DOI: 10.5061/dryad.r4xgxd2m1
title: Phenotypic and genetic divergence in a cold-adapted grasshopper may lead to lineage-specific responses to rapid climate change

description: Aim: Species responses to global warming will depend on intraspecific
 diversity, yet studies of factors governing biogeographic patterns of
 variability are scarce. Here, we investigate the evolutionary processes
 underlying genetic and phenotypic diversity in the flightless and
 cold-adapted grasshopper Sigaus piliferus, and project its suitable space
 in time. Location: Te Ika-a-Māui Aotearoa—North Island of New Zealand.
 Methods: We used mitochondrial sequences to investigate population
 connectivity and demographic trends using phylogeographic tools and
 neutrality statistics. Metric data were used to document phenotypic
 variation using naïve clustering. We used niche metrics to assess
 intraspecific niche variation, and niche modelling to investigate
 suitability under past and future scenarios. Multiple matrix regressions
 with randomization explored the processes contributing to phenotypic
 differentiation among grasshopper populations. Results: Niche models and
 demographic analyses suggest suitable space for this grasshopper was more
 restricted during glacial than interglacial stages. Genealogical
 relationships among ND2 haplotypes revealed a deep north-south split
 partly concordant with phenotypic and niche variation, suggesting two
 ecotypes that have mixed during recolonisation of the central volcanic
 region. Multiple matrix regressions with randomization indicate a link
 between climate and phenotypic differentiation inferred from leg and
 pronotum dimensions but not pronotum shape. Niche projections predict
 severe habitat reduction due to climate warming. Main conclusions: The
 current distribution and intraspecific diversity of S. piliferus reflect
 complex biogeographical scenarios consistent with Quaternary climates and
 volcanism. Phenotypic divergence appears adaptive. Current levels of
 genetic and phenotypic variation suggest adaptive potential, yet the pace
 of anthropogenic warming over the next 50 years could result in small
 populations that may collapse before adapting. Differences in niche
 features between diverging intraspecific lineages suggest distinct
 responses to climate change, and this has implications for prioritising
 conservation actions and management strategies.
description: ## README: Phenotypic and genetic divergence in a cold-adapted grasshopper
 may lead to lineage-specific responses to rapid climate change: Open
 Source Data We have submitted the data used for (1) genetic (Genetic
 data.zip), (2) morphological (Morphological data.zip), and (3) ecological
 niche modelling (ENM data.zip). ### Description of the data and file
 structure **##1. Genetic data.zip** This dataset consists of a 747 bp
 alignment of mitochondrial ND2 sequence for 186 individual *Sigaus
 piliferus* plus one sequence of *Sigaus villosus*, which was used as an
 outgroup in the phylogenetic analysis. Whole genomic DNA was extracted
 from leg muscle using a solvent-free Proteinase K and salting-out method
 (Trewick & Morgan-Richards, 2005). Extracted DNA was amplified by PCR
 for the mitochondrial protein-coding NADH-dehydrogenase 2 (ND2) gene under
 standard conditions, using the primers HopND2_147F and HopND2_1286R
 (Carmelet-Rescan et al., 2021). Sequencing reactions used BigDye
 Terminator v.3.1 (Life Technologies) with signal capture on an ABI-3730xl
 System (ThermoFisher). DNA sequences were edited and aligned in GENEIOUS
 vR10 (Kearse et al., 2012), and then exported in Phylip format as Sigaus
 piliferus.phy. The mtDNA sequences used to generate this file are openly
 available in Genbank, accession numbers PP277795–PP277981. **#1.1. Sigaus
 piliferus.phy** Phylip file of the alignment of ND2 partial gene used for
 the population genetics and phylogenetic analysis. This file consists of
 187 lines (rows), one for each sequence in the alignment. Each row
 consists of a sequence identifier (ID) followed by characters in the
 sequence (747 bp in this case). The sequnce indentifier (ID) followed a
 fiexd format: population_geographic region_specimen identifier. **##2.
 Morphological data.zip** This dataset consists of the two files used for
 metric and geometric morphometrics of phenotypic variation. For metric
 analysis we used four non-independent body dimensions (hind femur length,
 FL; hind femur width, FW; pronotum length, PL; pronotum width, PW) and two
 ratios (FL/FW and PW/PL) were recorded from 156 adult grasshoppers (n =
 100♀, 56♂) using an Olympus SZX7 stereomicroscope with image capture and
 Olympus cellSens Dimension v1.6 software (Olympus Corporation, Japan).
 Geometric analysis of pronotum shape used 14 digitalised landmarks on the
 dorsal perimeter of the pronotum in images from 161 adult grasshoppers (n
 = 101♀, 60♂) using TPSDIG2 2.29 (Rohlf, 2015). Images were taken using a
 Canon EOS 600D with EF100 mm f2.8 USM macro lens (Canon Inc., Japan)
 mounted on a vertical stand (Kaiser Fototechnik, Germany). Only females
 were analysed hereafter due to better sample size and more homogeneous
 geographic sampling compared to males. **#2.1 body shape.TPS** Coordinates
 and scale of the landmarks used for geometric morphometrics analysis. This
 file consists of 100 entries, one for each image. The first line (row)
 indicates the number of landmarks (14 in this case), following for 14
 lines containing the pairs of coordinates for each landmark. The next line
 (IMAGE) indicates the name of the image in this case denoting the specimen
 identifier (ID), followed for a line including specimen information (ID):
 specimen ID, sex, and genetic lineage based on genetic analyses. The last
 row indicates the image scale. LM=14 1355.00000 2173.00000 1410.00000
 2650.00000 2019.00000 2508.00000 2342.00000 2436.00000 2535.00000
 2379.00000 2899.00000 2356.00000 2942.00000 2015.00000 2895.00000
 1682.00000 2527.00000 1655.00000 2336.00000 1622.00000 2013.00000
 1534.00000 1402.00000 1384.00000 1351.00000 1865.00000 1460.00000
 2037.00000 IMAGE=GH002.JPG ID=GH002-F-North SCALE=0.005263 **#2.2 body
 size.xls** Linear dimensions used for traditional morphometrics analysis.
 This file consists of 156 entries (n = 100♀, 56♂), yet only females were
 analysed. For each entry, there are 13 rows: *Code:* specimen identifier
 (ID) *Femur_length:* femur length (mm) *Femur_width:* femur width (mm)
 *Pronotum_length:* pronotum length (mm) *Pronotum_width:* pronotum width
 (mm) *Latitude:* absolute latitude (degrees) *Longitude:* absolute
 longitude (degrees) *Elevation:* elevation (m) *Population:* population
 name *Region:* geographic region *Year:* collection year *Sex:* male (M)
 or female (F) *Lineage:* North or South **##3. ENM data.zip** This dataset
 consists of a presence-absence matrix (geodetic datum WGS84) including
 data for 14 related, endemic New Zealand grasshopper species, spanning
 1967 to 2016 (Koot et al., 2022), plus recent sample collections and
 verified, high resolution observations (uncertainty &lt;1km2) from
 iNaturalist NZ. We also included target-group absences (Mateo et al.,
 2010) consisting of sites where sampling effort has produced other
 Orthoptera (Gryllidae and Tettigoniidae), but not *S. piliferus*.
 Duplicate records were removed ensuring one datum per pixel (i.e. ~1 km2
 habitat area). The resulting database consisted of 1,188 site records,
 including 1,053 absences and 135 presences, representing the realised
 niche of *S. piliferus.* **#3.1. Sigaus piliferus.csv** Presence/absence
 data used for niche modelling and analysis. This file consists of 1,188
 entries, and for each entry, there are 3 rows: *Latitude:* latitude
 (degrees) *Longitude:* longitude (degrees) *Spiliferus:* presence (1),
 absence (0) As potential niche proxies we used 19 bioclimatic layers from
 WorldClim v1.4 for five periods (MIROC-ESM and NCAR-CCSM climate models):
 last interglacial at ∼120–140 kyr (LIG), last glacial maximum at ~28 kyr
 (LGM), mid-Holocene at ∼6 kyr (MH), current (1960–1990), and future
 (2061–2080). We projected two future scenarios with mean temperature
 increases of 1.4 °C (RCP4.5) and 3.0 °C (RCP8.5) in New Zealand by 2090
 (Ministry for the Environment, 2018). Layer files were at 30 arc-seconds
 resolution (~1 km2), except LGM which was at 2.5 arc minutes (~5 km2). The
 climate data for ecological niche modelling and niche analyses are freely
 available at
 [https://www.worldclim.org/data/worldclim21.html](https://www.worldclim.org/data/worldclim21.html). **##4. References** Trewick, S. A., & Morgan‐Richards, M. (2005). After the deluge: mitochondrial DNA indicates Miocene radiation and Pliocene adaptation of tree and giant weta (Orthoptera: Anostostomatidae). *Journal of Biogeography*, *32*(2), 295-309. Carmelet‐Rescan, D., Morgan‐Richards, M., Koot, E. M., & Trewick, S. A. (2021). Climate and ice in the last glacial maximum explain patterns of isolation by distance inferred for alpine grasshoppers. *Insect Conservation and Diversity*, *14*(5), 568-581. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., ... & Thierer, T. (2012). Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. *Bioinformatics*, **28**(12), 1647–1649. Rohlf, F. (2015). The Tps series of software. *Hystrix, the Italian Journal of Mammalogy*, *26*, 1–4. Koot, E. M., Morgan-Richards, M., Trewick, S. A. (2022). Climate change and alpine adapted insects: Modelling environmental envelopes of a grasshopper radiation. *Royal Society Open Science*, *9*: 211596. Mateo, R. G., Croat, T. B., Felicísimo, A. M. & Muñoz, J. (2010). Profile or group discriminative techniques? Generating reliable species distribution models using pseudo-absences and target-group absences from natural history collections. *Diversity and Distributions*, *16*(1), 84–94.