The new stonefly species, Neoperla mindoroensis sp. nov. (Perlidae), from Mindoro island is described. The new species is assigned to the N. recta species complex of the N. montivaga group on account of its obvious T7 and T8 with pointed processes and the presence of basolateral lobes in the everted aedeagal sac. The male adult is distinguishable by its aedeagus with a slightly raised mediodorsal lobe, fully covered with fine spinules, while the female adult has comparably small eggs (240 × 220 μm) with a punctate, chorionic surface with punctae arranged in polygonal FCIs. The life stages and sexes were assigned using COI mtDNA barcodes (2.2% maximum intraspecific genetic distance), which were compared with available barcodes of congeners, which had interspecific genetic distances varying by at least 23.5%. Biogeographic aspects, ecological habitat requirements, and suitability as potential bioindicator of the species are also briefly discussed.

DNA barcode, integrative taxonomy, Mt Hinundungan key biodiversity area, new species

Plecoptera (stoneflies) is a basal, aquatic order of Neopteran insects known for their intolerance to organic pollution (Fochetti and Tierno de Figueroa 2008). Their presence and abundance are important in rapid assessments of water quality in freshwater ecosystems, especially rivers and streams, as the order occurs worldwide, except in Antarctica (DeWalt and Ower 2019).

One of the most diverse stonefly genera in the Oriental Realm and Southeast Asia is Neoperla Needham, 1905. In fact, in the Philippines alone, there are already 23 recorded Neoperla species even if these are only known from few major islands (Jewett 1958; Kawai 1969; Sivec 1984; Zwick 1986; Sivec and Stark 2011; Dela Cruz et al. 2018) and have not been comprehensively sampled yet. Interestingly, some closely related species occur syntopically (Sivec 1984).

Usually, only the male adult stages of stoneflies were formally described from the Philippines (Zwick 1982, 1986; Sivec 1984; Sivec and Stark 2011). Conspecificity with female adults and nymphs were ambiguous due to a lack of material or suitable matching tools, which made it difficult to use them as bioindicators in freshwater assessments. In recent decades, DNA barcoding has increasingly been used in associating life stages of aquatic insects with their adults (Freitag 2013; Dela Cruz et al. 2016; Garces et al. 2018). For the first time, a new set of modified primers is applied here to Philippine Plecoptera.

This study focuses in the Baroc River Catchment, which is in the Key Biodiversity Area “69 Hinunduang Mt.” (sensu Ong et al. 2002) on Mindoro island. Here, several interesting aquatic insects have been discovered during the comprehensive assessment of the Ateneo Biodiversity Laboratory (Freitag 2013; Mey and Freitag 2013; Komarek and Freitag 2014; Vidal et al. 2017; Garces et al. 2018). This study also aims to address the lack of taxonomic studies of stoneflies from this biogeographically interesting island, where no stonefly records have been known until now, and specifically to describe one new Neoperla species based on male and female adults along with the associated nymphs identified by COI mtDNA barcodes.

As part of a freshwater biodiversity assessment project, the stonefly fauna of the Baroc River Catchment, Roxas, Oriental Mindoro, was sampled in 2018–2019. Nymphs were collected by manual collection from rock surfaces, submerged wood, and trapped leaf packs in riffle sections of the Baroc River and its tributaries, while the adults were collected by the use of black-light traps and emergence traps as described by Freitag (2004). The collections were preserved in 95% ethanol and stored at –20 °C at the Biodiversity Laboratory, Ateneo de Manila University until scientific treatment. The following codes were used to identify the sampling sites (all belong to the Baroc River System and are within the area of Barangay San Vicente, Roxas Municipality, Oriental Mindoro, the Philippines); maps indicating collections sites and notes are provided by Mey and Freitag (2013: 301) and Vidal et al. (2017: 2–5):

HBT: Quirao Buhay Creek tributary Tagugoy Creek, disturbed secondary forest; ca 12°36'30"N, 121°22'38"E, 200 m a.s.l.

HOC: Hinundungan River tributary Quianao Creek, secondary forest; ca 12°35'20"N, 121°21'40"E, 280 m a.s.l.

HR3: upper Hinundungan River, secondary forest; ca 12°35'10"N, 121°21'36"E, 280 m a.s.l.

TBC: Taugad Daka River tributary Batuwayang Creek, secondary forest; ca 12°38'09"N, 121°19'45"E, 490 m a.s.l.

TDR1: Taugad Daka River near Sitio Taugad Diit, rural extensive farmland and secondary vegetation; ca 12°37'33"N, 121°21'18"E, 180 m a.s.l.

TDR3: upper Taugad Daka River, secondary forest; ca 12°38'05"N, 121°19'33"E, 530 m a.s.l.

THC: Taugad River tributary Hiyong Creek, rural extensive farmland and secondary vegetation; ca 12°37'27"N, 121°22'48"E, 147 m a.s.l.

TIR: Taugad Diit River near Sitio Taugad Diit, rural extensive farmland and secondary vegetation; ca 12°37'32"N, 121°22'17"E, 180 m a.s.l.

TR2: Taugad River downstream Sitio Taugad Diit, secondary vegetation; ca 12°37'18"N, 121°22'58"E, 140 m a.s.l.

The external morphology of the specimens was studied under a Leica EZ4 stereomicroscope. The cold maceration technique (Zwick 1982) was employed to properly observe the aedeagus. The aedeagus, aedeagal sac, female inner genitalia, and the nymphal mouthparts were examined as wet mounts on microscopic slides under an Olympus CX21 compound microscope. Digital imaging of dissected parts was done using these microscopes with a DinoEye Eyepiece camera, then stacked using CombineZP software (Hadley 2010). The female inner genitalia were drawn in Adobe Illustrator 2020. The images of habitus and male terminalia were produced using a Canon EOS 650D and a Canon EOS 6D, respectively, with macro lens and a stack rack operated by Helicon Remote, and then stacked using Helicon Focus. Stacked images were enhanced with Adobe Lightroom and Adobe Photoshop 2020. Preparation of eggs was done following the procedure of Sivec et al. (1988) and examined and photographed using a Hitachi TM-1000 Table Top Scanning Electron Microscope (SEM) at the Materials Physics Laboratory, Ateneo de Manila University. Terminologies follow Murányi et al. (2015).

All type material is stored in alcohol and has been deposited at the Museum of Natural History of the National Museum of the Philippines, Manila, Philippines (NMP); Biodiversity Laboratory, Ateneo de Manila University, Quezon City, Philippines (AdMU); Collection Arthien Pelingen, Philippines (CAP), currently deposited in AdMU; Museum für Naturkunde Berlin, Germany (ZMB).

DNA was extracted from the legs using Qiagen DNeasy kit (Qiagen, Hilden, Germany) following the protocol for animal tissues (Qiagen 2002). The 5'-end of the cytochrome c oxidase subunit I (COI) region was then amplified using the primers LCO1490_mod (5'-TTTCAACAAACCATAAGGATATTGG-3') and HCO2198_mod (5'-TAAACTTCAGGATGRCCAAAAAATCA-3') (Garces et al. 2018). In a 25 µl Polymerase Chain Reaction (PCR) mix, it includes 17.8 µl ddH₂O, 2.5 µl 10× buffer, 1 µl Mg (25 mM), 0.5 µl dNTP mix (10 mM), 0.5 µl of each primer (10 mM), 0.2 µl Taq Polymerase (NEB), and 2 µl template DNA of unknown concentration. The PCR program was set as follows: 180 s at 94 °C; 30 s at 94 °C, 30 s at 47 °C, 60 s at 72 °C (× 35 cycles); 300 s at 72 °C. The amplification success was then checked in a 1.5% agarose gel using gel electrophoresis. The successfully amplified PCR products were sent to MACROGEN for cleaning and sequencing. The forward and reverse sequences were then manually traced and aligned (CLUSTALW) using BIOEDIT v. 7.2.5 (Hall 1999) along with the corresponding partial COI sequences of Neoperla clymene (Newman, 1839) and Neoperla obliqua Banks, 1913 retrieved from GenBank as seen in Table 1 (Pilgrim et al. 2011; Dela Cruz et al. 2018). A statistical parsimony analysis was conducted with TCS (Clement et al. 2002), and the haplotype network was visualized using POPART v. 1.7 (Leigh and Bryant 2015) and edited in Adobe Illustrator 2020.

The pairwise genetic distance analysis was performed in MEGA 7 (Kumar et al. 2016) using Kimura-2-parameter (K2P) model with bootstrap method in 1000 replicates.

Among the four stonefly genera which are known from the Philippines, Neoperla is the best documented genus in the country, with currently 24 species as listed above. For the first time, an integrative taxonomic approach was applied to describe a new Philippine Neoperla species using a newly designed primer. The same primer has proved to be efficient in generating DNA barcodes of Ephemeroptera species (Garces et al. 2018, 2020). In a preliminary analysis of a comprehensive assessment of Swiss stoneflies (Gattolliat et al. 2016), one of the factors pointed out is the need to explore more specific primers other than the standard COI primers (Folmer et al. 1994) for better output. In this study, the barcodes have been instrumental to associate the different stages of the new species and differentiate them from congeners of which mtDNA sequences are available. As a result, the female adult and the nymph were also properly described.

In biodiversity surveys of aquatic insects and ecological assessments of rivers (e.g. Junqueira et al. 2010), the nymphs, which are usually difficult to be identified to species level, are commonly collected and not the adult forms. Among all Oriental Neoperla recorded, the only nymphal stage described from the Philippines is Neoperla obliqua Banks, 1913 (Dela Cruz et al. 2018). Because of the very limited material and literature, the distinctive features of the nymphs are still unclear. In addition, when adults are collected, the females, which were not given proper taxonomic identification aside from its ootaxonomy, are also hard to identify. Describing the nymphs and female adults together with conspecific males using barcoding would somehow aid in overcoming the impediments to species identification in macroinvertebrate assessments. With the rise in popularity of eDNA technology in biodiversity surveys nowadays, the need of barcode references has never been more important than now (Balke et al. 2013; Lim et al. 2016; Fernández et al. 2018).

In this study, the 3.5% intraspecific divergence threshold was followed as observed in several EPT sequence divergence analyses (Zhou et al. 2010; Gattolliat et al. 2016). The maximum intraspecific divergence of 2.2% (Table 2) clearly contrasts from the minimum interspecific distance of 23.5%. The haplotype network association (Fig. 8) of the specimens does also support the morphological species concept. Barcoding of stonefly species in the Philippines has just started recently (Dela Cruz et al. 2016, 2018). Clearly, additional efforts must be done in collection and identification of stoneflies using integrative taxonomy to further advance the building of a comprehensive reference library which would aid studies on Plecoptera systematics and zoogeography.

Figure 8. 

Statistical parsimony haplotype network of Neoperla samples and Genbank records from aligned sequences of 567 bp.

Neoperla mindoroensis sp. nov. is a member of the N. recta Banks, 1913 species complex within the N. montivaga Zwick, 1977 species group (sensu Zwick 1983), which is recognized for its T7 and T8 with pointed processes, presence of basolateral lobes in the everted aedeagal sac, concentric and lateral folds visible around and in front of the receptacle attachment, and punctate, chorionic egg surface (Zwick 1983; Sivec 1984). With closest similarity to N. nishidai Sivec, 1984, this species complex now has six members, including N. andreas Sivec & Stark, 2011, N. pseudorecta Sivec, 1984, N. recta Banks, 1913, and N. zwicki Sivec, 1984 (Sivec and Stark 2011). The N. recta species complex has been proposed to have taken the “Formosa-Luzon migratory track” instead of the “Sumatra track” due to low species similarity with Borneo, Java, and Sumatra (Zwick 1986). The presence of N. mindoroensis sp. nov. on Mindoro Island, a large land mass between Luzon and Palawan, helps now to hypothesize how the species complex reached down to Palawan as well as to the oceanic islands of the eastern Philippines as some of its representatives have also been recorded in Cebu, Negros, Samar, and Mindanao (Sivec 1984; Sivec and Stark 2011).

The material treated here was exclusively retrieved from the Baroc River catchment as the main field research locality of the work group. During the long-term sampling program, the disturbed lower river reaches as well as various major and minor tributaries were accessed repeatedly. The new species was only found in rather undisturbed, clean tributaries (see Ecology), which suggests it has value as bioindicator for such habitats. However, to further assess its suitability and potential, an intensive ecological assessment is recommended.

This study was made possible with the Gratuitous Permits (GP 0133-17 and renewals) for the collection of aquatic wildlife as kindly issued by the Bureau of Fisheries and Aquatic Resources (BFAR). Prerequisite permissions were given by the respective local government units. We especially thank Barangay San Vicente, particularly Nonoy and Ronel Sescar and families, as well as all barangay kagawads and tanods and the Buhid communities of Tagaskan and Tauga Diit for their continued support. We are thankful to the members of the Ateneo Biodiversity Laboratory (Jhoana Garces, Emmanuel Delocado, and Clister Pangantihon) and Ms Maria Katrina Constantino for the technical assistance in the laboratory and in the field. The first author extends his sincere appreciation to Dr Thomas von Rintelen and Robert Schreiber for the laboratory training in DNA taxonomy as well as to Dr Benjamin Chan and Kim Macarasig for their training in SEM imaging. We are most grateful to Dr Dávid Murányi and Dr Marco Gottardo for their valuable comments and suggestions to the manuscript.

The graduate study of the first author is supported by the Advanced Science and Technology Human Resource Development Program (ASTHRDP) of the Department of Science and Technology – Science Education Institute (DOST-SEI). Parts of the academic training and course expeditions were kindly enabled through Biodiversity teaching modules funded by the German Academic Exchange Service (DAAD project BIO-PHIL 57393541). Additional fieldwork and the management of aquatic macroinvertebrate material at the Ateneo Biodiversity Laboratory were partly supported by the School of Science and Engineering Industry 4.0 Research Fund (SI4-013) and an LS Scholarly Work Faculty Grant (SOSE012018), Ateneo de Manila University.