DOI: 10.5061/dryad.3bk3j9kx6
title: Network structure variation across scales offers clues to the macroevolutionary persistence of specialised mutualisms

description: 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 &gt;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.
description: # Network structure variation across scales offers clues to the
 macroevolutionary persistence of specialised mutualisms Dataset DOI:
 [10.5061/dryad.3bk3j9kx6](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
 &gt;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 &gt;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](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/](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)