DOI: 10.5061/dryad.xwdbrv1k5
title: Evolution repeats itself in replicate long-term studies in the wild

description: The extent to which evolution is repeatable remains debated. Here we study
 changes over time in the frequency of cryptic color-pattern morphs in 10
 replicate long-term field studies of a stick-insect, each spanning at
 least a decade (across 30 years of total data). We find predictable
 ‘up-and-down’ fluctuations in stripe frequency in all populations,
 representing repeatable evolutionary dynamics based on standing genetic
 variation. A field experiment demonstrates that these fluctuations involve
 negative frequency-dependent natural selection (NFDS). These fluctuations
 rely on demographic and selective variability that pushes populations away
 from equilibrium, such that they can reliably move back towards it via
 NFDS. Finally, we show that the origin of new cryptic forms is associated
 with multiple structural genomic variants such that which mutations arise
 affects evolution at larger temporal scales. Thus, evolution from existing
 variation is predictable and repeatable, but mutation adds complexity even
 for traits evolving deterministically under natural selection.
description: We compiled data on morph frequencies in <em>Timema
 cristinae</em> using samples collected in the spring using sweep
 nets between 1990 and 2023. All individuals were scored as
 'striped', 'unstriped', or 'melanistic', or
 occasionally when it was difficult to distinguish between the first two
 categories as 'intermediate-striped'.  Samples from 1990 to 1999
 were collected and scored by Cristina Sandoval, who then trained PN in
 2000. PN collected and scored most samples from 2000 to 2023. The
 host-plant collected on was recorded for all individuals. 
 Datasets from two field experiments are included as
 well. <strong>Experiment 1</strong>. A
 total of 513 individuals were collected from populations LA (latitude
 34.41, longitude -119.80), PRC (latitude 34.53, longitude -119.86), PRNC
 (latitude 34.53, longitude -119.85),  VPC (latitude 34.53, longitude
 -119.85), and WTA (latitude 34.52, longitude -119.78) between April 30th
 and May 4th, 2021. Numbers were as follows: green-unstriped morphs, LA 17,
 PRC 64, PRNC 58, VPC 71, WTA 48; striped morphs, LA 136, PRC 0, PRNC 3,
 VPC 26, WTA 90. Individuals were randomly assigned to one of 21 treatments
 (n = 20 stick insects per treatment), which varied the initial stripe
 frequency from 0% (0 striped, 20 unstriped) to 100% (20 striped, 0
 unstriped) in 5% increments. Each of these treatments of 20 individuals
 was then randomly assigned to one of 21 experimental bushes (near site N2,
 in the general area of latitude 34.51 and longitude -119.80). Each bush
 was cleared of existing <em>T. cristinae</em>  by sampling it
 before release. Individuals were released on the morning of May 5th by
 dumping them from containers; the insects cling to the branches when
 released in this way because of a strong clinging reflex. Individuals were
 recaptured using visual surveys and sweep nets on the evening of May 7th
 and scored as striped or unstriped.
 <strong>Experiment 2</strong>. We conducted a second
 field transplant experiment to ask whether striped <em>T.
 cristinae</em> morphs exhibit higher fitness on
 <em>Adenostoma</em> than melanic morphs. For this experiment,
 stick insects were collected from a single location, PRNC (latitude 34.53,
 longitude -119.85). Stick insects for each pair of eight experimental
 blocks were captured on a single day in 2023: May 9th for blocks 1 and 2,
 May 11th for blocks 3 and 4, May 13 for blocks 5 and 6, and May 15 for
 blocks 7 and 8. For each block, 10 striped and 10 melanic stick insects (n
 = 20 total stick insects per block) were released onto a single
 <em>Adenostoma</em> bush at the experimental transplant site
 (latitude 34.51, longitude = -119.80, as in the first experiment) the day
 after they were collected. Two (blocks 1-6) or three (blocks 7 and 8) days
 after release, surviving stick insects were recaptured using visual
 surveys and sweep nets and scored as striped or melanic. 
 We used genome-wide association (GWA) mapping to identify regions
 of the genome associated with color-pattern (striped vs. unstriped)
 variation in <em>T. cristinae</em>. For this, we used a
 previously published DNA sequence data set comprising partial genome
 sequences (genotyping-by-sequencing or GBS data) for 602 <em>T.
 cristinae</em> from a single population (FHA, latitude 34.52,
 longitude -119.80).
description: All datasets are provided as ASCII text files. 
description: # Dataset: Evolution repeats itself in replicate long-term studies in the
 wild --- We compiled data on morph frequencies inTimema cristinae using
 samples collected in the spring using sweep nets between 1990 and 2023.
 All individual were scored as 'striped', 'unstriped',
 or 'melanistic', or occasionally when it was difficult to
 distinguish between the first two categories as
 'intermediate-striped'. Samples from 1990 to 1999 were collected
 and scored by Cristina Sandoval, who then trained PN in 2000. PN collected
 and scored most samples from 2000 to 2023. The host-plant collected on was
 recorded for all individuals. Datasets from two field experiments are
 included as well. Experiment 1. A total of 513 individuals were collected
 from populations LA (latitude 34.41, longitude -119.80), PRC (latitude
 34.53, longitude -119.86), PRNC (latitude 34.53, longitude -119.85), VPC
 (latitude 34.53, longitude -119.85), and WTA (latitude 34.52, longitude
 -119.78) between April 30th and May 4th, 2021. Numbers were as follows:
 green-unstriped morphs, LA 17, PRC 64, PRNC 58, VPC 71, WTA 48; striped
 morphs, LA 136, PRC 0, PRNC 3, VPC 26, WTA 90. Individuals were randomly
 assigned to one of 21 treatments (n = 20 stick insects per treatment),
 which varied the initial stripe frequency from 0% (0 striped, 20
 unstriped) to 100% (20 striped, 0 unstriped) in 5% increments. Each of
 these treatments of 20 individuals was then randomly assigned to one of 21
 experimental bushes (near site N2, in the general area of latitude 34.51
 and longitude -119.80). Each bush was cleared of existing T. cristinae by
 sampling it before release. Individuals were released on the morning of
 May 5th by dumping them from containers; the insects cling to the branches
 when released in this way because of a strong clinging reflex. Individuals
 were recaptured using visual surveys and sweep nets on the evening of May
 7th and scored as striped or unstriped. Experiment 2. We conducted a
 second field transplant experiment to ask whether striped T. cristinae
 morphs exhibit higher fitness on Adenostoma than melanic morphs. For this
 experiment, stick insects were collected from a single location, PRNC
 (latitude 34.53, longitude -119.85). Stick insects for each pair of eight
 experimental blocks were captured on a single day in 2023: May 9th for
 blocks 1 and 2, May 11th for blocks 3 and 4, May 13 for blocks 5 and 6,
 and May 15 for blocks 7 and 8. For each block, 10 striped and 10 melanic
 stick insects (n = 20 total stick insects per block) were released onto a
 single Adenostoma bush at the experimental transplant site (latitude
 34.51, longitude = -119.80, as in the first experiment) the day after they
 were collected. Two (blocks 1-6) or three (blocks 7 and 8) days after
 release, surviving stick insects were recaptured using visual surveys and
 sweep nets and scored as striped or melanic. We used genome-wide
 association (GWA) mapping to identify regions of the genome associated
 with color-pattern (striped vs unstriped) variation in T. cristinae. For
 this we used a previously published DNA sequence data set comprising
 partial genome sequences (genotyping-by-sequencing or GBS data) for 602 T.
 cristinae from a single population (FHA, latitude 34.52, longitude
 -119.80). ## Description of the data and file structure The dataset
 consists of the following plain text files: Tcris_master_32.txt = Timema
 cristinae stripe frequency time-series dataset. Column headers describe
 each field. The core data are the number of each color morph counted as
 given in the columns unstriped (green), striped, melanistic and
 intermediate. Other columns give basic collection information: location
 IDs, collection year, and latitude and longitude coordinates in decimal
 degrees, elevation in meters (elevation_Clarissa_3), host plant ID (host),
 and whether the site is from Refugio mountain (1) or not (0) (refugio_yn);
 climatic data (seasonal mean temperatures and precipitation, in degrees
 Fahrenheit and inches, respectively); and summaries calculated from the
 raw count data (total, proportion_melanistic, and proportion_striped).
 Empty cells correspond with data entries that are not applicable (NA).
 TcristinaeGradient.csv = data for experiment 1. This text file gives the
 number of striped (Str) and green (Uns) stick insects released (0) and
 recaptured (1), along with block numbers and the total number of stick
 insects recaptured for each block. SelectionMelanicStripe.txt = data for
 experiment 2. This text file gives the number of stripe and melanic stick
 insects recaptured for each numbered replicate. fha2013_gs_bin.pheno =
 stripe phenotypic data for association mapping. There is one row per stick
 insect with each row containing a 0 or 1 to denote unstriped vs striped
 morphs respectively. This was used for analyses with the striped genome.
 fha2013_gus_bin.pheno = stripe phenotypic data for association mapping.
 There is one row per stick insect with each row containing a 0 or 1 to
 denote unstriped vs striped morphs respectively. This was used for
 analyses with the green genome. fha2013_gen_gs.geno and
 fha2013_gen_gus.geno = genotype files for genome wide association mapping
 with gemma. The files differ in terms of the reference genome use, with gs
 referring to the striped genome and gus the green unstriped genome. These
 files have one row per SNP, with the SNP ID, two arbitrary characters, and
 the Bayesian genotype estimates for each individual. The order of
 individuals matches the pheno file. ## Sharing/Access information
 Information on data processing and associated scripts will be available
 from GitHub:
 [https://github.com/zgompert/RepeatableEvolution](https://github.com/zgompert/RepeatableEvolution)