. Blahnik (University of Minnesota, USA) and S. Pauls (Senckenberg Research Institute and Natural History Museum Frankfurt, Germany) made valuable suggestions on a previous version of this manuscript. We are also grateful to R. J. Blahnik for sharing information on the C. odonta complex. P. D. Moreira had a scientific initiation (PIBIC, UFRJ) fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Proc.148340/2020-2) and this paper was a requisite for obtaining her B.Sc. in Biological Sciences (CEDERJ). Some of the data generated herein was part of M. P. Rozo’s doctoral dissertation at Programa de Pós-graduação em Zoologia (UFRJ). G. R. Desidério is a post-doctoral fellow from Programa de Capacitação Institucional (PCI/DB, Proc. 317780/2021-2) and is grateful to Dr Beatriz Ronchi Teles (
Table S1
Data type: .xlsx
Explanation note: Pairwise K2P divergences of 48 COI sequences of Chimarra species.