Network structure variation across scales offers clues to the macroevolutionary persistence of specialised mutualisms
Data files
Sep 11, 2025 version files 629.15 KB
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8sites_epicephala.nexus
472.16 KB
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8sites_epicephala.nexuss.con.tre
113.99 KB
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Bawang.csv
144 B
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betalink_subreg_figures_supplementary.R
8.60 KB
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betalink_turnover.R
5.06 KB
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China.csv
651 B
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Chinese_networks_H2fun.R
1.36 KB
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Chinese_networks_NODF.R
3.08 KB
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continent.csv
219 B
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Daming.csv
90 B
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Diaoluo.csv
95 B
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Dinghu.csv
64 B
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H2fun_randomizations.R
2.13 KB
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Hainan.csv
461 B
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Jianfeng.csv
152 B
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latlong.xlsx
9.14 KB
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Nonghua.csv
104 B
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README.md
10.79 KB
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site_distances.csv
599 B
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Xinglong.csv
127 B
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Yingge.csv
135 B
Abstract
This is a study of the diversity and species-specificity of a plant-insect mutualism that uses multilocus molecular phylogenetic and species interaction network analyses. Field sampling collected larvae of leafflower moths (Lepidoptera: Gracillariidae: Epicephala spp.) from different host species of leafflower trees (Phyllanthaceae: Glochidion) at eight sites in southern China (Guangzhou and Hainan provinces and Guangxi Zhuang Autonomous Region). Larvae were preserved in ethanol or reared to adulthood (and the resulting adults preserved in ethanol) for DNA extractions; collection site and host plant species were recorded for each individual moth collected. DNA was Sanger-sequenced for three loci (mitochondrial cytochrome oxidase I, COI; nuclear arginine kinase, ArgK; nuclear elongation-factor 1-alpha, EF1-α). Phylogenetic inference was conducted on these samples. Minimally monophyletic clades with posterior probabilities >0.90 identified in the phylogenetic analyses were treated as candidate “species” of leafflower moths in the subsequent network analyses. Local quantitative species interaction networks for each site were manually drawn based on the phylogenetic tree, in which each OTU (tip on phylogeny) represented a single interaction event between that Epicephala moth “species” and its host Glochidion tree species. Network structural analyses were then performed with the resulting local networks and the combined regional networks for the five sites on the island of Hainan, the three sites in continental China, and all eight sites together. Turnover analyses on the dataset were also performed.
Dataset DOI: 10.5061/dryad.3bk3j9kx6
Description of the data and file structure
Files and variables
File: 8sites_epicephala.nexus
Description: Nexus file containing the complete DNA sequence alignment generated by and used in this study for phylogenetic analysis in MrBayes (Ronquist and Huelsenbeck 2003), representing 241 individual Epicephala moth specimens (of which 240 specimens were newly collected and sequenced for this study). Data were Sanger-sequenced for three loci (mitochondrial cytochrome oxidase I, COI; nuclear arginine kinase, ArgK; nuclear elongation-factor 1-alpha, EF1-alpha). Each OTU (or “taxlabel”) in the alignment corresponds to an individual Epicephala specimen. The labeling system for the OTUs provides collection information about the specimens. For example, the DNA sequence name “ex_Glochidion_acuminatum_12_2_ZLJ039_Jianfeng_Hainan_China” indicates the host Glochidion species (Glochidion_acuminatum), the individual tree from which larvae were collected (voucher specimen number ZLJ039, which is also tree number 12 from that site), the individual larva from which DNA was extracted (e.g., 12_2 means larva number 2 from tree number 12), and the sampling site (Jianfengling, Hainan).
All sequence data are new to this study except the outgroup, an Epicephala moth collected from Kirganelia reticulata (previously known as Phyllanthus reticulatus) in a past study (Kawakita and Kato 2009). Novel sequence data generated by this study have also been uploaded to GenBank.
Following the data alignment, the first MrBayes block specifies the outgroup and data partition (by locus and codon), the second block specifies the model of sequence evolution identified by PartitionFinder (Lanfear et al. 2012), and the third block specifies the Bayesian phylogenetic analysis parameters for analysis in MrBayes.
File: betalink_subreg_figures_supplementary.R
Description: R code for analyzing and plotting turnover of species and interaction links among sites within the two subregions (continental China and Hainan) examined in this study.
File: betalink_turnover.R
Description: R code for analyzing and plotting turnover of species and interaction links among the eight sites examined in this study.
File: Chinese_networks_H2fun.R
Description: R code for calculating reciprocal specialization (H2’) sensu Blüthgen et al. (2006) of networks generated in this study.
File: Chinese_networks_NODF.R
Description: R code for calculating nestedness (NODF; Almeida-Neto et al. 2008) of networks generated in this study.
File: 8sites_epicephala.nexuss.con.tre
Description: Bayesian consensus tree generated in MrBayes (Ronquist and Huelsenbeck 2003) from the provided sequence alignment (“8sites_epicephala.nexus”). Tips represent individual Epicephala specimens. Node labels represent posterior probabilities. This tree was used to generate minimally monophyletic clades (posterior probabilities >0.90) which were taken as Epicephala “species” for the purposes of the subsequent network analyses.
File: H2fun_randomizations.R
Description: R code for calculating p-values for H2’ for networks generated in this study; code provided by Ryosuke Nakadai (R. Nakadai, Yokohama National University, pers. comm., 2022).
File: site_distances.csv
Description: Distances between each pair of sites examined in this study, used in turnover analyses.
Variables
- name_i: First site
- name_j: Second site
- distij: Distance in kilometers
File: latlong.xlsx
Description: Latitudes and longitudes for the eight study sites in this study, used to calculate distances between sites.
Variables
- latitude and longitude
General information about .csv network files:
Eleven .csv files are provided containing local and regional species interaction networks generated by and used in this study. Rows are labeled by species epithets of Glochidion species, as described by current taxonomy (leftmost column contains these names). Note that G. zeylanicum and G. hirsutum (also called G. zeylanicum var. hirsutum by some authors) are considered conspecific in our study. Columns represent Epicephala taxa, corresponding to minimally monophyletic groups with Bayesian posterior probabilities >0.90 recovered in this study, and treated as “species” of Epicephala in the network analyses. Epicephala taxa are identified by numbers, following the numbers used in the figures in the Chinese-language master’s thesis by Lian-Jie Zhang (2017). Please note that our numbering system for these Epicephala clades follows that of the figures in Zhang (2017) and bears no relationship to any numbers assigned to undescribed Epicephala species in prior works by the present authors or anyone else. In a few cases, the Zhang (2017) analysis found clades that the present analysis has split into two based on the posterior probabilities recovered in the present study’s phylogenetic analysis (e.g., clade 26 in Zhang 2017 is clades 26A and 26B in the present study) or the Zhang (2017) analysis found separate clades that form a single minimally monophyletic clade in the present study (e.g., clades 10 and 11 in Zhang 2017 is clade 10_11 in the present study, using an underscore to combine the two numbers from Zhang 2017).
All networks in this folder are quantitative, with numbers in each cell recording the number of interaction events between the two taxa. For instance, a number of 4 means that 4 Epicephala larvae from that Epicephala clade were collected from that host plant species.
File: Bawang.csv
Description: Quantitative network from Bawangling, Hainan.
File: China.csv
Description: Quantitative network from all eight sites in this study, created by summing all eight networks together.
File: continent.csv
Description: Quantitative network from the three sites on the Asian continent (Daming, Dinghu, and Nonghua; in Guangdong Province and Guangxi Zhuang Autonomous Region), created by summing all three of those constitutive networks together.
File: Daming.csv
Description: Quantitative network from Daming Shan, Guangxi.
File: Diaoluo.csv
Description: Quantitative network from Diaoluo Shan, Hainan.
File: Dinghu.csv
Description: Quantitative network from Dinghu Shan, Guangdong.
File: Hainan.csv
Description: Quantitative network from the five sites on the island province of Hainan (Bawang, Diaoluo, Jianfeng, Xinglong, and Yingge), created by summing all five of those constitutive networks together.
File: Jianfeng.csv
Description: Quantitative network from Jianfengling, Hainan
File: Xinglong.csv
Description: Quantitative network from Xinglong Qiaoxiang National Forest Park, Hainan
File: Nonghua.csv
Description: Quantitative network from Nonghua Water Source Forest Reserve, Guangxi
File: Yingge.csv
Description: Quantitative network from Yinggeling, Hainan
References:
Almeida-Neto M, Guimarães P, Guimarães PR Jr, Loyola RD, Ulrich W. 2008 A consistent metric for nestedness analysis in ecological systems: reconciling concept and measurement. Oikos 117, 1227–1239. (doi:10.1111/j.0030-1299.2008.16644.x)
Blüthgen N, Menzel F, Blüthgen N. 2006 Measuring specialization in species interaction networks. BMC Ecol. 6, 9. (doi:10.1186/1472-6785-6-9)
Dormann CF, Fründ J, Blüthgen N, Gruber B. 2009 Indices, graphs and null models: analyzing bipartite ecological networks. Open Ecol. J. 2, 7–24.
Dormann CF, Gruber B, Fründ J. 2008 Introducing the bipartite Package: Analysing Ecological Networks. R News 8, 8–11.
Kawakita A, Kato M. 2009 Repeated independent evolution of obligate pollination mutualism in the Phyllantheae-Epicephala association. Proc. Biol. Sci. 276, 417–426. (doi:10.1098/rspb.2008.1226)
Lanfear R, Calcott B, Ho SYW, Guindon S. 2012 Partitionfinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Mol. Biol. Evol. 29, 1695–1701. (doi:10.1093/molbev/mss020)
Marquitti FMD, Guimarães PR Jr, Pires MM, Bittencourt LF. 2014 MODULAR: software for the autonomous computation of modularity in large network sets. Ecography 37, 221–224. (doi:10.1111/j.1600-0587.2013.00506.x)
Oksanen J et al. 2019 vegan: Community Ecology Package. See https://CRAN.R-project.org/package=vegan.
Poisot T, Canard E, Mouillot D, Mouquet N, Gravel D. 2012 The dissimilarity of species interaction networks. Ecol. Lett. 15, 1353–1361. (doi:10.1111/ele.12002)
Ronquist F, Huelsenbeck JP. 2003 MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. (doi:10.1093/bioinformatics/btg180)
Zhang, L. 2017 Variation in network structure of a specialized pollination mutualism between leafflower (Phyllanthaceae, Glochidion) and leafflower moths (Gracillariidae, Epicephala) across different communities. M.Sc., University of the Chinese Academy of Sciences, South China Botanical Garden.
Code/software
Code and package versions:
Phylogenetic model selection: PartitionFinder v. 1 (Lanfear et al. 2012)
Phylogenetic inference: MrBayes v. 3.1 (Ronquist and Huelsenbeck 2003)
Modularity analyses: MODULAR (Marquitti et al. 2014)
Reciprocal specialization (H2’) analyses: Package bipartite v. 2.15 (Dormann et al. 2008, 2009) implemented in R v. 3.6.2
Nestedness analyses: Package vegan v. 2.5-5 (Oksanen et al. 2019) implemented in R v. 3.6.2
Turnover analyses: Function betalinkr_multi (Poisot et al. 2012) in package bipartite v. 2.15 (Dormann et al. 2008, 2009) implemented in R v. 3.6.2
Access information
Other publicly accessible locations of the data:
- Sequence data newly generated for this study will be submitted to GenBank.
- Species interaction network data generated for this study will be submitted to a suitable species interaction network repository as well (to be determined)
Data was derived from the following sources:
- DNA sequence data from one OTU (an Epicephala moth collected from the plant Kirganelia reticulata, formerly known as Phyllanthus reticulatus) was obtained from the following study and from GenBank (https://www.ncbi.nlm.nih.gov/genbank/; accession numbers FJ235506, FJ235407, and FJ235383).
- Kawakita A, Kato M. 2009 Repeated independent evolution of obligate pollination mutualism in the Phyllantheae-Epicephala association. Proc. Biol. Sci. 276, 417–426. (doi:10.1098/rspb.2008.1226)