DOI: 10.5061/dryad.mpg4f4r53
title: Data from: Inferring the evolution of reproductive isolation in a lineage of fossil threespine stickleback, Gasterosteus doryssus

description: Darwin attributed the absence of species transitions in the fossil record
 to his hypothesis that speciation occurs within isolated habitat patches
 too geographically restricted to be captured by fossil sequences. Mayr’s
 peripatric speciation model added that such speciation would be rapid,
 further explaining missing evidence of diversification. Indeed, Eldredge
 and Gould’s original punctuated equilibrium model combined Darwin’s
 conjecture, Mayr’s model, and 124 years of unsuccessfully sampling the
 fossil record for transitions. Observing such divergence, however, could
 illustrate the tempo and mode of evolution during early speciation. Here,
 we investigate peripatric divergence in a Miocene stickleback fish,
 Gasterosteus doryssus. This lineage appeared and, over ~8,000 generations,
 evolved significant reduction of twelve of sixteen traits related to
 armor, swimming, and diet, relative to its ancestral population. This was
 greater morphological divergence than we observed between reproductively
 isolated, benthic-limnetic ecotypes of extant Gasterosteus aculeatus.
 Therefore, we infer that reproductive isolation was evolving. However,
 local extinction of low-armoured G. doryssus lineages shows how young
 isolate populations often disappear, supporting Darwin’s explanation for
 missing evidence and revealing a mechanism behind morphological stasis.
 Exctinction may also account for limited sustained divergence within the
 stickleback species complex and help reconcile speciation rate variation
 observed across time scales.
description: (a) Fossil specimen data To quantify
 morphological divergence in the fossils, we compiled published data from
 the D (Bell et al. 1985), L (Bell et al. 2006), and K series (Stuart et
 al. 2020, Voje et al. 2022). See Figure 1A for stratigraphic correlations
 among series. Series D consisted of 26 samples spaced at ~5,000-year
 intervals over an estimated 108,275 years (Figure 1A). Six traits were
 measured from D: standard length, pelvic score, and the number of
 pre-dorsal pterygiophores, dorsal spines, anal-fin rays, and dorsal-fin
 rays (Bell et al. 1985). Series L comprised a section starting ~4,500
 years before the replacement event until ~16,500 years after and was
 sampled continuously. Three armour traits were measured for series L:
 pelvic score, number of dorsal spines, and number of touching pre-dorsal
 pterygiophores. This series confirmed replacement of lineage I by lineage
 II within ~125 years (Bell et al. 2006) and that subsequent evolution of
 lineage II was probably caused by directional natural selection (Hunt et
 al. 2008). Our analysis focuses on series K (Stuart et al. 2020; Voje et
 al. 2022) because 16 traits were measured for K (Table S1), allowing
 estimates of multivariate divergence of traits that should reflect
 swimming, feeding, and defense. These traits are also divergent between
 benthic and limnetic ecotypes in the species-pair lakes as well as among
 allopatric generalist populations of <em>G. aculeatus</em>
 (Walker 1997; Spoljaric and Reimchen 2007; Willacker et al. 2010). Series
 K consisted of 18 samples taken at ~1000-year intervals, and mean sample
 times span ~16,363 years. Series K starts at the replacement, when lineage
 I and lineage II specimens occurred in a single sample. We removed lineage
 I fish from this sample for morphological analysis. Traits measured and
 their sample sizes are provided in Table S1 and Table S2, respectively.
 Finally, we note that a parapatric, highly armored form with a benthic
 diet (Bell 2009) was collected from Quarry E (of Brown 1986),
 approximately 1.7km from the depositional environment studied here. This
 site was dated to roughly the same time as the K series (Brown 1986) and
 appears to have been a nearshore sample, based on abundant terrestrial
 plant fossils and thick clastic layers embedded within the diatomite (Bell
 2009). This contrasts the open water habitat that characterized the
 depositional environment of series D, L, and K (Bell 2009). 
 (b) Extant specimen data For comparison to
 fossil divergence, we measured benthic and limnetic ecotypes from five
 species-pair lakes (Table 2) for the same 16 traits that were scored in
 the K-series fossils (Table S1). Specimens were loaned by D. Schluter’s
 lab (University of British Columbia). They collected from Enos Lake in
 1988 and from Emily, Little Quarry, Paxton, and Priest Lakes in 2018. The
 Enos specimens were fixed in formalin and stored in 40% isopropanol. The
 other specimens were initially preserved in 95% ethanol in the field
 before being transferred to water then formalin in the lab and stored in
 40% isopropanol. In 2019, we stained these specimens for bone using
 Alizarin Red. Standard length as well as pelvic-spine lengths were
 measured with calipers. We used a dissection microscope to count dorsal
 spines, pelvic spines, dorsal-fin rays, and anal-fin rays. Right and
 left-side pelvic girdle and ectocoracoid lengths were measured from
 ventral photographs (Canon EOS Rebel T7, Tamron 16-300 mm MACRO lens,
 Kaiser RS1 copy stand). Lateral X-rays were used to measure dorsal spine
 length, number of pterygiophores anterior to the pterygiophore holding the
 third spine, length of the pterygiophore just anterior to the third spine,
 cleithrum length, and pre-maxilla ascending branch length. We also counted
 vertebrae from X-rays: abdominal vertebrae were counted anterior to the
 first vertebra with a haemal spine contacting an anal fin pterygiophore.
 Caudal vertebrae were posterior, including the first vertebra with the
 haemal spine contacting the anal fin pterygiophore (Aguirre et al. 2014).
 X-rays were taken with an AXR Hot Shot X-ray Machine (Associated X-ray
 Corporation) at the Field Museum of Natural History. Specimens were
 exposed at 35kV and 4mA for 7 to 10 seconds. We developed the film and
 scanned individual fish images using the B&W Negatives setting on
 an Epson Perfection 4990 Photo flatbed at 2400 dpi. Measurements from
 photographs and X-rays were taken with FIJI (Schindelin et al. 2012) and
 its plugin ObjectJ (https://sils.fnwi.uva.nl/bcb/objectj/). Table S3 reports sample sizes by population, ecotype, and trait.
description: Microsoft Excel is helpful to open the data files.
 R and R Studio are necessary to run the analysis
 scripts.
description: # Inferring the evolution of reproductive isolation in a lineage of fossil
 threespine stickleback, Gasterosteus doryssus Threespine stickleback data:
 Fossil and Species Pair Gasterosteus spp. --- The data are comprised of
 morphological traits (continuous, count, index) collected from samples of
 a fossil lineage of threespine stickleback (Gasterosteus doryssus) from a
 paleo lake in Nevada (Bell et al. 1985; Bell et al. 2006; Stuart et al.
 2020) and extant threespine stickleback (Gasterosteus aculeates) from five
 lakes on Vancouver Island. ## Description of the data and file structure
 RS.speciationPruned.for.sizecorrection.csv contains fossil data (Fos in
 the universal ID column) structured as temporal samples where T0 is time
 zero, k.19 is the next oldest sample, proceeding to K.03 as the youngest
 sample closest to the present. The mean age of each sample in years,
 relative to the first sample, is available in the the second column of
 KSampleMeanTimes.csv. RS.speciationPruned.for.sizecorrection.csv also
 contains data from extant stickleback (any of the non Fos fish in the data
 frame). They are organized by species pair (Emily, Enos, Little Quarry,
 Paxton, Priest), and by ecotype (Benthic or Limnetic). There is also
 information on sex (M or F). The column codes for
 RS.speciationPruned.for.sizecorrection.csv are: univID: specimen ID where
 first three characters are population, fifth character is ecotype. stl:
 standard length in mm ips: pelvic score index (described in further detail
 in Stuart et al. 2020) mds: number of dorsal spines mdf: number of fin
 rays in the dorsal fin maf: number of fin rays in the anal fin lps: length
 of the pelvic spine (mm) Ips.prop: ips divided by the number of elements
 in the ips score ect: length of the ectocoracoid (mm) tpg: length of the
 pelvic girdle (mm) mav: number of abdominal vertebrae mcv: number of
 caudal vertebrae mpt: number of pre dorsal pterygiophores cle: length of
 the cleithrum (mm) pmx: length of the ascending branch of the pre maxilla
 (mm) ds1: length of the first dorsal spine (mm) ds2: length of the second
 dorsal spine (mm) ds3: length of the third dorsal spine (mm) lpt: length
 of the pterygiophore anterior to the pterygiophore containing the 3rd
 spine (mm) Further detail is provided in Stuart et al. 2020 and the
 manuscript accompanying these data on datadryad.org.
 l.series_from_FossilIntervalData_recreatesAppendix1Bell2006.csv has data
 on three armor traits from a higher resolution sample of G. doryssus from
 the same stretch of rock. It contains data for pelvic score index (ips),
 number of dorsal spines (mds), number of pre dorsal pterygiophores (mpt)
 and the number of pterygiophores that are touching (ptt). Interval denotes
 the temporal sample into which a sample was binned. specID is the fossil
 specimen number. Missing data code: NA ## Sharing/Access information Data
 was derived in part from the following sources: Bell, M.A., J.V.
 Baumgartner, and E.C. Olson. 1985. Patterns of temporal change in single
 morphological characters of a Miocene stickleback fish. Paleobiology 11:
 258-271. Bell, M.A., M.P. Travis, and D.M. Blouw. 2006. Inferring natural
 selection in a fossil threespine stickleback. Paleobiology 32: 562-577.
 Stuart, Y.E., M.P. Travis, and M.A. Bell. 2020. Inferred genetic
 architecture underlying evolution in a fossil stickleback lineage. Nature
 Ecology and Evolution 4: 1549-1557. ## Code/Software
 "FvE.RS.FINAL_forDataDryad_240201.R" contains the script that
 recreates figures in the accompanying manuscript. It should run in order
 because some objects created at the top of the script are necessary later.
 This file was updated with code to produce Figure 1 new Panel A and new
 figure S2. "FvE.RS.FIGURES_forDataDryad_240312.R" contains the
 script that recreates figures in the accompanying manuscript. It should
 run in order because some objects created at the top of the script are
 necessary later. This file was updated to reflect new journal policy
 requiring double-blind anonymity. We removed annotation that referred to
 author names.