DOI: 10.5061/dryad.t76hdr86q
title: High sexual display trait diversity without measured genetic divergence in a montane hybrid zone involving young species (Habronattus americanus subgroup, Araneae, Salticidae)

description: Genetic introgression, allele exchange across species boundaries, is a
 commonly recognized feature of animal evolution. Under such a paradigm
 contemporary contact zones provide first-hand and complementary insight
 into the geographic, phenotypic, and genetic details of introgression.
 Also, when mate choice phenotypes are conspicuous and variable in hybrids,
 contact zones provide potential insight into how sexual selection
 interacts with species boundary maintenance, particularly when postzygotic
 reproductive isolation is weak. The Habronattus americanus subgroup
 includes several recently evolved jumping spider species, with an
 estimated age of about 200,000 years, and substantial evidence for
 hybridization and introgression. We explored a contact zone involving H.
 americanus (Keyserling, 1885) and H. kubai (Griswold, 1979) on Mount
 Shasta, California, in alpine habitats that would have been unavailable
 (under ice) at the Last Glacial Maximum. We characterized morphological
 diversity within the contact zone, including the fine-scale geographic
 distribution of hybrid and parental individuals, and assessed genetic
 variation using ddRADseq data. Combined results indicate a lack of
 measured genomic differentiation between specimens with distinct
 morphologies, including individuals with phenotypes of the parental
 species. We identified a diverse array of hybrid morphologies, with
 phenotypic evidence for backcrossing, essentially forming a phenotypic
 bridge between parental taxa. The study area is characterized by more
 hybrid than parental individuals, with a significantly larger number of
 red-palped morphologies than white and/or yellow-palped morphologies; the
 novel, white-palped phenotype is perhaps transgressive. Overall, these
 results contribute to a better understanding of the expected ebb and flow
 of lineage interactions during the early stages of speciation.
description: <strong><em>Specimen Collection.
 </em></strong>The Mount Shasta study site is located just west
 of Panther Meadows in the Shasta-Trinity National Forest, at an elevation
 of approximately 2300 meters, on the south flank of Mount Shasta. We
 surveyed for spiders in an approximately 270,000 square meter area.
 Habitat included a southward extending open area of mostly treeless
 high-elevation pumice field, with herbs and low shrubs, and adjacent
 high-elevation coniferous forest. A small, paved road winds through the
 study site. <em>Habronattus americanus</em> subgroup members
 prefer open habitats, so we spent most survey time out of the coniferous
 forest. We did not sample the more easterly Panther Meadows proper, with
 meadow edge microhabitats that we typically associate with <em>H.
 kubai</em>. Special permits were not required to collect spiders
 from this location. Evidence of hybridization at this
 location was previously identified by the collection of male specimens
 with combined traits of both parental species. In July 2018 a team of
 three persons collected 89 specimens from across the study site, including
 78 adult males, 9 females, and two penultimate males. We visually scanned
 ground habitats at random and attempted to collect all adult males seen
 (hand-collected into glass vials); because females of the two different
 species cannot be morphologically separated, we limited our collection of
 adult females. All specimens were georeferenced where collected using a
 Garmin Etrex 20x handheld GPS device.
 <strong><em>Morphological Data Collection and
 Analysis. </em></strong>Eleven discrete morphological
 characters were scored for all 78 adult male specimens. Our sample
 includes individuals described as <em>H. kubai</em> and
 <em>H. americanus, </em>with specimens possessing character
 combinations typical for these species (see description above and Bougie
 et al. 2021). The remaining specimens are described as <em>H.
 americanus </em>x <em>H. kubai </em>hybrids. We scored a
 subset of morphological characters from Bougie et al. (2021), focusing on
 characters that varied within the Mount Shasta samples. Characters were
 scored by examining individuals in 100% ethanol under a dissecting
 microscope. We scored hue (not vibrancy) for some characters,
 distinguishing red from white from golden/yellow. Acknowledging that color
 can fade for specimens preserved in ethanol, the specimens that we scored
 were relatively fresh (2018 collections) and preserved in 100% EToH at
 -80°C; we found that most of the pigmentation hue was readily scorable for
 these specimens. To summarize morphological variation
 and identify morphological clusters we performed a non-metric
 multidimensional scaling (NMDS) analysis of Bray-Curtis dissimilarity
 values, using the metaMDS function in the R package Vegan v2.5-6 (Oksanen
 et al. 2020). <strong><em>Morphological
 Characterization of the Contact Zone. </em></strong>To
 statistically determine if there are more hybrids than parental morphs, we
 performed a corrected one-sided one-proportion z-test to compare the
 proportion of hybrids to a target proportion of 0.5, using the function
 ‘prop.test()’ in R v4.0.3 (R Core Team 2020). The null hypothesis is that
 the proportion of hybrids is not different from 0.5; the alternative
 hypothesis is that the proportion of hybrids is significantly larger than
 0.5, indicating more males with mixed characters than males with parental
 characters. To test if the number of differently colored palped
 individuals was skewed in any direction, we performed two
 Χ<sup>2</sup> goodness of fit tests. The first test included
 all male individuals with red, white, or yellow palps, while the second
 test only included hybrid phenotypes – parental phenotypes were excluded.
 Both tests shared a null hypothesis that there are equal numbers of red,
 yellow, and white-palped individuals across the study site. To further
 explore spatial characteristics of the zone we mapped the spatial
 distribution of both parental forms and each hybrid type. For ease of
 visualization, we binned hybrid morphs into red, white, or yellow
 categories. Since the contact zone that we sampled is
 oriented along an elevational gradient from the south (2270 m) to the
 north (2424 m), we tested for a relationship between palp color and mean
 elevation. We performed a one-way analysis of variance (ANOVA) using the
 function ‘aov()’ in R v4.0.3 (R Core Team 2020), with elevation as the
 dependent variable and palp color as the categorical independent variable
 with red, white, and yellow categories. We also performed a post hoc Tukey
 HSD test to identify if any comparison of means between specific pairs of
 palp color groups was statistically significant, using the ‘TukeyHSD()’
 function in R v4.0.3 (R Core Team 2020). To test for a
 relationship between palp color and habitat type we recorded the number of
 red, yellow, and white-palped individuals in two (barren and shrub) of
 four recorded land cover types (barren, evergreen forest, developed, and
 shrub), classified according to the National Land Cover Database (NLCD)
 2019 Land Cover dataset (Wickham et al. 2021). We only used the barren and
 shrub types because there are very few samples in the evergreen forest and
 developed types. Additionally, while the land cover shows individuals
 collected on a developed road, these individuals were not actually
 collected on the pavement, but nearby. Individuals collected in either
 evergreen forest or “developed” were re-coded to barren or shrub depending
 on which of the two land types was closest to them. Since the observations
 are binary (barren or shrub), we performed a Χ<sup>2</sup>
 goodness of fit test with our expected counts of red = 23.5, yellow = 9,
 and white = 5.5. The null hypothesis was that there is no relationship
 between palp color and habitat type. To test for a
 correlation between morphology and geographical location we performed a
 Mantel test using the Bray-Curtis dissimilarity matrix and a Haversine
 geographic distance matrix, using the R v 4.0.3 function ‘mantel()’ with
 999 permutations in the Vegan v 2.5-7 package (Oksanen et al. 2020).
 Haversine distances were calculated using the R v 4.0.3 function ‘distm()’
 in the geosphere package, version 1.5.10 (Hijmans 2019).
 <strong><em>Molecular Data Collection and Analysis.
 </em></strong>The molecular sample included 72 adult male
 samples, two penultimate males, and eight females collected at the focal
 site. We also included a handful of individuals collected from nearby
 <em>H. americanus</em> (Gumboot Lake, ~30 kilometers distant)
 and <em>H. kubai</em> (Grass Lake, ~31 kilometers distant)
 populations. These non-focal <em>H. kubai</em> and
 <em>H. americanus</em> populations are distant enough that it
 is unlikely they have contributed to the current dynamic near Panther
 Meadows. At both non-focal locations, we only found a single species,
 without evidence for hybrids, but acknowledge that our sample sizes here
 (less than 20 total individuals per site) may not be large enough to
 detect rare hybrids. Two to three legs were used for
 DNA extraction, performed using the Qiagen DNeasy Blood & Tissue
 protocol (Qiagen, Valencia, CA). DNA quality and quantity were evaluated
 using gel electrophoresis and a Qubit Fluorometer, respectively. We used
 double digest restriction-site associated DNA sequencing (ddRADseq) to
 gather genomic-scale data. We used the protocol described in Brelsford et
 al. (2017), using SbfI and MseI enzymes – a combination that increases
 sequencing depth while accounting for large <em>Habronattus
 </em>genome sizes (~5.6 Gb, Gregory and Shorthouse 2003). Sequencing
 was completed at Novogene using 150PE reads on an Illumina Hiseq 4000
 platform. Raw sequence read data were demultiplexed
 using Stacks v2.5.0 under default settings. After demultiplexing, the
 reads were processed using Stacks with the --bound_high flag set to 0.05,
 --min-maf flag set to 0.05, and --min-samples-overall to 90 to require 90%
 of individuals across the dataset to process a locus. All other parameters
 were left as default. We conducted a Structure 2.3.4
 (Pritchard et al. 2000) analysis under the admixture model using 2182
 unlinked SNPs, including both focal and non-focal site samples. Structure
 was run from K 1 to 4, each replicated three times using the package
 Rrunstruct (Anderson 2015) and function ‘structure_runs()’ in R v4.0.3. To
 identify possible genetic divergence between different morph types we
 estimated pairwise F<sub>ST </sub>between palp color types
 using all ddRAD loci (5873 SNPs), conducted in Arlequin v3.5.2.2
 (Excoffier and Lischer 2010). We calculated F<sub>ST
 </sub>using different palp-colored individuals as “populations” and
 a separate analysis using the three different palp-color hybrid groups and
 the two parental morphs as “populations.” Females, two intermediate-palped
 males, and individuals from non-focal sites were excluded from these
 analyses. We tested for a correlation between Haversine
 geographical distances and Euclidean genetic distances using a Mantel
 test, with the R v 4.0.3 function ‘mantel()’ with 999 permutations in the
 Vegan v 2.5-7 package (Oksanen et al. 2020). Euclidean genetic distances
 were calculated on a matrix of 6359 unlinked SNPs using the ‘dist()’
 function in the R package stats (R Core Team 2020). To test for a
 correlation between genetic and morphological distance, we performed an
 additional Mantel test using the same R function as above between the
 Bray-Curtis dissimilarity matrix of morphological characters and Euclidean
 genetic distances.
description: # High sexual display trait diversity without measured genetic divergence
 in a montane hybrid zone involving young species (Habronattus americanus
 subgroup, Araneae, Salticidae)
 ## Description of the data
 and file structure Supp. Table S1. Collection information and sequencing
 statistics for all specimens. Sample ID = voucher number for individual
 specimens; Morphological Analysis (x=included in analysis); ddRADSeq
 (x=included in analysis); palp color = intermediate highlighted in yellow;
 Raw reads & Loci - "n/a" specifies that RADSeq failed for
 specific specimen. Supp. Table S2. Scored morphology matrix. Morphological
 scorings for individual specimens (see Supp Table S1), as follows:
 **Character A** (Iridescent scales on clypeus). * **States** 0 = no
 iridescent scales; 1 = full rectangular; 2 = ‘m’ shaped; 3 = ‘m’ shaped in
 middle, extends low to sides; 4 = four low semi-circle broken segments; 5
 = two irregular iridescent patches separated at the center; 6 = very low
 iridescent rectangle; 7 = two broken semi-circle segments; 8 = four
 connected semi-circle segments; 9 = iridescent rectangle halfway up the
 clypeus and expanding at ends. **Character** **B** (Clypeal covering
 emarginate: clypeus covered with two scale types and/or colors forming a
 well-marked white transverse band). * **States** 0 = absent/no white band;
 1 = spans entire length of AER; 2 = present only under AMEs. **Character**
 **C** (Color of non-iridescent setae on clypeus, NOT including white
 transverse band if present). * **States:** 0 = all of clypeus covered in
 iridescence; 1 = brown; 2 = black. **Character** **D** (Clypeal covering
 divided in center). * **States:** 0 = absent/not divided; 1 = divided.
 **Character** **E** (Color of hair pencils/hairs covering chelicerae). *
 **States:** 0 = blue; 1 = pale/white; 2 = yellow/gold. **Character** **F**
 (Leg I femur: Color of ventral side). * **States:** 0 = brown/dark; 1 =
 white/pale; 2 = speckled tan and black; 3 = white stripe/patch, rest
 brown. **Character** **G** (Leg I femur: pattern). * **States:** 0 =
 longitudinally striped; 1 = banded at the patella joint, no stripe.
 **Character** **H** (Leg I tibia: Color of ventral side). * **States:** 0
 = brown/dark; 1 = pale; 2 = speckled pale and black; 3 = white patch, rest
 brown/dark. **Character** **I** (Leg I tibia: pattern). * **States**: 0 =
 longitudinally striped; 1 = speckled; 2 = plain; 3 = banded at joints, no
 stripe; 4 = cross between speckled and striped. **Character** **J**
 (Palpal patella color). * **States:** 0 = uniform, yellow/gold; 1 =
 uniform, white/pale; 2 = uniform, red; 3 = nonuniform, mostly white/pale,
 some red. **Character** **K** (Color of hairs covering dorsal cymbium). *
 **States:** 0 = uniform, yellow/gold; 1 = uniform, white/pale; 2 =
 uniform, red; 3 = nonuniform, pale white with some pale red hairs. Text
 highlights are only meant to demarcate particular individuals or character
 states, and are included for visual purposes only. Supp. Table S3. Hybrid
 versus parental proportions, based on one sided proportion z-test. Supp.
 Table S4. Χ2 goodness of fit test for palp color counts. Supp. Table S5.
 Χ2 goodness of fit test for palp color counts of only hybrid types. Supp.
 Table S6. Χ2 goodness of fit test for land cover type. Supp. Table S7.
 Pairwise FST estimated using all ddRADseq loci. Supp. Figure S1. Digital
 images of specimens collected from the Mount Shasta hybrid zone, including
 parental H. americanus and H. kubai morphs, representatives of red-palped
 hybrids, a representative yellow-palped hybrid, and a representative
 white-palped hybrid. Not all 19 diagnosable morphs are included. All
 photos by M. Hedin. Images not to scale. Supp. Figure S2. Land cover
 classifications as defined by the National Land Cover Database.