DOI: 10.5061/dryad.7sqv9s4x1
title: Discovery of facultative parthenogenesis in a New World crocodile

description: Over the past two decades, there has been an astounding growth in the
 documentation of vertebrate facultative parthenogenesis (FP). This unusual
 reproductive mode has been documented in birds, non-avian
 reptiles—specifically lizards and snakes—, and elasmobranch fishes. Part
 of this growth among vertebrate taxa is attributable to awareness of the
 phenomenon itself and advances in molecular genetics/genomics and
 bioinformatics, and as such our understanding has developed considerably.
 Nonetheless, questions remain as to its occurrence outside of these
 vertebrate lineages, most notably in Chelonia (turtles) and Crocodylia
 (crocodiles, alligators, and gharials). The latter group is particularly
 interesting because unlike all previously documented cases of FP in
 vertebrates, crocodilians lack sex chromosomes and sex determination is
 controlled by temperature. Here, using whole-genome sequencing data, we
 provide the first evidence of FP in a crocodilian, the American Crocodile,
 Crocodylus acutus. The data support terminal fusion automixis as the
 reproductive mechanism; a finding which suggests a common evolutionary
 origin of FP across reptiles, crocodilians, and birds. With FP now
 documented in the two main branches of extant archosaurs, this discovery
 offers tantalizing insights into the possible reproductive capabilities of
 the extinct archosaurian relatives of crocodilians and birds, notably
 members of Pterosauria and Dinosauria.
description: DNA extracted from the mother and fetus using a Qiagen DNeasy
 Blood & Tissue kit was sent to Novogene (Sacramento, CA) for whole
 genome sequencing on an Illumina platform (NovaSeq 6000 PE150). Raw
 sequences were mapped by Novogene to the Saltwater crocodile,
 <em>Crocodylus</em> <em>porosus</em>, reference
 genome with single nucleotide polymorphisms (SNPs) identified by Novogene
 using the following the command in SAMtools: mpileup -m 2 -F 0.002 -d
 10. Following the parameters used in Card et al. as a
 guideline, variants were filtered using VCFtools v. 0.1.16, the R package
 vcfR, and bedtools v2.30.0, with the following criteria: (1) indels were
 excluded; (2) individuals with a read depth of less than 5 were excluded;
 (3) variants with a Phred quality score below 30 were excluded; (4)
 non-biallelic SNPs were excluded; (5) SNPs with significant statistical
 biases were removed using the hard filter ‘MQ &lt; 40.0’; (6) SNPs
 were thinned to avoid the potential effects of linkage by randomly
 selecting one variant per 10 kb, 25 kb, and 50 kb region of the saltwater
 crocodile (<em>Crocodylus</em> <em>porosus</em>)
 genome, resulting in three sets of filtered VCFs. Prior to thinning, SNPs
 that were not found in both individuals were removed using the bedtools
 v2.30.0 ‘intersect’ function. For each set of VCFs,
 files were prepared for input to program ParthenoGenius. First, maternal
 and offspring VCFs were merged into a single VCF with BCFtools. PGDspider
 was then used to convert each merged VCF into Structure file format.
 Structure files were modified to remove column two which contained
 Structure PopData values, such that the resulting files contained only the
 sample IDs (mother = M2, offspring = P1) and genotypes at each SNP
 retained after filtering. These files were then converted into CSV
 format. ParthenoGenius was used to test for evidence
 and mode of parthenogenesis for each of the three thinned SNP data sets.
 Briefly, ParthenoGenius is a python program that first compares the number
 of the mother’s homozygous loci for which the offspring does not have
 identical genotypes to the mother against the number expected due to
 genotyping error alone based on a per-base genotyping error rate to
 determine whether discordance between maternal and offspring genotypes at
 maternal homozygous loci is more likely due to sources of genotyping error
 or the presence of paternal alleles. If the number of maternal homozygous
 loci for which the offspring has non-identical genotypes to the mother is
 less than the number expected due to genotyping error alone (i.e., the
 offspring is homozygous at all or nearly all of the mother’s homozygous
 loci), the offspring is called as a parthenogen and the proportion of
 maternal homozygous loci for which the offspring’s genotypes differ is
 recorded by ParthenoGenius as an updated estimated per-base error rate for
 the following heterozygosity scan. This assumes that genotyping error rate
 is consistent across the genome. If parthenogenesis is supported,
 ParthenoGenius then scans maternal heterozygous loci to identify those at
 which the offspring has retained heterozygosity for maternal alleles. If
 the number of maternal heterozygous loci at which the offspring is
 heterozygous is less than the number expected based on genotyping error
 alone assuming a null hypothesis of gametic duplication (i.e., the
 offspring is homozygous for maternal alleles at all or nearly all of the
 maternal heterozygous loci), the mode of parthenogenesis is called as
 gametic duplication. Alternatively, if the number exceeds that expected
 due to genotyping error alone (i.e., the offspring has retained
 heterozygosity at some or all maternal heterozygous loci), the mode of
 parthenogenesis is called as automixis (although there is no test to parse
 terminal from central fusion automixis). A per-base error rate of 0.1% was
 used for the initial homozygosity scan of the three thinned data sets,
 representing a conservative estimate of genotyping error rate on par with
 that of a filtered SNP array.