Zootaxa 3964 (1): 044–062 ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ Article ZOOTAXA Copyright © 2015 Magnolia Press ISSN 1175-5334 (online edition) http://dx.doi.org/10.11646/zootaxa.3964.1.2 http://zoobank.org/urn:lsid:zoobank.org:pub:634BDD12-A3F5-4A81-A84A-FDFB0409191C Complete mitochondrial genomes of two Oriental , Neoneuromus tonkinensis (van der Weele) and Nevromus exterior (Navás) (: ), and phylogenetic implications of Corydalinae

YUNLAN JIANG1, YAJUN ZHOU1, YIRAN WANG1, LU YUE1, YAN YAN 1, MENGQING WANG2 & XINGYUE LIU1,3 1Department of Entomology, China Agricultural University, Beijing 100193, China 2Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100081, China 3Corresponding author. E-mail: [email protected]

Abstract

The complete mitochondrial (mt) genomes of two Oriental endemic species, Neoneuromus tonkinensis (van der Weele) and Nevromus exterior (Navás), were determined and analyzed, which represent the first mt genomes of the genera Neoneuromus van der Weele, 1909 and Nevromus Rambur, 1842. The mt genome of N. tonkinensis is a typical circular DNA of 15776 bp with A+T content being 76.3%, while that of N. exterior is 15763 bp with A+T content being 77.5%. Both mt genomes are composed of 37 genes with an ancestral gene arrangement of the mt genome. Eleven of the 13 protein coding genes (PCGs) start with codon ATT and ATG, except for cox1 and nad1 respectively having ATC and ATA as the start codons in the mt genome of N. tonkinensis. Complete termination codons TAG and TAA were found in nine PCGs, while the remaining four genes are supposed to end with a single T. Most tRNAs are folded into the typical clover-leaf structure except for the trnS1 whose dihydrouridine arm is a simple loop. The secondary structure of rrnL con- sists of five structural domains and 50 helices, while the rrnS includes three domains and 34 helices. In the phylogenomic analysis, both Bayesian inference (BI) and maximum likelihood (ML) approaches, based on sequence data of all 13 PCGs and two rRNA genes of the mt genomes, suggested that Neoneuromus and Nevromus form a monophyletic group, which is the sister group of the lineage including Corydalus and Acanthacorydalis but not the sister group of Acanthacorydalis van der Weele, 1907 as previously reported based on morphological data.

Key word: Corydalinae, Neoneuromus, Nevromus, mitochondrial genome, phylogeny

Introduction

Corydalinae (dobsonfly) is one of the subfamilies of the megalopteran family Corydalidae. Adult dobsonflies are characterized by the head with well-developed postocular plane (usually bearing a pair of postocular spines), and by the male genitalia having callus cerci not fused with ectoprocts and having a pair of well-developed ninth gonostyli, while the dobsonfly larvae can be easily distinguished by the presence of ventral abdominal tufts as a respiratory structure for adaptation of aquatic habitats (Glorioso 1981; Yang & Liu 2010). Currently, there are ca. 160 dobsonfly species sorted into nine genera which are vicariously distributed in America, eastern and southern Asia, and southern Africa (Yang & Liu 2010). Neoneuromus van der Weele, 1909 and Nevromus Rambur, 1842 are Oriental endemic genera of Corydalinae respectively including nine and six valid described species (Yang & Liu 2010; Liu et al. 2012). Adults of these two genera are relatively large-sized with acute postocular spines and the male genitalia are characterized by the male ninth tergum medially not separated anteriorly with an ovoid internal fossa and by the male ninth sternum which is attenuate and much narrower than ninth tergum (Liu & Yang 2004; Liu et al. 2012). Previous phylogenetic studies on the intergeneric phylogeny of Corydalinae using morphological data suggested that Neoneuromus and Nevromus are sister groups (Glorioso 1981; Penny 1993; Contreras-Ramos 1998, 2011). However, the position of these two genera in Corydalinae has not been resolved. Neoneuromus +

44 Accepted by B. Kondratieff: 28 Apr. 2015; published: 29 May 2015 Nevromus was assigned to be the sister lineage of the clade including Acanthacorydalis van der Weele, 1907, Corydalus Latreille, 1802, Chloronia Banks, 1908, and Platyneuromus van der Weele, 1909 in Glorioso (1981) and Penny (1993). Whereas, Neoneuromus + Nevromus was recovered to be the sister group of Acanthacorydalis in Contreras-Ramos (2011). These hypotheses were based on morphological evidence, but no molecule-based phylogenetic study is currently available to resolve these questions. In this study, we determined the first complete mitochondrial (mt) genomes of two representative species of Neoneuromus and Nevromus, i.e. Neoneuromus tonkinensis (van der Weele, 1907) and Nevromus exterior Navás, 1927. We analyzed the genomic organization, gene arrangement, codon usage, and the secondary structure of tRNAs and rRNAs, and we compared the mt genomes of N. tonkinensis and N. exterior with other sequenced mt genomes of Corydalinae. The present phylogenomic analysis did not support the sister group relationships between Neoneuromus + Nevromus and Acanthacorydalis, but recovered these two genera as the sister lineage of Acanthacorydalis + Corydalus.

Material and methods

Specimens and DNA extraction. A specimen of N. tonkinensis was collected at Tam Dao National Park, Vinh Phuc Province, Vietnam on May 17, 2012. The specimen of N. exterior was collected at Bac Kan City, Bac Kan Province, Vietnam on May 19, 2012. Both specimens were collected by Xingyue Liu using a light trap. The specimens were preserved in 95% ethanol and stored at -20°C in the Entomological Museum of China Agricultural University (CAU), Beijing, China. Total genomic DNA of N. tonkinensis and N. exterior were extracted by using the TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China) from the mesothoracic muscle. PCR amplification and sequencing. The mt genomes of N. tonkinensis and N. exterior were generated by amplification of overlapping PCR fragments. Primers for the present PCR are provided in Tables 1–2. All PCRs used NEB LongAmp Taq DNA polymerase (New England BioLabs, Ipswich, MA, USA) under the following amplification conditions: 30s at 95°C, 40 cycles of 10s at 95°C, 50s at 40–55°C, 1kb/min at 65°C depending on the size of amplicons, and the final elongation step at 65°C for 10 min. The quality of PCR products was evaluated by 1% agarose gel electrophoresis.

TABLE 1. Primer sequences of Neoneuromus tonkinensis mt genome used in this study. Number Primer ID Nucleotide sequence (5’-3’) Reference 1 TF210 AATTAAGCTACTAGGTTCATACCC Simon et al. 2006 TR1284 ACARCTTTGAAGGYTAWTAGTTT Simon et al. 2006 2 F20 (SPB-586) CCATTCCATTTYTGATTTCC Simon et al. 2006 R20 (SPB-1738) TTTATTCGTGGAAATGCTATGTC Simon et al. 2006 3 S2F TGGTACCTCAAGGAACACCTC Present study S2R TGCTCCTAAAGCTCCGGTTA Present study 4 F01 (SPA-2756) ACATTTTTTCCTCAACATTT Simon et al. 2006 R01 (SPA-3665) CCACAAATTTCTGAACACTG Simon et al. 2006 5 F02 (SPA-3399) TCTATTGGTCATCAATGGTACTG Simon et al. 2006 R02 (SPA-4061) GAAAATAAATTTGTTATCATTTTCA Simon et al. 2006 6 F03 (SPA-3790) CATTAAGTGACTGAAAGCAAGTA Simon et al. 2006 R03 (SPA-4552) ATGACCTGCAATTATATTAGC Simon et al. 2006 7 TF4463 TTTGCCCATCTWGTWCCNCAAGG Simon et al. 2006 TR5460 TCAACAAAATGTCARTAYCA Simon et al. 2006 8 F05 (SPA-4792) GTAGATGCAAGCCCTTGACC Simon et al. 2006 R05 (SPA-5731) ATTGGATCAAATCCACATTC Simon et al. 2006 9 TF5470 GCAGCTGCYTGATAYTGRCA Simon et al. 2006 ...... continued on the next page

MITOCHONDRIAL GENOMES OF TWO ORIENTAL DOBSONFLIES Zootaxa 3964 (1) © 2015 Magnolia Press · 45 TABLE 1. (Continued) Number Primer ID Nucleotide sequence (5’-3’) Reference TR5731 TTAGGGTCAAATCCRCAYTC Simon et al. 2006 10 S3F CCATTTTGGATTTGAAGCAG Present study S3R GCTCCTTGATTTCATTCGTGA Present study 11 F06 (SPA-5747) CCATTTGAATGTGGRTTTGATCC Simon et al. 2006 R06 (SPA-6384) AAAATTAAAAGCATAATATTGAAG Simon et al. 2006 12 S4F CACGAATGAAATCAAGGAGCTT Present study S4R GGAATATGGATTGTATGAG Present study 13 F07 (SPA-6172) AGAGGCAATTTATTGTTAATAA Simon et al. 2006 R07 (SPA-7211) TTAAGGCTTTATTATTTATATGTGC Simon et al. 2006 14 S13F GAATAACATACAGTTAATCCTGTAG Present study S13R GATTTGGTTATTTTTATTGAATGGG Present study 15 F08 (SPA-7077) TTAAATCCTTTGAGTAAAATCC Simon et al. 2006 R08 (SPA-7793) TTAGGTTGAGATGGTTTAGG Simon et al. 2006 16 S14F CCCAGAATAAATTTTTCCATGTTGT Present study S14R GCTCCTATTTCCGGGTCTATAATTT Present study 17 TF-J7806 GAMACAARACCTAACCCATCYCA Simon et al. 2006 TR-N8727 AAATCTTTRATTGCTTATTCWTC Simon et al. 2006 18 TF-J8641 CCAGAAGAACATAANCCRTG Simon et al. 2006 TR-N9629 GTTTGTGAGGGWGYTTTRGG Simon et al. 2006 19 TF-J9648 ACCTAAAGCTCCCTCACAWAC Simon et al. 2006 TR-N10608 CCAAGTARTGAWCCAAARTTTCA Simon et al. 2006 20 S5F AACTTTACGAACAACACACCCTCT Present study S5R GCAAACCCTCCTCAAACTCA Present study 21 F12 (SPB-11335) CATATTCAACCAGAATGATA Simon et al. 2006 R12 (SPB-12067) AATCGTTCTCCATTTGATTTTGC Simon et al. 2006 22 F13 (SPB-11876) CGAGGTAAAGTACCACGTACTCA Simon et al. 2006 R13 (SPB-12595) GTTGGATTTCTAACTTTATTRGARCG Simon et al. 2006 23 F14 (SPB-12261) TACCTCATAAGAAATAGTTTGAGC Simon et al. 2006 R14 (SPB-13000) TTACCTTAGGGATAACAGCGTAA Simon et al. 2006 24 F15 (SPB-12888) CCGGTCTGAACTCAGATCATGTA Simon et al. 2006 R15 (SPB-13889) ATTTATTGTACCTTTTGTATCAG Simon et al. 2006 25 F16 (SPB-13342) CCTTTGCACAGTCAAAATACTGC Simon et al. 2006 R16 (SPB-14220) TTATGCACACATCGCCCGTC Simon et al. 2006 26 F17 (SPB-14197) GTAAAYCTACTTTGTTACGACTT Simon et al. 2006 R17 (SPB-14745) GTGCCAGCAAYCGCGGTTATAC Simon et al. 2006 27 F23 (SPC-10621) CTCATACTGATGAAATTTTGGTTC Simon et al. 2006 R23 (SPC-11526) TTCTACTGGTCGTGCTCCAATTCA Simon et al. 2006 28 S10F GTGGACCATCGATTATGGAACAGAT Present study S10R ATCTGTCCATAATCGATGGTCCACT Present study

46 · Zootaxa 3964 (1) © 2015 Magnolia Press JIANG ET AL. TABLE 2. Primer sequences of Nevromus exterior mt genome used in this study. Number Primer ID Nucleotide sequence (5’-3’) Reference 1 F17 (SPB-14197) GTAAAYCTACTTTGTTACGACTT Simon et al. 2006 R17 (SPB-14745) GTGCCAGCAAYCGCGGTTATAC Simon et al. 2006 2 F18 (SPB-14610) ATAATAGGGTATCTAATCCTAGT Simon et al. 2006 R18 (SPB-200) ACCTTTATAAATGGGGTATGAACC Simon et al. 2006 3 ZF6 CTCTAATTTTTCCTCAAAAACTTCA Present study ZR6 GATTCTCTAATTAGTATGGCTCTTA Present study 4 ZF7 TCAGCTTTGAAATACTTTTTAGCTC Present study ZR7 AGATTCTCTAATTAGTATGGCTCTT Present study 5 F20 (SPB-586) CCATTCCATTTYTGATTTCC Simon et al. 2006 R20 (SPB-1738) TTTATTCGTGGAAATGCTATGTC Simon et al. 2006 6 ZF1 AACTATTAGCCTTCAAAGCTGAAAA Present study ZR1 GAGCTTAAATCCATGGCACTAATCT Present study 7 F01 (SPA-2756) ACATTTTTTCCTCAACATTT Simon et al. 2006 R01 (SPA-3665) CCACAAATTTCTGAACACTG Simon et al. 2006 8 F2 (SPA-3399) TCTATTGGTCATCAATGGTACTG Simon et al. 2006 R2 (SPA-4061) GAAAATAAATTTGTTATCATTTTCA Simon et al. 2006 9 F3 (SPA-3790) CATTAAGTGACTGAAAGCAAGTA Simon et al. 2006 R3 (SPA-4552) ATGACCTGCAATTATATTAGC Simon et al. 2006 10 TF3790 CATTAGATGACTGAAAGCAAGTA Simon et al. 2006 TR4908 CGAGTTAYATCTCGTCATCATTG Simon et al. 2006 11 TF4463 TTTGCCCATCTWGTWCCNCAAGG Simon et al. 2006 TR4908 CGAGTTAYATCTCGTCATCATTG Simon et al. 2006 12 F5 (SPA-4792) GTAGATGCAAGCCCTTGACC Simon et al. 2006 R5 (SPA-5731) ATTGGATCAAATCCACATTC Simon et al. 2006 13 TF5470 GCAGCTGCYTGATAYTGRCA Simon et al. 2006 TR6384 TATATTTAGAGYATRAYAYTGAAG Simon et al. 2006 14 F07 (SPA-6172) AGAGGCAATTTATTGTTAATAA Simon et al. 2006 R07 (SPA-7211) TTAAGGCTTTATTATTTATATGTGC Simon et al. 2006 15 F08 (SPA-7077) TTAAATCCTTTGAGTAAAATCC Simon et al. 2006 R08 (SPA-7793) TTAGGTTGAGATGGTTTAGG Simon et al. 2006 16 ZF4 AGAAGAAAATGGAATTTGAGCTCTT Present study ZR4 GTGGTGTCAAAGATGTAATTTTTAC Present study 17 ZF2 GGAATTTGAGCTCTTTTTGTAATAG Present study ZR2 TGGATCAATAATTTTAGCTGGAATT Present study 18 F10 (SPA-8641) CCAGAAGAACATAGCCCATG Present study R10 (SPA-9629) GTTTGTGAAGGTGTGTTGGG Present study 19 TF-J9172 CGCTCAGGYTGRTACCCYCA Simon et al. 2006 TR-N10608 CCAAGTARTGAWCCAAARTTTCA Simon et al. 2006 20 TF-J9648 ACCTAAAGCTCCCTCACAWAC Simon et al. 2006 TR-N11010 TATCTACAGCRAATCCYCCYCA Simon et al. 2006 21 ZF5 ACAATATTAATAGAAATCAACCCTT Present study ZR5 GGCTGTTCCTATTACTACAAAAAGT Present study ...... continued on the next page

MITOCHONDRIAL GENOMES OF TWO ORIENTAL DOBSONFLIES Zootaxa 3964 (1) © 2015 Magnolia Press · 47 TABLE 2. (Continued) Number Primer ID Nucleotide sequence (5’-3’) Reference 22 F23 (SPC-10621) CTCATACTGATGAAATTTTGGTTC Simon et al. 2006 R23 (SPC-11526) TTCTACTGGTCGTGCTCCAATTCA Simon et al. 2006 23 F12 (SPB-11335) CATATTCAACCAGAATGATA Simon et al. 2006 R12 (SPB-12067) AATCGTTCTCCATTTGATTTTGC Simon et al. 2006 24 F13 (SPB-11876) CGAGGTAAAGTACCACGTACTCA Simon et al. 2006 R13 (SPB-12595) GTTGGATTTCTAACTTTATTRGARCG Simon et al. 2006 25 F14 (SPB-12261) TACCTCATAAGAAATAGTTTGAGC Simon et al. 2006 R14 (SPB-13000) TTACCTTAGGGATAACAGCGTAA Simon et al. 2006 26 F15 (SPB-12888) CCGGTCTGAACTCAGATCATGTA Simon et al. 2006 R15 (SPB-13889) ATTTATTGTACCTTTTGTATCAG Simon et al. 2006 27 F16 (SPB-13342) CCTTTGCACAGTCAAAATACTGC Simon et al. 2006 R16 (SPB-14220) TTATGCACACATCGCCCGTC Simon et al. 2006

All PCR products were sequenced in both directions using the BigDye Terminator Sequencing Kit (Applied Bio Systems) and the ABI 3730XL Genetic Analyzer (PE Applied Biosystems, San Francisco, California USA) with two vector-specific primers and internal primers for primer walking. Bioinformatics analysis. The complete mt genomes of N. tonkinensis and N. exterior are deposited in GenBank with accession numbers KP126231 and KP126232, respectively. Sequence assembly was done by using ContigExpress. tRNAs were identified by tRNAscan-SE Search Server v. 1.21 (Lowe & Eddy 1997). PCGs and rRNAs were identified by alignment with relevant genes of other species of Megaloptera. The nucleotide composition and codon usage were analyzed by MEGA 5.0 (Tamura et al. 2011). The secondary structure of tRNAs was generated by tRNAscan-SE as stated, while that of rRNAs was predicted through the relevant genes from a fishfly species Neochauliodes punctatolosus Liu & Yang and an giant dobsonfly species Acanthacorydalis orientalis (McLachlan) (Wang et al. 2012, 2014). The control regions were identified afterwards by the boundary of the rRNA genes and compared with other insect mt genomes. Phylogenetic analysis. To test the taxonomic positions of Neoneuromus and Nevromus within Corydalinae, five dobsonfly species with available complete mt genomes were included as the ingroup taxa. The selected outgroup taxa were: Neochauliodes punctatolosus Liu & Yang (Megaloptera: Corydalidae: Chauliodinae), Sialis hamata Ross (Megaloptera: Sialidae), Thyridosmylus langii (McLachlan) (Neuroptera: Osmylidae) and Mongoloraphidia harmandi (Navás) (Raphidioptera: Raphidiidae) (Table 3).

TABLE 3. Taxa used in the present phylogenetic analysis. Order Family/Subfamily Species Accession number Megaloptera Corydalidae/Corydalinae Acanthacorydalis orientalis KF840564 Corydalidae/Corydalinae Corydalus cornutus NC_011276 Corydalidae/Corydalinae Protohermes concolorus NC_011524 Corydalidae/Chauliodinae Neochauliodes punctatolosus NC_018772 Sialidae Sialis hamata NC_013256 Corydalidae/Corydalinae Neoneuromus tonkinensis KP126231 Corydalidae/Corydalinae Nevromus exterior KP126232 Neuroptera Osmylidae Thyridosmylus langii NC_021415 Raphidioptera Raphidiidae Mongoloraphidia harmandi NC_013251

Alignment of the sequences of the 13 PCGs was inferred from the amino acid alignment using ClustalW in MEGA 5.0 (Tamura et al. 2011). The rRNA alignments were conducted by G-blocks Server (http:// molevol.cmima.csic.es/castresana/Gblocks_server.html). A Bayesian inference was performed in MrBayes Version

48 · Zootaxa 3964 (1) © 2015 Magnolia Press JIANG ET AL. 3.1.2 (Ronquist & Huelsenbeck, 2003) with the GTR+I+G model estimated by Modeltest 3.7 (Posada & Crandall 1998). Two simultaneous runs of 2,000,000 generations were conducted. The dataset was sampled every 200 generations with a burn-in of 25%. A maximum likelihood analysis was conducted with PHYML online web server using the GTR model (Guindon & Gascuel 2003; Guindon et al. 2005). In the ML algorithms, bootstrap values (BP) (Felsenstein, 1985) were calculated with 100 replicates. Abbreviations. mt: mitochondrial; PCG: protein-coding gene; atp6 and atp8: genes for the ATPase subunits 6 and 8; cox1-cox3: genes for cytochrome c oxidase subunits I–III; cytB: a gene for apocytochrome b; nad1-nad6 and nad4l: genes for NADH dehydrogenase subunits 1–6 and 4L; rrnL: large (16S) rRNA subunit (gene); rrnS: small (12S) rRNA subunit (gene); trnX (where X is replaced by one letter amino acid code of the corresponding amino acid): transfer RNA.

FIGURE 1. Mitochondrial map of Neoneuromus tonkinensis and Nevromus exterior. The tRNAs are denoted by the color blocks and are labeled according to the IUPACIUB single-letter amino acid codes. Gene name without underline indicates the direction of transcription from left to right, underlining indicates right to left.

Results and discussion

Genome organization and structure. The complete mt genomes of N. tonkinensis and N. exterior are both typical circular DNAs with 15776 bp and 15763 bp respectively, both of whom are similar in length with the other megalopteran mt genomes sequenced, ranging from 15608 bp to 15851 bp. Both mt genomes consist of 37 genes, including 22 tRNAs, 13 PCGs, two rRNAs and a non-coding region, which is rich in A+T as the putative control

MITOCHONDRIAL GENOMES OF TWO ORIENTAL DOBSONFLIES Zootaxa 3964 (1) © 2015 Magnolia Press · 49 region (Fig. 1, Tables 4–5). The gene order is identical to that of Drosophila yakuba (Burla) (Clary & Woletenholme, 1985) that has been hypothesized as ancestral for . Twenty-three genes are coded on the majority strand (J-strand), while the other 14 genes are on the minority strand (N-strand) in the both mt genomes. Gene overlaps were observed at 20 locations and involved a total of 64 bp in the N. tonkinensis mt genome. There were gene overlaps at 16 locations involving 46 bp in the N. exterior mt genome. In both mt genomes, two gene pairs including atp8-atp6 and nad4-nad4l overlap 7 nucleotides, i.e. ATGATAA and ATGTTAA which are reported in many other insect mt genomes. Moreover, there are two overlappings with a length of 8 bp respectively found in trnW-trnC and trnY-cox1, representing the longest overlaps in the mt genomes of N. tonkinensis and N. exterior.

FIGURE 2. Inferred secondary structure of 22 tRNAs in the Neoneuromus tonkinensis and Nevromus exterior mt genome. The tRNAs are labeled with the abbreviations of their corresponding amino acids. Dash (-) indicates Watson-Crick bonds and dot (·) indicates GU bonds.

50 · Zootaxa 3964 (1) © 2015 Magnolia Press JIANG ET AL. TABLE 4. Organization of the Neoneuromus tonkinensis mt genome. Negative numbers indicate overlapping nucleotides between adjacent genes. Numbers listed in anticodon indicate the anticodon position of each tRNA gene in the whole genome. Gene Direction Location Size Anticodon Start Stop Intergenic (bp) codon codon nucleotides (bp) trnI J 1–68 68 30–32GAT trnQ N 69–137 69 105–107TTG 0 trnM J 137–205 69 167–169CAT -1 nad2 J 206–1222 1017 ATT TAA 0 trnW J 1221–1287 67 1252–1254TCA -2 trnC N 1280–1343 64 1312–1314GCA -8 trnY N 1343–1408 66 1374–1376GTA -1 cox1 J 1401–2949 1549 ATC T -8 trnL1 J 2942–3005 64 2971–2973TAA -8 cox2 J 3007–3690 684 ATG TAA 1 trnK J 3691–3761 71 3721–3723CTT 0 trnD J 3761–3827 67 3789–3791GTC -1 atp8 J 3828–3986 159 ATT TAA 0 atp6 J 3980–4654 675 ATG TAA -7 cox3 J 4654–5443 790 ATG T -1 trnG J 5441–5502 62 5470–5472TCC -3 nad3 J 5503–5856 354 ATT TAG 0 trnA J 5855–5917 63 5884–5886TGC -2 trnR J 5932–5995 64 5961–5963TCG 14 trnN J 5995–6060 66 6026–6028GTT -1 trnS1 J 6060–6127 68 6086–6088GCT -1 trnE J 6128–6195 68 6158–6160TTC 0 trnF N 6194–6258 65 6224–6226GAA -2 nad5 N 6256–7984 1729 ATT T -3 trnH N 7985–8047 63 8015–8017GTG 0 nad4 N 8045–9386 1342 ATG T -3 nad4l N 9380–9676 297 ATG TAA -7 trnT F 9679–9741 63 9709–9711TGT 2 trnP N 9742–9808 67 9774–9776TGG 0 nad6 F 9811–10326 516 ATT TAA 2 cytB F 10326–11462 1137 ATG TAA -1 trnS2 F 11461–11527 67 11490–11492TGA -2 nad1 N 11540–12496 957 ATA TAA 12 trnL2 N 12495–12556 62 12525–12527TAG -2 rrnL N 12557–13869 1313 0 trnV N 13870–13939 70 13905–13907TAC 0 rrnS N 13940–14728 789 0 Control 14729–15776 1048 0 region (A+T- rich region)

MITOCHONDRIAL GENOMES OF TWO ORIENTAL DOBSONFLIES Zootaxa 3964 (1) © 2015 Magnolia Press · 51 TABLE 5. Organization of the Nevromus exterior mt genome. Negative numbers indicate overlapping nucleotides between adjacent genes. Numbers listed in anticodon indicate the anticodon position of each tRNA gene in the whole genome. Gene Direction Location Size Anticodon Start Stop Intergenic (bp) codon codon nucleotides(bp) trnI J 1–65 65 30–32 GAT 0 trnQ N 66–134 69 102–104 TTG 0 trnM J 134–202 69 164–166 CAT -1 nad2 J 203–1219 1017 ATT TAA 0 trnW J 1218–1283 66 1248–1250 TCA -2 trnC N 1276–1339 64 1308–1310 GCA -8 trnY N 1339–1403 65 1370–1372 GTA -1 cox1 J 1396–2935 1540 ATT T -8 trnL1 J 2937–3000 64 2966–2968 TAA 1 cox2 J 3003–3686 684 ATG TAA 2 trnK J 3687–3757 71 3717–3719 CTT 0 trnD J 3757–3823 67 3785–3787 GTC -1 atp8 J 3824–3982 159 ATT TAA 0 atp6 J 3976–4650 675 ATG TAA -7 cox3 J 4650–5436 787 ATG T -1 trnG J 5437–5498 62 5466–5468 TCC 0 nad3 J 5499–5852 354 ATT TAG 0 trnA J 5851–5913 63 5880–5882 TGC -2 trnR J 5928–5990 63 5957–5959 TCG 14 trnN J 5990–6054 65 6020–6022 GTT -1 trnS1 J 6054–6121 68 6080–6082 GCT -1 trnE J 6121–6186 66 6151–6153 TTC -1 trnF N 6185–6246 62 6215–6217 GAA -2 nad5 N 6247–7972 1726 ATT T 0 trnH N 7973–8036 64 8003–8005 GTG 0 nad4 N 8037–9375 1339 ATG T 0 nad4l N 9369–9665 297 ATG TAA -7 trnT J 9668–9730 63 9698–9700 TGT 2 trnP N 9731–9795 65 9763–9765 TGG 0 nad6 J 9798–10313 516 ATT TAA 2 cytB J 10313–11449 1137 ATG TAA -1 trnS2 J 11448–11514 67 11477–11479 TGA -2 nad1 N 11527–12480 954 TTG TAA 12 trnL2 N 12482–12543 62 12512–12514 TAG 1 rrnL N 12544–13888 1345 0 trnV N 13889–13958 70 13924–13926 TAC 0 rrnS N 13959–14746 788 0 Control region 14747–15763 1017 0 (A+T-rich region)

52 · Zootaxa 3964 (1) © 2015 Magnolia Press JIANG ET AL. TABLE 6. Start codons of seven megalopteran mt genomes. Nt.: Neoneuromus tonkinensis; Nn.: Nevromus exterior; Cc.: Corydalus cornutus; Np.: Neochauliodes punctatolosus; Pc.: Protohermes concolorus; Sh.: Sialis hamata; Ao.: Acanthacorydalis orientalis. Gene Start codon Nt. Nn. Cc. Np. Pc. Sh. Ao. nad2 ATT ATT ATT ATT ATT ATT ATT cox1 ATC ATT ATT ATC ATT ATT ATT cox2 ATG ATG ATG ATG ATG ATG ATG atp8 ATT ATT ATT ATC ATT ATC ATA atp6 ATG ATG ATG ATG ATG ATG ATG cox3 ATG ATG ATG ATG ATG ATG ATG nad3 ATT ATT ATT ATT ATT ATT ATT nad5 ATT ATT ATT ATA ATT ATT ATT nad4 ATG ATG ATG ATG ATG ATG ATG nad4l ATG ATG ATG ATG ATG ATG ATG nad6 ATT ATT ATT ATA ATT ATT ATT cytB ATG ATG ATG ATG ATG ATG ATG nad1 ATA TTG TTG TTG TTG ATG TTG

TABLE 7. Stop codons of seven megalopteran mt genomes. Nt.: Neoneuromus tonkinensis; Nn.: Nevromus exterior; Cc.: Corydalus cornutus; Np.: Neochauliodes punctatolosus; Pc.: Protohermes concolorus; Sh.: Sialis hamata; Ao.: Acanthacorydalis orientalis. Gene Stop codon Nt. Nn. Cc. Np. Pc. Sh. Ao. nad2 TAA TAA TAA TAA TGT TAA TAA cox1 T- T- T- T- TAA T- T- cox2 TAA TAA T- TAA T- T- T- atp8 TAA TAA TAA TAA TAA TAA TAA atp6 TAA TAA TAA TAA TAA TAA TAA cox3 T- T- T- TAA T- TAA T- nad3 TAG TAG TAG TAG T- TAA TAG nad5 T- T- T- T- T- T- T- nad4 T- T- T- T- T- T- T- nad4l TAA TAA TAA TAA TAA TAA TAA nad6 TAA TAA TAA TAA TAA TAA TAA cytB TAA TAA TAA TAA T- T- TAA nad1 TAA TAA TAA TAA TAA TAA TAA

Transfer RNAs. All the 22 typical tRNAs that have been detected in the mt genomes were found in the mt genomes of N. tonkinensis and N. exterior, each with length ranging from 62 to 71 bp. Fourteen genes are located on the J-strand and the other eight are located on the N-strand. Most tRNAs could be folded into the typical clover-leaf structure (Fig. 2), with the exception of trnS1 for its dihydorouridine (DHU) arm forming a simple loop. Some mismatched base pairs were found, such as G-U and U-U, although most follow the classical A-U and G-C based on the predicted secondary structures. Protein-coding genes. All 13 PCGs in the N. exterior mt genome and most PCGs of the N. tonkinensis mt genome use the start codons ATT and ATG whereas only cox1 and nad1 start with ATC and ATA, respectively. Eight PCGs use the termination codons TAA (nad2, atp8, atp6, nad4l, nad6, cytB, nad1) or TAG (nad3 only),

MITOCHONDRIAL GENOMES OF TWO ORIENTAL DOBSONFLIES Zootaxa 3964 (1) © 2015 Magnolia Press · 53 while the other five PCGs have an incomplete stop codon (T). The stop codons TAA and TAG always have an overlap comprising several nucleotides with the downstream tRNAs. In general, the start and stop codons of PCGs in the seven sequenced mt genomes of Megaloptera do not have particular difference from each other (Tables 6–7). The start and stop codons of all PCGs in the mt genomes of N. exterior and C. cornutus are exactly the same. The start codons of atp6 and nad4 in the seven megalopteran species all share the start codon ATG and nad3 all start with ATT. Additionally, atp6, nad5, nad4 and nad4l use the same start and stop codons in the seven megalopteran species. Ribosomal RNAs. The length of rrnL in the mt genomes of N. tonkinensis and N. exterior is 1313 bp and 1345 bp, respectively. The length of rrnS of the N. tonkinensis mt genome is 789 bp which is just 1 bp longer than that of the N. exterior mt genome. Considering the secondary structures, both of the rrnL and rrnS in N. tonkinensis and N. exterior are generally congruent with the secondary structure models proposed for the giant dobsonfly Acanthacorydalis orientalis (Wang et al., 2014). The structure of rrnL was considered to be a typical characteristic in , which consists of five structural domains (I–II, IV–VI) and 50 helices with domain III absent (Figs. 3–4). The inferred structure of the rrnS includes three domains and 34 helices, in accordance with that of other Neuropterida species (Fig. 5). The nucleotides and secondary structures of the rrnS are much similar, but there are more differences in the rrnL between these two species. Non-coding regions. The longest non-coding region (control region), which is located between the rrnS and trnI, is 1048 bp in the N. tonkinensis mt genome, and is 1017 bp in the N. exterior mt genome. They are both AT rich, with 87.5% A+T content in the N. tonkinensis mt genome and 88.7% A+T content in the N. exterior mt genome. It has been reported that the control region plays the most important role in the regulation of mt genome transcription and replication, and one possible explanation is that the control region comprises four different motifs in some arthropods including tandem repeated sequences, a long sequence of Ts, a sub-region of even higher A+T content and the stem-loop structures (Wang et al., 2014). However, we did not find any obvious tandem repeat sequences in both mt genomes presently described. In addition, the control region, five small non-coding intergenic spacers, were found in the N. tonkinensis mt genome with a range of 1–14 bp in size, whereas seven non-coding intergenic spacers were found in the N. exterior mt genome, ranging from 1 bp to 14 bp.

TABLE 8. Nucleotide composition of the Neoneuromus tonkinensis mt genome. Feature A% T% C% G% A+T% AT-skew GC-skew Whole genome 38.4 37.9 14.6 9.1 76.3 0.006 -0.23 Protein-coding genes 30.9 43.6 12.7 12.8 74.5 -0.17 0.45 1st codon position 32.5 36 12.4 19.3 68.5 -0.05 0.22 2ndcodon position 19.5 47 18.8 14.5 66.5 -0.41 -0.13 3rdcodon position 40.5 48 6.9 4.6 88.5 -0.08 -0.03 Protein-coding genes-J 31.0 41.5 16.1 11.5 72.5 -0.14 -0.17 1stcodon position 32.7 32 16.1 19 64.7 0.01 0.08 2ndcodon position 19.9 45 21.7 13.2 64.9 -0.39 -0.24 rd 40.2 47 10.5 2.4 87.2 -0.08 -0.63 3 codon position 30.7 47.1 7.3 14.9 77.8 -0.21 0.34 Protein-coding genes-N 32.2 41 6.5 19.8 73.2 -0.12 0.51 1stcodon position nd 18.8 50 14.3 16.7 68.8 -0.45 0.08 2 codon position 40.9 50 1.2 8.1 90.9 -0.10 0.74 rd 3 codon position 38.8 37.3 13.1 10.9 76.1 0.02 -0.09 tRNA genes 38.8 37.3 11.3 12.6 76.1 0.02 0.05 tRNA-J 37.3 38.9 7.4 16.4 76.2 -0.02 0.38 tRNA-N 41.5 38.6 13.6 6.3 80.1 0.04 -0.37 rRNA genes 42.0 38.8 13.1 6.0 80.8 0.04 -0.37 rrnL 40.6 38.3 14.3 6.8 78.9 0.03 -0.36 rrnS 44.1 43.4 7.8 4.7 87.5 0.008 -0.25 Control region

54 · Zootaxa 3964 (1) © 2015 Magnolia Press JIANG ET AL. FIGURE 3. Predicted secondary structure of the rnnL in the Neoneuromus tonkinensis mt genome. Roman numerals denote the conserved domain structure. Dash (-) indicates Watson-Crick base pairing and dot (•) indicates G-U base pairing.

FIGURE 4. Predicted secondary structure of the rnnL in the Nevromus exterior mt genome. Roman numerals denote the conserved domain structure. Dash (-) indicates Watson-Crick base pairing and dot (•) indicates G-U base pairing.

MITOCHONDRIAL GENOMES OF TWO ORIENTAL DOBSONFLIES Zootaxa 3964 (1) © 2015 Magnolia Press · 55 FIGURE 5. Predicted secondary structure of the rrnS in the Neoneuromus tonkinensis and Nevromus exterior mt genome. Roman numerals denote the conserved domain structure. Dash (-) indicates Watson-Crick base pairing and dot (•) indicates G- U base pairing.

Base composition. The mt genomes of N. tonkinensis and N. exterior show a strong bias toward A and T (N. tonkinensis: A=38.4%, T=37.9%, C=14.6%, G=9.1%; N. exterior: A=38.6%, T=38.9%, C=13.5%, G=9.0%) (Tables 8–9). The A+T content is also much higher than the G+C content in all gene categories of the mt genomes of N. tonkinensis and N. exterior, respectively, i.e. PCGs (74.5%, 75.8%), tRNAs (76.1%, 76.5%), rRNAs (80.1%, 81.0%), and the control region (87.5%, 88.7%). The AT- and GC-Skew indicate the strand bias in the base composition. AT-Skew=(A-T)/(A+T), GC-Skew=(G-C)/(G+C). The plus or minus of AT-Skew represents that A is more than T or the contrary, which is the same in GC-Skew. The AT- and GC-Skew are 0.006 and -0.230 in the N. tonkinensis mt genome, whereas they are -0.005 and -0.199 in N. exterior. Codon usage. All the genetic codons in the mt genomes of N. tonkinensis and N. exterior use standard invertebrate mitochondrial genetic code (Table 10). It is obvious that the preferred codon usage is A/T in their third position rather than G/C. The four-fold degenerate codon usage in PCGs prefers T over A. The missing codons in

56 · Zootaxa 3964 (1) © 2015 Magnolia Press JIANG ET AL. these two mt genomes are the GC-rich codons, for example CGC (Arg) and AGG (Ser) in N. tonkinensis, and CUG (Leu) and AGG (Ser) in N. exterior. The top six most frequently used codons in these two mt genomes are the AT- rich codons, i.e. UUU (Phe), UUA (Leu), AUU (Ile), AUA (Met), UAU (Tyr) and AAU (Asn). NNU are the most frequently used codons and NNG are the most infrequently used ones.

TABLE 9. Nucleotide composition of the Nevromus exterior mt genome. Feature A% T% G% C% A+T% AT-skew GC-skew Whole genome 38.6 38.9 9.0 13.5 77.5 -0.005 -0.199 Protein-coding genes 31.6 44.2 12.6 11.6 75.8 -0.166 0.038 1st codon position 32.8 36.1 19.6 11.5 68.9 -0.049 0.258 2nd codon position 19.5 47.1 14.7 18.6 66.6 -0.415 -0.118 3rd codon position 42.5 49.3 3.4 4.8 91.8 -0.074 -0.164 Protein-coding genes-J 31.6 42.5 11.5 14.4 74.0 -0.147 -0.113 1st codon position 33.1 33.1 19.1 14.6 66.3 0.000 0.132 2nd codon position 20.0 45.0 13.4 21.5 65.1 -0.385 -0.232 3rd codon position 41.5 49.3 2.0 7.2 90.8 -0.085 -0.562 Protein-coding genes-N 31.6 46.9 14.3 7.2 78.6 -0.194 0.329 1st codon position 32.2 40.9 20.3 6.6 73.1 -0.119 0.509 2nd codon position 18.7 50.5 16.8 14.1 69.2 -0.460 0.088 3rd codon position 44.1 49.4 5.6 0.9 93.5 -0.057 0.725 tRNAs 38.9 37.6 13.1 10.4 76.5 0.016 0.112 tRNA-J 39.2 37.0 12.6 11.2 76.2 0.029 0.059 tRNA-N 38.4 38.8 13.8 9.0 77.2 -0.005 0.210 rRNAs 39.6 41.4 12.7 6.2 81.0 -0.022 0.342 rrnL 42.2 39.6 6.0 12.2 81.8 0.033 -0.345 rrnS 40.0 39.7 6.7 13.6 79.7 0.003 -0.338 Control region 43.1 45.6 3.4 7.9 88.7 -0.028 -0.393

FIGURE 6. Phylogenetic relationships among the dobsonfly genera inferred from mt genome sequences. Numbers at the nodes are Bayesian posterior probabilities (left) and ML bootstrap values (right).

MITOCHONDRIAL GENOMES OF TWO ORIENTAL DOBSONFLIES Zootaxa 3964 (1) © 2015 Magnolia Press · 57 ……continued on the next page page next on the ……continued ; N, total innumber all proteins; N+, total commonly used codon for the amino acid. acid. amino the for codon used commonly N. exterior N. ; Nn.: ; Nn.: N. tonkinensis N. mt genomes. Nt.: Nevromus exterior exterior Nevromus and and N RSCU N+ RSCU N- RSCU N- RSCU N+ N RSCU 317 333 1.81 1.91 197 1.71 1.86 134 183 136 2 1.97 422 451 4.3 4.76 235 3.72 4.34 212 210 216 5.33 5.09 74 69 1.67 1.52 43 40 1.77 1.65 31 125 119 2.91 2.81 29 1.36 68 67 1.55 2.47 2.82 57 52 69 2.79 3.32 81 1.6 1.91 50 55 1.82 2.32 19 26 1.4 1.11 359 362 1.85 1.88 240 1.89 1.85 122 237 122 1.94 1.53 227 253 1.81 1.87 129 0.92 1.91 112 115 124 1.84 1.41 88 105 1.99 2.31 42 52 1.73 2.14 46 53 2.3 2.49 Neoneuromus tonkinensis Neoneuromus 24 17 0.19 0.13 7 6 1 17 0.09 11 1 0.16 33 15 0.19 0.09 31 15 0.29 0.14 2 0 0 0.03 58 29 0.59 0.31 27 56 0.5 0.99 2 2 0.05 0.05 30 23 0.15 0.12 19 25 0.2 5 0.15 4 0.06 0.06 UUU UUC UUG UUG 42 28 CUU 0.43 57 0.3 53 10 6 CUC 0.58 8 0.56 53 0.18 0.11 32 50 7 CUA 0.94 0.92 4 22 0.54 0.77 0.08 0.07 3 8 7 0.1 0.07 0.14 0.13 0 0 0 0 GUG 7 0.05 6 UCU 3 1 0.17 0.16 0.13 UCC 0 0.08 2 4 29 0 4 15 0.07 UCA 0.67 0.16 3 0.35 3 3 0.07 26 15 UCG 2 0.95 0.63 3 0.09 0.15 0 0 0.17 UUA 0 0 0 0 0.04 0 2 2 0 0.02 CUG AUU AUC AUA AUG GUU GUC 8 2 GUA 0.18 0.04 8 1 0.33 0.04 0 1 0 0.05 Codon usage of the usage Codon Underlined codons stand for the cognate codon of tRNA for each amino acid. amino for each tRNA of codon cognate the for stand codons Underlined number in J-strand; N-, total number in N-strand; RSCU, relative synonymous codon usage. Values in bold type stand for the most for the stand type bold in Values usage. codon synonymous relative RSCU, N-strand; in number total N-, J-strand; in number TABLE 10. TABLE Nt. Nt. Nn. acid Codon Nn. Nt. Nn. Nt. Nn. Nt. Nn. Nn. Nt. Amino (F) Phe Ser (S) (S) Ser Leu (L) (L) Leu Met (M) Ile (I) Val(V)

58 · Zootaxa 3964 (1) © 2015 Magnolia Press JIANG ET AL. ……continued on the next page 78 91 1.78 2.09 52 68 1.48 1.92 26 23 2.88 3.06 66 68 1.83 1.86 47 48 1.88 1.92 19 154 155 1.69 1.76 20 1.74 95 98 1.73 1.56 1.66 59 57 1.97 1.97 106 114 2.52 2.73 61 66 2.22 2.42 45 48 3.1 3.31 135 135 1.78 1.71 62 62 1.61 1.51 73 73 57 1.92 1.95 62 1.5 1.61 43 49 1.39 1.56 14 13 2 1.86 80 82 1.65 1.78 48 47 1.85 1.84 32 35 1.71 1.42 17 10 0.35 0.22 4 4 0.15 0.16 13 6 0.29 0.58 69 71 1.58 1.63 62 62 1.76 1.75 7 9 1.13 0.82 39 35 0.93 0.84 29 27 1.05 0.99 10 8 17 0.55 0.69 23 0.22 0.29 15 20 0.39 0.49 2 3 0.08 0.05 28 21 0.31 0.24 27 20 0.44 0.34 1 1 0.03 0.03 89 77 2.7 2.3 64 57 2.56 2.26 25 20 2.42 3.13 24 41 1.22 0.73 31 20 4 0.8 1.23 10 0.5 1.21 19 15 0.5 0.39 19 14 0.61 0.44 0 1 0 0.14 N CCU RSCU N+ RSCU N- RSCU CCC 15 CCC 13 0.39 0.45 11 15 0 0.6 0.44 CCA 2 0 0.24 CAG CAG 6 5 AAU 0.17 0.14 AAC 3 2 0.12 0.08 3 3 0.26 0.27 ACG ACG 3 0.14 3 1 2 0.07 0.07 0.07 GCC 0 0.04 1 2 22 3 0 15 0.07 GCA 0.52 0.06 0.08 1 0.36 1 3 0.02 20 15 GCG 0 0.73 0.55 2 0 0.12 0 UAU 0 0.14 UAC CAU CAC AAA AAG CCG CCG 4 3 ACU 0.12 0.09 1 2 0.04 0.08 3 1 0.12 0.38 CAA ACC ACC 25 9 ACA 0.57 0.21 25 9 0.71 0.25 0 0 0 0

(continued) (continued) TABLE 10. Nt. TABLE Nt. Nn. acid Codon Nn. Nt. Nn. Nt. Nn. Nt. Nn. Nn. Nt. Amino Pro (P) Thr (T) Thr (T) (Q) Gln Asn (N) Asn His (H) His (K) Lys Ala (A) GCU (Y) Tyr

MITOCHONDRIAL GENOMES OF TWO ORIENTAL DOBSONFLIES Zootaxa 3964 (1) © 2015 Magnolia Press · 59 58 61 1.68 1.77 36 39 1.53 1.66 22 22 2 2 73 73 1.85 1.82 46 45 2 1.96 27 28 1.64 1.65 1.64 27 28 2 45 1.96 46 1.82 1.85 73 33 36 3 25 30 1.71 3 64 1.69 1.91 1.89 65 10 13 1.84 1.76 1.33 1.73 23 90 94 23 2 1.92 34 36 2.43 2.57 27 28 2.53 3.11 7 8 1.6 0.62 106 119 1.88 2.07 73 79 2.18 2.31 33 40 1.72 1.43 76 79 1.77 1.86 28 31 2.63 1.31 48 80 76 48 2.58 1.42 4.24 1.32 41 44 1.22 1.28 39 32 1.7 1.38 11 8 0.32 0.23 11 8 0.47 0.34 0 0 6 0 0 2 0.31 0.11 5 2 0.67 0.27 1 0 0 0.08 5 5 0.12 0.12 4 4 0.15 0.17 1 1 0.05 0.06 GAC N GAU RSCU N+ RSCU N- RSCU GAG 6 7 0.15 0.17 0 1 0 0.04 6 6 0.36 0.35 6 6 0.36 0 0.04 1 0 0.17 6 7 0.15 GAG 0.29 5 1 UGU 10 1 0.09 3 UGC 2 0.16 8 0.24 12 UGG 0 0 0 0 0.2 0 0.22 2 CGU 16 0 0 5 0 2 0 1 0.44 0.14 17 CGC 1.14 0.09 0 1.21 1 8 0.07 6 CGA 6 1 0.43 0.75 0.67 8 CGG 11 2.2 0.71 AGU 37 37 AGC 0.86 0.87 17 16 0.62 0.67 20 21 1.13 1.17 GAA UGA AGA 0 0 0 0 0 0 0 0 AGG GGU GGC 2 2 GGA 0.04 0.03 2 2 0.06 0 0 0 0 (continued) (continued) TABLE 10. Nt. TABLE Nt. Nn. acid Codon Nn. Nt. Nn. Nt. Nn. Nt. Nn. Nn. Nt. Amino (D) Asp Cys (C) (C) Cys (R) Arg (S) Ser GGG 38 33 0.67 0.57 18 12 0.54 0.35 20 21 0.9 0.87 Gly (G) Gly Glu (E) Glu (W) Trp

60 · Zootaxa 3964 (1) © 2015 Magnolia Press JIANG ET AL. Phylogeny. The BI and ML analyses generated trees with the same topology (Fig. 6). Within Megaloptera, all five dobsonfly genera formed a monophyletic group. Protohermes was assigned to be the sister of the remaining dobsonflies which is generally consistent with the previous phylogenetic results based on the morphological data (Glorioso 1981; Penny 1993; Contreras-Ramos 2011). Neoneuromus + Nevromus was herein corroborated to be a sister group. However, Neoneuromus + Nevromus was not recovered to be the sister of Acanthacorydalis as suggested in Contreras-Ramos (2011). This lineage was assigned to be the sister of Acanthacorydalis + Corydalus. The presently recovered relationships among Neoneuromus, Nevromus, Acanthacorydalis, and Corydalus are somewhat concordant with the previous morphology-based phylogenetic results in Glorioso (1981) and Penny (1993). However, in all previous works, Acanthacorydalis that is the only genus in the Corydalinae with sexually dimorphic adult mandibles and has not been indicated as a sister taxon of Corydalus. Hence, the future mt phylogenomic study should include samples from all genera of Corydalinae to reconstruct a more robust phylogenetic framework of this subfamily.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (Nos. 31322501 and 31320103902) and the Foundation for the Author of National Excellent Doctoral Dissertation of PR China (No. 201178).

References

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