Research Article |
Corresponding author: Xiaolei Huang ( huangxl@fafu.edu.cn ) Academic editor: Brian Lee Fisher
© 2019 Junaid Ali Siddiqui, Zhilin Chen, Qiang Li, Jun Deng, Xiaolan Lin, Xiaolei Huang.
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:
Siddiqui JA, Chen Z, Li Q, Deng J, Lin X, Huang X (2019) DNA barcoding of aphid-associated ants (Hymenoptera, Formicidae) in a subtropical area of southern China. ZooKeys 879: 117-136. https://doi.org/10.3897/zookeys.879.29705
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As one of the most abundant and complex groups of terrestrial insects, ants have associations with many other organismal groups, such as hemipteran insects producing honeydew. With the aim of expanding the knowledge base of ant species associated with aphids, this study analyzed mitochondrial COI barcodes of 301 ant samples for 37 aphid-associated ant species in a subtropical area of southern China. Sequence analyses revealed that the intraspecific and interspecific distances ranged from zero to 7.7%% and 0.2 to 31.7%, respectively. Three barcoding approaches – Automatic Barcode Gap Discovery, Bayesian Poisson Tree Processes and Generalized Mixed Yule-coalescent – were used to help delimit ant species based on COI sequences, and their results corresponded well with most of the morphospecies. All three approaches indicate cryptic diversity may exist within Tetramorium bicarinatum and Technomyrmex albipes, with intraspecific genetic distances of 7.7% and 6.24%, respectively. Our analyses also reported five species for the first time from Fujian Province of China, and the COI sequences of nine species are newly added into the GenBank. This study provides information about species diversity of aphid-associated ants in subtropical China and compiles a DNA barcode reference library for future ant barcoding work.
cryptic diversity, DNA barcode, genetic distance, myrmecophily
Relationships between various organisms are crucial for upholding the ecological function of natural communities. The interactions between ants and aphids are classic examples of mutualism and are important to support ecosystem function (
Ants (Hymenoptera, Formicidae) are a dominant terrestrial insect group. They have colonized almost the entire world except Antarctica, especially in the tropical regions (
Biological classification based on morphological characters has been a routine practice to identify biodiversity on the Earth. Nowadays, biodiversity quantification is a challenge for taxonomist if only based on morphological identification. The recognition of minute anatomical differences between closely related species sometimes is complicated morphologically (
The present study aimed to investigate the subtropical ant fauna associated with aphids with the help of DNA barcoding. Both the morphological and DNA barcoding approaches were used and results were compared. Our study provides information of species composition and species diversity of ants in a subtropical region, and a DNA library for future ant barcoding work.
The ant specimens were collected from four localities (Fuzhou, Quanzhou, Shouning, Wuyishan) (Fig.
The ant species were identified by Dr Chen Zhilin and Dr Zhou Shanyi (Guangxi Normal University, Guilin 541004, China). Both of them have described more than 100 ant species up till now. Their knowledge and expertise help guarantee the reliability of the morphological identification. The voucher specimens have been stored at the Insect Systematics and Diversity lab at Fujian Agriculture and Forestry University.
DNA was isolated from the leg or whole ant body using the Qiagen DNeasy kit following the manufacturer’s protocols. Mainly a non-destructive DNA extraction method was used. In cases where numerous individuals from a colony were available, a destructive technique (entire ant crushed) was preferred. Polymerase chain reactions (PCR) were carried out in a total reaction volume of 50 µL containing 8 µL of dNTP mixture (2.5 mM), 5 µL of 10× PCR buffer (25 mM Mg2), 10 pmol of each primer and I unit of Taq DNA polymerase (TaKaRa Bio Inc., Otsu, Japan). The reaction conditions for the COI gene include: initial denaturation at 95 °C for 5 min; 35 cycles of 94 °C for 1 min, 50 °C for 1 min (denaturing) and 72 °C for 1 min (extension); a final elongation at 72 °C for 7 min reactions were done using the ProFlex PCR system. Standard primers used were: forward primer LepF1 (ATTCAACCAATCATAAAGATATTGG) and reverse primer LepR1 (TAAACTTCTGGATGTCCAAAAAATCA) (
A total of 301 sequences from our study (Suppl. material
Automatic Barcode Gap Discovery (ABGD) (
The maximum likelihood (ML) (
A BI tree was reconstructed under the GTR+I+G (
A total of 37 ant species associated with aphids were identified morphologically, belonging to 19 genera of three subfamilies, viz., Dolichoderinae (8), Formicinae (16) and Myrmicinae (13) (Suppl. material
The specimens collected from the Wuyishan Nature Reserve showed maximum species diversity up to 21 species, whereas the other two localities, Shouning and Fuzhou, had almost similar species diversity with 18 and 16 species respectively. The subfamily Myrmicinae had highest number of taxa in our study, with seven genera and 13 species occupying 55.48% of the total 301 samples. The genus Crematogaster was the most dominate group representing 23% of total samples. Moreover, three ant species P. punctatus, C. egidyi and P. noda showed the mostaphid associations with 17, 16 and 12 aphid species respectively (Suppl. material
Intraspecific divergences were calculated for all species except those with only one sequence while interspecific distances were calculated for subfamilies and genera using p-distance and K2P model (Table
COI K2P genetic distances for aphid-associated ant species in this study. Intraspecific distances were calculated within the same species and interspecific distances between species of same genus. Only species with two or more sequences were included.
Taxon name | Number of sequences | Intraspecific distance | Interspecific distance | Number of haplotypes | ||
---|---|---|---|---|---|---|
min. | max. | mean | range | |||
Dolichoderinae | 0.137–0.27 | |||||
Iridomyrmex anceps | 7 | 0 | 0 | 0 | 1 | |
Liometopum sinense | 2 | 0 | 0 | 0 | 1 | |
Ochetellus glaber | 6 | 0 | 0.005 | 0.002 | 2 | |
Tapinoma melanocephalum | 9 | 0 | 0.034 | 0.008 | 0.139–0.258 | 2 |
Technomyrmex albipes | 8 | 0 | 0.062 | 0.033 | 2 | |
Formicinae | 0.048–0.316 | |||||
Camponotus japonicus | 9 | 0 | 0.007 | 0.003 | 0.195–0.251 | 5 |
Camponotus mitis | 2 | 0.008 | 0.008 | 0.195–0.251 | 2 | |
Camponotus nicobarensis | 5 | 0 | 0.002 | 0.001 | 0.195–0.251 | 2 |
Formica japonica | 14 | 0 | 0 | 0 | 0.002–0.005 | 1 |
Formica sinae | 12 | 0 | 0.005 | 0.002 | 0.002–0.005 | 6 |
Lepisiota rothneyi | 2 | 0 | 0 | 0 | 0.002–0.005 | 1 |
Nylanderia flavipes | 10 | 0 | 0.022 | 0.010 | 0.139–0.222 | 2 |
Nylanderia bourbonica | 11 | 0 | 0.002 | 0.001 | 0.139–0.222 | 2 |
Nylanderia flaviabdominis | 6 | 0 | 0.013 | 0.007 | 0.139–0.222 | 2 |
Polyrhachis dives | 13 | 0 | 0.015 | 0.006 | 0.175–0.184 | 3 |
Polyrhachis illaudata | 3 | 0 | 0.002 | 0.001 | 0.175–0.184 | 2 |
Prenolepis emmae | 6 | 0 | 0.003 | 0.001 | 3 | |
Myrmicinae | 0.056–0.317 | |||||
Aphaenogaster smythiesii | 2 | 0 | 0 | 0 | 1 | |
Crematogaster egidyi | 53 | 0 | 0.015 | 0.007 | 0.056–0.229 | 6 |
Crematogaster osakensis | 5 | 0 | 0.003 | 0.001 | 0.056–0.229 | 3 |
Crematogaster rogenhoferi | 10 | 0 | 0.005 | 0.002 | 0.056–0.229 | 2 |
Monomorium chinense | 3 | 0 | 0 | 0 | 0.175 | 1 |
Pheidole fervida | 2 | 0 | 0 | 0 | 0.167–0.199 | 1 |
Pheidole noda | 30 | 0 | 0.020 | 0.009 | 0.167–0.199 | 5 |
Pristomyrmex punctatus | 46 | 0 | 0.058 | 0.016 | 8 | |
Tetramorium wroughtonii | 2 | 0 | 0 | 0 | 0.169–0.218 | 1 |
Tetramorium bicarinatum | 9 | 0 | 0.077 | 0.043 | 0.169–0.218 | 2 |
Tetramorium caespitum | 3 | 0 | 0.020 | 0.013 | 0.169–0.218 | 2 |
Total | 0.048–0.345 |
The ABGD approach produced 39 molecular operational taxonomic units (MOTUs) or genetic groups. Among them, 35 MOTUs matched with the morphospecies identification, which represented 89.7% of the morphospecies in total. The other four MOTUs might indicate species differentiation for some morphospecies (Fig.
Maximum likelihood haplotype tree for the COI gene. Bootstrap values higher than 50 are displayed. Color strips on the right side represent the MOTUs produced by ABGD, bPTP and GMYC methods; extreme right one indicates the morphologically identified species. Black square around some bars indicates differences between the MOTUs and morphospecies. Values inside the square indicate the number of MOTUs produced by different approaches.
ML and Bayesian Inference analysis applied to all 303 sequences along with two outgroups created monophyletic groups. As the phylogenetic trees (Figs
Ants are eusocial insects having the high degree of caste polymorphism with various distinct anatomical characters and size variations (
Most of the morphospecies identified were supported by DNA barcoding approaches. According to the
In the present study, three ant species (P. punctatus, C. egidyi and P. noda) were found associated with a maximum number of aphid species on various host plants (Suppl. material
Species delineation and identification on the basis of DNA sequence distance analysis, like the DNA barcoding gap (
The comparative performance of different algorithms to species delineation has been studied previously. ABGD considered as the most computationally effective approach. It needs a priori specification of an intraspecific distance threshold, and this method is based on the genetic distances calculated from a single locus (
The ML and BI phylogenetic analysis produced almost same topologies on the basis of the COI sequences and produced two discrete clades. One clade included two putative sister clades representing the subfamilies Myrmicinae and Formicinae (Figs
We would like to thank help from Junjie Li, Qian Liu, Muhammad Qasim, Habib Ali and Mubasher Hussain during this work. This work was supported by National Key R&D Program of China (2016YFE0203100) and Open Fund of Fujian Provincial Key Laboratory of Insect Ecology, Fujian Agriculture and Forestry University.
Table S1. Ant and associated aphid species collected from different localities of Fujian Province of southern China
Data type: species data
Table S2. Analyzed samples of ant species with information on sampling location, GenBank accession number, BOLD process ID, morphological species identification, collection date, voucher specimen number, latitude and longitude
Data type: species data
Figure S1. Line chart of genetic distance of different taxonomic level based on p-distance and K2P model
Data type: molecular data
Figure S2. Bayesian Inference tree of the 301 COI sequences from our study
Data type: phylogenetic tree
Explanation note: The values besides the branches indicate Bayesian posterior probabilities. The dotted lines indicate the outgroups.