DOI: 10.5061/dryad.z08kprrcz
title: Mixed mating in a multi-origin population suggests high potential for genetic rescue in North Island brown kiwi, Apteryx mantelli

description: Reinforcement translocations are increasingly utilised in conservation
 with the goal of achieving genetic rescue. However, concerns regarding
 undesirable results, such as genetic homogenisation or replacement, are
 widespread. One factor influencing translocation outcomes is the rate at
 which the resident and the introduced individuals interbreed.
 Consequently, post-release mate choice is a key behaviour to consider in
 conservation planning. Here we studied mating, and its consequences for
 genomic admixture, in the North Island brown kiwi Apteryx mantelli
 population on Ponui Island which was founded by two translocation events
 over 50 years ago. The two source populations used are now recognised as
 belonging to two separate management units between which birds differ in
 size and are genetically differentiated. We examined the correlation
 between male and female morphometrics for 17 known pairs and quantified
 the relatedness of 20 pairs from this admixed population. In addition, we
 compared the genetic similarity and makeup of 106 Ponui Island birds,
 including 23 known pairs, to birds representing the source populations for
 the original translocations. We found no evidence for size-assortative
 mating. On the contrary, genomic SNP data suggested that kiwi of one
 feather did not flock together, meaning that mate choice resulted in
 pairing between individuals that were less related than expected by random
 chance. Furthermore, the birds in the current Ponui Island population were
 found to fall along a gradient of genomic composition consistent with
 non-clustered representation of the two parental genomes. These findings
 indicate potential for successful genetic rescue in future Apteryx
 reinforcement translocations, a potential that is currently underutilised
 due to restrictive translocation policies. In light of our findings, we
 suggest that reconsideration of these policies could render great benefits
 for the future diversity of this iconic genus in New Zealand.
description: Blood sampling and
 morphometric measurements of the birds were conducted in accordance with
 the Kiwi Best Practice Manual (Robertson & Colbourne 2017), the
 Massey University Animal Ethics Committee (MUAEC) permits 06/05, 07/144,
 16/92, and 18/83, and the Department of Conservation Wildlife permits
 AK-14969-RES, AK-21519-FAU, 50249-FAU and 70875-RES. Samples from the
 parental populations were collected in 2020 and 2021. For birds fitted
 with transmitters (most of the Ponui Island birds analysed, all the
 studied pairs, and most of the Hauturu birds from the Remutaka Forest and
 the Pūkaha National Wildlife Centre), blood sampling and measuring
 occurred together with the annual transmitter replacement. A licenced kiwi
 dog assisted with catching the remaining birds, which were caught
 specifically for this study. The density of kiwi has been estimated to be
 around one kiwi per three ha both in Trounson and on Hauturu (in 2007 and
 2008, respectively; (Holzapfel<i> et al.</i>
 2008)(Craig<i> et al.</i> 2011, Craig 2019)). Age of the birds was mostly
 unknown, hence individuals were considered adults if they were known to
 have bred or, when the breeding status was unknown, based on their sex,
 size, and weight combined; females were considered adult if weighing
 &gt; 2000 g, or &gt; 1700 g if having a tarsus width (TW) &gt;
 11 mm or a bill &gt; 113 mm; males were considered adults if weighting
 &gt; 1700 g, or &gt; 1400 g if having a tarsus length (TL)
 &gt; 90 mm or a bill &gt; 90 mm. Bill length,
 tarsus length, and the ratio between these measurements were used to
 explore differences in size distribution among adult birds from Hauturu,
 Trounson, and Ponui Island. Linear modelling (lm) in R (R core team
 version 3.6.2) was used for this analysis. To analyse size assortative
 mating, Pearson correlations between female and male morphometric values
 were investigated. The measurements analysed were TL, TW, bill length,
 weight to TL ratio, bill length to TL ratio, and body condition was used
 for 17 Ponui Island pairs. Body condition was calculated based on weight
 and TW following Taborsky and Taborsky (1999). DNA was
 extracted from 10-50 µl thawed <i>A. mantelli</i> blood using
 a High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland). The
 manufacturer’s instructions were followed with the exception that the DNA
 was eluted twice using 50 µl of elution buffer for each centrifugation
 round. The DNA extraction success and quality were validated using agarose
 gel electrophoresis (1% (w/v) agarose in 1x TAE buffer) and the
 concentration of DNA was measured with a Qubit 2.0 fluorometer using the
 dsDNA High Sensitivity assay (Life Technologies, CA, USA).
 Pair-ended Genotype-by-sequencing (GBS) library, sequencing
 preparation, and associated quality checks were done by The Elshire Group
 Limited. GBS libraries were constructed using 100 ng of genomic DNA, 1.44
 ng of adapters, the restriction enzyme EcoT22i, and 18 PCR cycles, and
 otherwise following the protocol presented in Elshire<i> et
 al.</i> (2011). For this study, sequencing of 150 unique birds
 across three 96-well plates were analysed. Sample location within plates
 was randomised and each plate contained one positive and one negative
 control. Sequencing was performed on an Illumina HiSeq XTen with 2 x 150bp
 paired-end reads. The previously published <i>A.
 mantelli</i> genome (Le Duc<i> et al.</i> 2015) was
 deemed suboptimal as a reference genome for our study based on being (1)
 highly fragmented and (2) a composite genome of multiple individuals
 representing two separate management units within the species. Raw
 short-read data generated by Le Duc <i>et al.</i> (2015) was
 accessed from the European Nucleotide Archive (Study Assession RJEB6383)
 and the ERR519283_1.fastq.gz date file was used to re-assemble a reference
 genome based on a single individual. Raw reads were aggressively trimmed
 using trim_galore (https://github.com/FelixKrueger/TrimGalore) before
 assembly with Meraculous 2.2.5.1 (Chapman<i> et al.</i> 2011)
 using a kmer size of 35 and heterozygous mode 1. The resulting reference
 genome utilised for our analyses was haploid, with heterozygous regions
 collapsed into a single haplotype chosen at random between the two
 possible sequences. Processing of raw read data,
 including filtering, trimming, alignment, and SNP calling was conducted by
 Tea Break Bioinformatics. The 1 538 639 658 raw sequencing reads were
 demultiplexed using Axe (axe-demux; Murray & Borevitz 2018),
 adapters and barcodes were trimmed using the batch_trim.pl script
 (https://github.com/Lanilen/GBS-PreProcess) using default parameters.
 Forward and reverse reads were pair-matched and aligned to the reference
 genome using Bowtie 2 (Langmead & Salzberg 2012) using default
 parameters. SNP-calling was conducted in
 STACKS 2.5 (Catchen<i> et al.</i> 2013) using the populations
 program set for the EcoT22i enzyme, bootstrapping, and site merging.
 Initially, this was done for each plate separately. The graphical output
 from Kinship-using-GBS-with-Depth-adjustment program (KGD; Dodds<i>
 et al.</i> 2015) and Tensorflow Projector
 (https://projector.tensorflow.org/) were then used to verify the absence
 of bias or batch effects after which Stacks 2.5 was rerun for the combined
 dataset of all three plates using the following command line: --vcf -r 0.1
 --min-maf 0.1 -e ecoT22I --ordered-export --bootstrap --merge-sites
 --genepop --structure --fasta-loci --fasta-samples --fasta-samples-raw
 --write-single-snp. These settings allowed for up to 90% missing data per
 locus (-r 0.1) to maximise the number of individuals included in the
 resulting dataset. SNPs derived from different cut sites that had
 overlapping read coverage were combined into single loci (--merge-sites).
 To ensure robust SNP identification and to restrict noise, the minimum
 minor allele frequency was set to 10% (min-maf 0.1). The output was filtered to only
 include the first variable site per locus
 (--write-single-snp) resulting in 51691 SNPs that were
 utilised for analyses with an average depth of 24 reads (with a standard
 deviation of 12), and an average call rate of 0.6 (with a standard
 deviation of 0.07). The generated output provided as text
 files formatted for genepop (see below), and fasta files for loci and
 samples (--genepop --fasta-loci --fasta-samples). For relatedness analyses, known
 first-degree relatives (known offspring and siblings) were excluded and
 these analyses contained 20 known pairs and a total of 74 Ponui Island
 individuals. Unscaled pairwise relatedness values were derived using KGD
 (Dodds<i> et al.</i> 2015). This matrix of relatedness values
 is based on pairwise proportional allelic similarity but accounts for read
 depth (including missing data) which can lead to values greater than one
 and below zero. These values were used to evaluate whether paired
 individuals were more or less related than expected under a scenario of
 random mating in two ways. Test one explored whether the relatedness of
 paired individuals to their partner was higher or lower than the average
 relatedness of the paired individuals to all other Ponui Island birds in
 the data set. This approach provided an indication of whether paired
 individuals were more closely related than expected by chance (a
 prediction of assortative mating) or more distantly related than expected
 by chance (a prediction of disassortative mating). To account for possible
 bias caused by cryptic clusters (in particular if a few paired individuals
 belonged to a cluster very distantly related to the rest), test one was
 complemented with test two. Test two involved ranking the relatedness of
 the female to the male and <i>vice versa</i> for each pair and
 then comparing these results to expectations for ranked relatedness from a
 computer simulation scenario of random mating. A ranking of 1 suggested
 that the partner represented the least related bird within the dataset and
 a ranking of 73 that the partner was the most related bird in the dataset.
 Random mating was simulated using 10*40 randomly drawn ranks between 1 and
 73. T-tests were used for
 both these tests. In addition, the average
 relatedness of the male and female in each pair on Ponui Island
 (<i>n</i> = 23 Ponui Island pairs) to the birds from Hauturu,
 was used to categorise pairs as being ‘similar’ or ‘different’ in their
 genetic makeup). Based on the average relatedness to the Hauturu birds for
 106 Ponui Island, the relatedness space was split into four quadrants to
 categorise pairs. Birds were classified as ‘similar’ when both male and
 female were found to be less related or both more related to Hauturu than
 the average value. Birds were classified as ‘different’ when the male was
 more and the female less related than the average or <i>vice
 versa</i>.
description: The following data files accompany the publication “Mixed mating
 in a multi-origin population suggests high potential for genetic rescue in
 North Island brown kiwi, Apteryx mantelli”. <br> The data are from
 the A. mantelli populations in the Trounson Kauri Park, on Hauturu-o-Toi
 (also known as Little Barrier Island), and on Ponui Island.<br>
 Provided as CSV files are:  Morphometric
 data compared between adults of three populations. This file identifies
 sample origin, taxon identity, sex, tarsus length, tarsus width, bill
 length, and bill length to tarsus length ratio.
 Morphometric data compared among 17 pairs of birds. This file
 identifies 17 pairs and the female and male bill length to tarsus length
 ratio, the tarsus length, the weight to tarsus length ratio, the bill
 length, the weight, the tarsus width, and the body condition score using
 sex specific or non-sex specific conversion.  A
 relatedness matrix including all 150 individuals analysed. This file is
 the output from Kinship-using-GBS-with-Depth-adjustment program, with
 metadata added to indicate known firstdegree relatives, taxon identity,
 population origin, and birds ID. Relatedness values
 and ranked relatedness values among 20 analysed pairs. This file includes
 the allelic relatedness between paired birds, the ranked relatedness
 between each paired birds and its partner, the average relatedness of each
 bird to all birds in the dataset, the ranked relatedness based on random
 simulation, the average value for all paird relatedness in the population,
 the average ranked relatedness based on simulation. 
 Requests for raw reads from the underlying GBS
 sequence library should be made to Richard Witehira:
 richardwitehira@xtra.co.nz or Andre Witehira: andre.witehira@gmail.com. 
 Permission would then be sought from the kaitiaki (guardians) representing
 hapu (sub-tribes) that affiliate with the areas of sample collection. Kiwi
 are taonga (treasures) to the indigenous Māori people of Aotearoa New
 Zealand. All individuals, samples, or genomic data obtained from taonga
 species have whakapapa (genealogy, connections, and belonging) and are
 considered taonga in their own right. Tikanga Māori (Māori customary
 practices) determines their use.